Biophysics of Electron Transfer

and Molecular Bioelectronics

ELECTRONICS AND BIOTECHNOLOGY
ADVANCED (EL.B.A.) FORUM SERIES

Volume 1 FROM NEURAL NETWORKS AND BIOMOLECULAR

ENGINEERING TO BIOELECTRONICS
Edited by Claudio Nicolini
Volume 2 MOLECULAR MANUFACTURING
Edited by Claudio Nicolini
Volume 3 BIOPHYSICS OF ELECTRON TRANSFER AND
MOLECULAR BIOELECTRONICS
Edited by Claudio Nicolini

A Continuation Order Plan is available for this series. A continuation order will bring delivery
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Biophysics of Electron Transfer
and Molecular Bioelectronics

Edited by

Claudio Nicolini
Institute of Biophysics
University ofGenoa
Genoa, Italy

Springer Science+Business Media, LLC

 Library of Congress Cataloging-1n-Publicat1on Data

81ophysics of electron transfer and molecular b1oelectronics 1 edited

by Claudio N1col1ni.
p. cm. -- CElectronics and biotechnology advanced CEL.B.A. 1
forum series , v. 31
Includes b1bl1ographical references and index.
ISBN 978-1-4757-9518-9 ISBN 978-1-4757-9516-5 (eBook)
DOI 10.1007/978-1-4757-9516-5
1. Metalloproteins. 2. Charge exchange. 3. Molecular
electronics. I. Nicolinl. Claudio A. I!. Ser1es, Electronics and
biotechnology advanced foum ser1es , v. 3.
RC552.M46856 1998
572 · .43--dc21 98-31320
CIP

Proceedings of the 1997 International Workshop on Biophysics of Electron Transfer: Fundamental

Aspects and Applications, held October 8 ~ I 0, 1997, in Bressanone, ltaly
ISBN 978-1-4757-9518-9

© 1998 Springer Science+Business Media New York

Originally published by Plenum Press, New York in 1998
Softcover reprint of the bardeover 1st edition 1998
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PREFACE

The present volume is a continuation ofthe EL.B.A. Forum Series which was initiated
in the spring of 1992 in Marciana Marina (Italy), with the first volume entitled From Neural
Networks and Biomolecu/ar Engineering to Bioelectronics published by Plenum Press in
1995.
Bioelectronics-miginally introduced in April, 1987, at a symposium hosted by
CIREF, a research consortium among leading high tech industries in Novara (Italy)---was
later defined in two successive consensus reports at the first (Bruxelles, 1991) and second
(Frankfurt, 1994) European Union Workshops on this widely interdisciplinary field, as "the
use ofbiological materials and biological architectures for information processing and sens-
ing systems and devices down to molecular Ievel." lt is worth noting that these workshops
gave birth to the first European research program on "lnterfacing Biology with Electronics"
during 1996-1999, following the !arge Programma Nazionale Ricerca on "Technologies for
Bioelectronics" launched by the ltalian Ministry ofUniversities and Research in 1990.
In autumn, 1996, with the second volume, entitledMolecular Manufacturing, the em-
phasis was placed on the ernerging parallel area of nanotechnology, independently initiated
in Palo Alto, Zurich, Genova, Mainz, and Tokyo by various groups (i.e., IBM, Xerox, Polo
Nazionale Bioelettronica, Max Planck Institutes), universities (i.e., Stanford, Genova, Rice,
Tokyo), and organizations (i.e., Foresight, Erato, Fondazione EL.B.A., Frontiers Research,
MITI) of different sizes, scopes, and latitudes.
The present third volume ofthe series highlights the various aspects ofthe biophysics
of electron transfer which has been ernerging as an independent branch of research. This vol-
ume appears at a crucial moment, when significant progress is being made in this field, and
when technologies derived from it are being recognized as critical for the development ofbio-
technology, electronics, and material sciences.
The significant roJe, ofthe Fondazione EL.B.A., the Istituto Cultura Trentina, and the
Istituto Nazionale Biostruttura e Biosistemi in the organization ofthis sixth course ofthe Na-
tional School ofBiophysics in Bressanone, ltaly, October 8-10 1997, with the cooperation of
UNESCO and the ltalian Society ofBiophysics, are duly acknowledged as weil as forattract-
ing and supporting (within the framework of Copernicus project number
ERBIC l5CT9608l 0 sponsored by the European Union) top Ievel scientists for the XIV EL.
B.A. Forum summarized in this volume and part ofthis course.
Polo Nazionale Bioelettronica must be acknowledged for bringing to light this series
and the enormous industrial potential of electron transfer in biopolymers.
I would like to express my gratitude particularly to Mr. Fabrizio Nozza and Andrea
Rossi ofthe Fondazione EL.B.A.

Claudio Nicolini
Member of the National Science and Technology Council
President ofthe Fondazione EL.B.A.

V
CONTENTS

Metalloprotein Engineering for New Materials, Drugs and Nanodevices ........... .

C. Nicolini

Modulation of the Electron Transport System of Oxygenic Photosynthesis . . . . . . . . . . :n

G. Forti and G. Finazzi

Electron Transfer in Mitochondrial Steroid Hydroxylating Cytochrome P450

Systems: RoJe of Adrenodoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
R. Bernhardt

Preparation, Structural Characterization and Functional Coupling of Tethered

Membranes to Solid Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
W. Knoll, N. Bunjes, M. Denyer. C. Heibel, M. Matsuzawa, R. Naumann,
A. Offenhäusser, J. Rühe, E.-K. Schmidt, A. Sinner, and C. Sprößler

Targeted Expression of Mammalian Cytochromes P450scc and P4502b4 in Yeast

Saccharomvces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
M. A. Eldarov, V. E. Sidorovich, G. E. Pozmogova, and K. G. Skryabin

The Molecular Role of the Pufx Protein in Bacterial Photosynthetic Electron

Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 03
F. Francia, P. Turina, B. A. Melandri, and G. Venturoli

Single Electron and Quantum Phenomena in Ultra Small Particles . . . . . . . . . . . . . . . . I 17

V. Erokhin, S. Carrara, and C. Nicolini

Electron Correlation in Quantum Molecular Biophysics: The Case Study of

Hemocyanin ....................................................... 139
P. Fariselli and R. Casadio

Electron Transfer Reactions in Multicopper Oxidases ........................... 161

L. Calabrese

The Optical Biosensor Study of Protein-Protein Interactions within Cytochromcs

P450 Containing Monooxygenase Systems ............................... 173
A. I. Archakov and Y. D. Ivanov

Index ................................................................. 195

VII
METALLOPROTEIN ENGINEERING FOR NEW MATERIALS, DRUGS AND
NANODEVICES

Claudio Nicolini

Istituto di Biofisica, Universita di Genova

Corso Europa 30, 16132 Genova Italy
Polo Nazianale Bioelettronica,
Via A. Moro 17, 57030 Marciana Marina (LI), Italy
Fondazione Elba
Via del Babuino 181, 00187 Roma Italy

INTRODUCTION

The purpose of this chapter is to provide an overview of present efforts on the engineering
thin films of of various metalloproteins, likewise P450 and C cytochromes and Photosynthetic
Reaction Center {RC), from ab initio considerations on the individual proteins in solution up to
the assembly and characterization of monolayers and multilayers. Over the years
metalloproteins, namely P450, C and Photosynthetic Reaction Centers, have become the
proteins of choice among the many currently under study in my laboratories towards the
implementation of drugs, materials and devices for numerous industrial applications. It should
be noted that several molecular manipulation techniques have been recently introduced which
could be utilized in order to optimize the properties of the above cytochromes in a wide range
of applications, namely:
self-assembly (Morgan et al., 1992; Hoffinann et al., 1992; Nicolini et al, 1995);
Langmuir-Blodgett/Langmuir-Shaeffer techniques and their modifications (Nicolini et al.,
1993; Antolini et al 1995; Nicolini, 1996b, 1997), including utilisation of reverse Iipid
micelies (Erokhin et al., 1994) and derivatization (Riccio et al, 1996) to form ordered thin
protein films;
site-directed chernical modifications complementing the above two techniques (Bernhardt et
al, this volume; Paskievitch et al., 1996).
The choice of metalloproteins as the most plausible materials to be used in bioelectronics is
determined by several of their inherent well-documented properties, of which the most
important is that the meta! sites present in those proteins are redox centers across which electron
transfer occurs along highly selective multidirectional pathways (Nicolini, 1996a); this is not the
case for low-molecular-weight electron carriers in inorganic materials such as silicon, that are
unidirectional and more uniformly (i.e., less selectively) reactive with respect to electron

Bwphysics of Electron Transfer and Molecu/ar Bwelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998
transfer. These pathways are considered later in the chapter. Other considerations are as follows
for these classes of proteins:
metal sites are weil characterized by a number of spectral methods, so useful structural and
mechanistic information can be obtained using relatively inexpensive techniques;
• as a result of genetic engineering efforts, the genes for most of these systems are available
and can be expressed in large quantities in yeast or E. coli (Eldarov et al., 1996).
have well-studied biophysical, biochemical and enzymatic properties since they have served
as routine objects for many types of investigations;
have highly asymmetric distributions of electrostatic fields araund them. Same also have
asymmetric distribution of hydrophilic/hydrophobic amino acid residues over their surfaces.
Both of the two factors can be used for controlled association of these proteins into
supramolecular structures like thin films suitable for macroscopic characterization and/or
direct industrial applications.
from X-ray or NMR experiments, three-dimensional structures are known for most of them,
which are good starting points for designing or improving their properties, stabilities and
electron-transfer characteristics in several applications; for few others, likewise P450 sec and
P450 B24, modeling by homology might be the raute, until X ray crystallography and NMR
experiments still in progress will provide the needed informations.

METALLOPROTEINS SYSTEMS

Cytochrome C

Cytochrome Cis a small ["13 kDa, 104 amino-acids] soluble heme protein, which serves to
shuttle electrons between cytochrome reductase and cytochrome oxidase in the respiratory
electron-transfer chain. The high-resolution three dimensional structure of horse heart
Cytochrome C has been recently elucidated. The polypeptide chain is folded into a roughly
globular shape within which a heme packet is formed. The heme group, covalently linked to the
polypeptide chain via condensation of its vinyl peripheral substituents with cysteine in the 14 and
17 positions, is nearly completely buried within the surrounding protein matrix, but forms a
number of hydrogen bonds with nearby polar main-chain and side-chain groups and therefore
occupies a clearly hydrophilic local environment. Only four atoms of one edge of the
protoporphyrin IX heme prosthetic group are exposed to solvent (CMC, CAC, CBC, CMD
carbons), encircled by positively charged Arg and Lys. Two internally bound water molecules
seem to play apart in electron transfer. One internally bound water molecule (Watl25) mediates
a charged interaction between the heme propionate 01A and the guanidinium group of Arg-38.
The other water molecule (Watl12) is inside the protein molecule, centrally buried next to the
heme group.
Horse Cytochrome C has two surface tyrosines, in contact with the solvent, and two that
are buried. One of the latter, Tyr-67, is involved in H-bond donation to the sulphur atom of
Met-80 and a water molecule, which is in turn H-bonded to Thr-78. These are expected to be
relatively weak H-bonds. The other buried tyrosine, Tyr-48, donates and H-bond to a negatively
charged heme propionate group, which is expected to produce a strong interaction. The
pronounced evolutionary conservatism of aromaticity araund the heme group suggests that
these rings have an important roJe.

Photosynthetic Reaction Centre

Reaction Center from Rhodobactei. Sphaeroides. is a !arge (100 kDa) transmembrane

protein made of 3 subunits (L, M, and H) and is involved into the first stage of photosynthesis

2
providing photoinduced transmembrane electron transfer (I 00% quantum yield) in
photosynthetic bacteria (Branden and Tooze, 1991 ). Crystal structure of this protein has been
reported in the Iiterature (Allen et al., I987; Yeates et al., I987). Electron transfer chain formed
by a dimer ofBacteriochlorophyll (Bchl 2), Bacteriopheophytin (Bphe), and 2 Quinones (Qa and
Qb) is shown in figure I along with the schematic subunit structure. In natural conditions Bchl2,
primary donor in the RC electron transfer chain, is reduced by a multiheme cytochrome attached
at the periplasmatic side of the protein; cytochrome is lost during the extraction from the
membrane (Branden and Tooze, 1991). The RC solution here used contains protein molecules
surrounded, in the hydrophobic region, by the detergent used for the extraction, i.e. spreading
solution is made oftwo components.

P450scc Cytochrome

There is no data on seif-exehange in cytochromes P450. This is related to the fact that is,
unlike other cytochromes, a complex enzymatic system involving other coenzymes (Adx and
AdR) in which the electron transfer must proceed in concert with other functions like substrate
binding and oxygen activation, and is performed with the help of cofactors - the iron-sulphur
protein, reductase, and, in some cases, cytochrome B5 (Porter and Coon, I99I ). Also, it follows
from its 3D structure (Figure I) that there is no way of bringing its possible inner electron
transfer pathways close to those of another P450 molecule unless its conformation dramatically
changes. Accordingly, the estimated seif-exehange rate for cytochrome P450 is nearly zero. The
structure of this protein has not yet been determined by X-ray crystallography, so sequence
homology modelling has to be applied to provide a model for this structure. Such a model has
been developed at this Institute, but a model is present also in the Protein Data Bank
(Vijayakumar and Salerno, I992). The key feature of cytochrome P450-catalysed reactions is
the transfer of electrons from NAD(P)H to either NADPH-cytochrome P-450 reductase in the
microsomal system (P450d) or a ferredoxin reductase and a non-haem iron protein (in the case

Figure I. Proposed model for the heme environment of the P450scc. The heme is attached to Cys423, and
its carboxyls interact with Arg357 and Arg421.

3
ofP450scc, it is called 'adrenodoxin', i.e. adrenal ferredoxin) in the mitochondrial (P450scc) and
bacterial systems (p450cam), and then to the cytochrome P450; this Ieads to the activation of
molecular oxygen followed by the insertion of one oxygen atom into the substrate (camphor for
P450cam, cholesterol for P450scc). The action of cytochromes P450 comprises the following
stages (White and Coon, 1980):
1) Substrate binding which perturbs the spin equilibrium of the iron of cytochrome heme and
facilitates the transfer of the first electron.
2) Oxygen binding to the reduced P450 at the sixth position ofthe iron; if, instead of oxygen,
CO is bound to the heme iron, the characteristic absorbance band appears at around 450 nm.
3) Transfer of the second electron through P450. Note that, in liver microsomal P450s, this
electron, can be obtained from cytochrome B5 (Pompon and Coon, 1984).
4) Splitting of the oxygen-oxygen bond for which two protons are required, followed by
dissociation of the product from the cytochrome. This stage is the less studied, but, as with the
final stage of electron transfer in smaller metalloproteins, it can be disregarded for applications
in bioelectronics, while the first three stages can be utilized.
Although the reported electron transfer characteristic time observed spectrophotometrically
for interaction with adrenodoxin is only around 10-2 sec (Tsubaki et al., 1989), its use as a
material for bioelectronics is linked to its specificity (see later) and to its exceptional ability to
form films (Nicolini et al., 1995; Erokhin et al., 1997; and later in this chapter), as weil as a
variety of factors that can be monitored, controlled and engineered that govern its functions
(including that of electron transfer), the most important being substrate or cofactor binding and
related changes in spectral characteristics (Guengerich, 1991). Cytochrome P450scc, like other
P-450s involved in steroid transformations, is indeed very selective for its substrate which makes
it very promising for biosensor applications (Gilles-Gonzalez et al., 1994), namely for
cholesterol (see later).
The main feature of the structure is the residue Cys357 of P450cam (Cys423 in P450scc, as
shown by homology modeling in Figures I and 2) which contains the fifth heme Iigand (a
thiolate anion) determining the spectral and functional characteristics of cytochrome P-450
including electron transfer (Poulos et al., 1985). The peptide containing this invariant residue is
the single most highly conserved P-450 segment and can be readily recognised in P-450s from
all organisms, including P450scc (Black and Coon, 1987; Gonzales, 1990; Kalb and Loper,
1988). Possible electron transfer pathways ofthe protein (Figure 2) should be near this site. The
oxygen-binding pocket is centered on Thr-252 (P450scc analogue: Thr29l), and a mutation to
Ile291 (Paskievitch et al., 1996) confirmed the 280-300 region to be the substrate-binding
domain of P450scc. When the threonine in question wass replaced by bulky isoleueine of
phenylalanine residues, it appeared to cause steric hindrance to the oxygen or CO binding
(Paskievitch et al., 1996). This did also happen upon introduction of bulky residues into the
neighbouring Asn290 position, by a Asn290Ile mutant in which the 450 nm band is absent, in
contrast to the recombinant native protein.
Recently by homology modeling of P450scc.we insert a feature crucially important for
modeling electron transfer, namely, the heme. Accordingly, we inserted the heme into the
P450scc model using homology modeling on the basis of P450cam structure. To do that, we
superimposed (Sivozelhezov et al, in preparation) the structures of the rninimum possible
fragments of the sequences of both proteins, the parts of their heme-binding peptides containing
the highly conserved phenylalanine known to interact directly with the heme and to stabilize the
heme environment, and the cysteine by which the heme is attached to P450s. That was the
Phe416-Cys423 fragment for the P450scc, and the corresponding Phe350-Cys357 fragment for
P450cam. The result is shown in Figure 1. Although there are some weak van der Waals
violations (steric clashes between the heme and the neighboring arnino acid residues Ile 130,
Ser352, Arg357 and Arg421), those can easily be overcome by conformational flexibility of
those residues alone, without major rearrangements ofthe entire P450 structure. This makes the

4
 Figure 2. Equipotential surfaces of ± 1kT/e of electrostatic potential distribution around Cytochrome P450scc.
The two views differ by a 180° rotation around the vertical axis.

model seem adequate. Besides, the obtained model predicts formation of salt bridges between
each of the two negatively charged carboxyl groups of the heme and the positively charged
groups ofP450scc- Arg357 and Arg421 (bottom part ofthe picture). These sah bridges should
stabilize the P450scc heme environment, a prediction I have so far never encountered in the
literature. A critical experiment to show if this model is true would be to change those residues,
by site-directed mutagenesis, into uncharged ones. A reduced stability of the holoenzyme should
be expected. This feature seems to be present not only in P450scc alone, but also in other P450s
in most ofwhich these sequence positions are occupied by either an arginine or a histidine. Both
are able to form sah bridges by accepting protons from the heme carboxyls. This gives
additional support to the presented model. Furthermore the distribution of electrostatic potential
around P450scc. was calculated by solving the Poisson - Boltzmann equation. Charges were
assigned to the Iysine, arginine, glutamic acid, and asparatic acid residues. Histidine residues
were assumed uncharged which corresponds to pH3 7.5 . The heme iron was assigned acharge of
+ 1 i.e. assuming the P450scc to be in its oxidized form . The ionic strength of solution was 0.15
(physiological). The charges of Arg357 and Arg421 were assumed tobe zero. Indeed, if sah
bridges are formed by those residues, their charges would be canceled out by charges of their
sah-bridge partners which are equal in magnitude but opposite in sign (Figure 2).
Equipotential surfaces G=const) of the P450scc electrostatic potential are presented in
Figure 2 for const=+lkT/e (red) and -1kT/e (blue). The unit kT/e (equal to about 38 mV at
room temperature) is chosen to compare the energy of a unit charge in the calculated potential
field, ej, to the energy of thermal motion, kT. The overall electrostatic potential distribution
pattern ofp450scc is largely quadrupolar. The plane ofthe quadrupole is approximately parallel
to the viewing direction in the Figures 2, and tilted about 30°. This allows to tentatively predict
a pattern of P450scc orientation in thin films. Such a pattern is crucially important for
formulation ofupdate rules for cellular-automata modeling ofP450scc thin films. In this pattern,
the molecules should be oriented in such a way as to avoid exposure of their charged areas to
the air-water interface, so the quadrupole plane should be almost parallel to the interface plane.
To check adequacy ofthis pattern, I tried to locate the areas near the P450scc surface at which
the potential is near zero. Such areas could be located near the interface.
Figure 3 shows distribution of electrostatic potential around P450scc for a fixed distance
between the potential surface and the protein-water boundary (instead of fixed potential of
Figures 2). The distance was 5.5 A, which corresponds to the average distance of the surface
from the nearest P450scc charge of about 7 A. This is a good reference point because, at this
distance, the potential of a unit charge equals kT in water at room temperature. Two areas of

5
near-zero potential were located, one near the top and the other near the bottom ofthe molecule
in the chosen projection (Figure 3). To further check the proposed pattem, investigation will be
performed of the amino acid residues of P450scc located in the identified areas of P450scc
surface. Apart from P450scc behavior in the films, this will allow to identify the charged arnino
acid residues which contribute to the electrostatic potential distribution, to eventually predict
effects oftheir mutation on P450scc interaction with adrenodoxin.
For what concems cloning and expression, PCR technique has been used in order to
eliminate the arnino-terrninus sequence ofthe gene-precursor ofthe P450scc. The pTZ19 vector
with the complete P450scc sequence (1 ,8 kb) has been used as the PCR matrix. The following
primers were used as the PCR primers: universal primer with the sequence complementary to
the vectors sequence moredistal to gene cloning site as 3' and the olygonucleotyde (33 bp) 22
base of which are complementary to the initial region of the gene and 11 bp contains recognition
site of Ncol and start- codone ATG as 5'. The product of PCR was obtained by means of the
restriction with Ncol and Bglii of the fragment eluted from agarose gel. This fragment
contained the 5' region ofthe mature gene ofthe P450scc was cloned into pET21d (Novagene)
using compatible sites Ncol e BamHI. In parallel the 3'part (BamBI - EcoRI)of the cDNA of
P450scc was cloned into pGEMEX (Novagene) using BamBI "sticky" - Hindill "blunt" sites" .
To recreate the complete sequence of the mature gene, the 5'part from pET was cloned into
pGEMEX. The correct sequence of the insert was confirmed by the data of the dideoxy chain -
terminator sequence obtained on the automatic sequencer "Applied Biosystems" Model 373A
1.2.0 version. Olygonucleotide corresponded to T7promoter was used as the sequencing primer.
The E. coli strain BL21(DE3) was transformed following the CaC1 2 protocol (Sambrook et al.,
1989). The single colony was inoculated in 10 m1 of Luria - Bertani broth containing ampicilline
and was grown ovemight at 37° C with agitation (120 rpm). The 500 m1 of TB broth was
inoculated with ovemight culture (1/100 of the volume) in the presence of ampicilline. The
culture was grown for 3 h at 37° C at 180 rpm. When the culture achieves the optical density of
0,6 - 0,8 at 600 nm IPTG (final concentration 1 mM) and -aminolevulinic acid (1 mM) were
added. The expressionwas carried out for 24 hat 30 at 120 rpm. The biomasswas collected by
centrifugation at 3000 rpm per 3 minutes at 4° C. The pellet was resuspended in Tris-HCl

Figure 3. The equidistant electrostatic potential surface of Cytochrome P450scc. The distance is 5.5 Afrom the
protein-water boundaJy. Label 1, cp<-lkT/e; Iabel 3, -I <cp<lkT/e; Iabel 2, cp> lkT/e. The two views differ by a
180° rotation around the vertical axis.

6
0,01M (pH 8.0) and sucrose 0,75 M, was treated with lyzozime (100 ng/ml) and was
centrifuged at 3000 rpm per 3 minutes at 4° C. The pellet was resospended in Tris-HCL 20 mM
(pH 7.4), EDT A 1 mM and DTT 0.1 mM. The obtained suspension was sonicated in ice on
Artek sonicator (8 cycles of 15 sec with intervals of 30 sec at 0,5 W). The suspension was
centrifuged (15 minutes, 18000 x g at 4° C) and supernatant was centrifuged at 260000 x g for
1 h at 4° C. The obtained membrane fraction was solubilized in Tris-HCL 50 mM (pH 7.4),
EDTA 1 mM, PMSF 0.5 mM, 20% ofglycerol and 1% ofEmulgene 913 and was centrifuged
(105000 x g for 60 minutes).
KCL was added to the supernatant to the concentration of 50 mM and obtained solution
was transferred on the column with DEAE cellulose equilibrated with KCI 50 mM, Tris-HCL 50
mM, EDT A 0.1 mM, glycerol 20% and Emulgene 0,3%. The cytochrome P450scc was found in
flow-through.
After ovemight dialysis in 50 mM tris-HCL the solution was transferred on the column
with cholate- sefarose equilibrated with NaPi 10 mM. During washing with 10 mM NaPi and
Nacholate 20 mM the colored band was collected.

MONO LA YER ENGINEERING

Protein films, being organized molecular architectures, are objects of detailed investigations
toward the design and realization of new kinds of devices as weil as for the basic understanding
ofnew phenomena resulting from their dense molecular organization (Lvov et al., 1991; Lvov,
and Decher, 1994; Nicolini, 1996c).Among the available techniques allowing to form protein
thin films, Langmuir-Biodgett (LB) one (Roberts, 1990) seems to be the only which can provide
densely packed films with molecular resolution in its thickness and, if necessary, organized in
superstructures i.e. alternating layers offunctional molecules (Kuhn, 1981). This technique (for
a review see Nicolini, 1997 and Figure 4) has been successfully used for depositing films of
different kinds ofproteins such as antihoclies (Erokhin et al., 1990; Turko et al., 1991; Facci et
al., 1994; Tronin et al., 1995), enzymes (Langmuir and Schaefer 1938; Fromhertz and
Marcheva, 1975; Zhu et al .. 1989; Fujiwara et al., 1992; Antolini et al., 1995), cytochromes
(Turko et al., 1992; Erokhin et al., 1994; Tazi et al., 1994), bacterio- (Hwang et al., 1977,
Sukhorukov et al., 1992) and bovine (Maxia et al., 1995) rhodopsins, and of photosynthetic
Reaction Centres (RC) from different bacteria (Alegria and Dutton, 1991a,b; Erokhin et al.,
1992; Facci et al., 1994). In the most of the cases protein functional activity was shown to be
preserved in films (Erokhin et al., 1995). Besides, several new properties, connected with the
nature of the organized molecular assembly rather than related to the specific features of the
individual molecules have been found. Among them, film anisotropy (Facci et al., 1994),
mobility of protein molecules during the deposition (Facci et al., 1995), dramatic increased
thermal stability (Nicolini et al., 1993) ofthe proteins in the film were reported in the literature.
Particularly, RC from different photosynthetic bacteria have been organized in LB films to
study these proteins in an environment similar to biological membranes (Alegria and Dutton,
199a,b; Erokhin et al., 1992; Facci et al., 1994). On the other hand, these films are considered as
promising candidates for future opto-electronics development. For example, solid state
photovoltaic cells have been described and realized (Tamura et al., 1991; Yasuda et al., 1994).
In spite of the amount of works in the field of RC LB films, several questions remains
unanswered so far, namely the effect of surface tension on protein integrity and the roJe of
surface pressure on the anisotropy of the resulting films,
The aim of a recent work, therefore, has been (Facci et al , 1997) to study these aspects. As
it is not possible to use direct structural analyses such as x-ray or electron diffraction, being the
resulting film rather amorphous, the study required the use of several different experimental
techniques and conclusions were drawn basing on the concurrent contributions of each method.

7
 LB films of Cytochromes P450 seem to be also among best candidates for bioelectronic
reasons, P450scc particularly (Nicolini, 1995). The cytochrome P450scc is the hemecontaining
membrane protein structurally similar to cytochromes, but functionally is the typical
monooxigenase (Hanukoglu and Hanukoglu, 1986). The P450scc catalyse the first and rate-
limiting step in the biosynthesis of steroid hormones: conversion of cholesterol to pregnenolone.
Mini electron transport chain for conducting of this reaction with P450scc as key-point of this
enzymatic complex was found in the adrenal cortex mitochondria. Nevertheless, there is only
one publication connected with LB films ofthis protein (Turko et al., 1992). Authors developed
a technique of the formation of Cytochrome P450scc monolayers at the air/water interface and
their transfer to solid substrates. Surface density was estimated with 125 I Iabelied cytochrome
and turned out to be 17 pM/cm 2 Comparison of the value and the thickness of the monolayer,
estimated by interference microscopy, with known sizes of the protein allowed to authors to
conclude that densely packed layer of the protein was obtained at the substrate. The properties
ofthe formed film were not investigated. Moreover, no works on recombinant cytochrome P450
LB films were done, even if recombinant protein seems to be more adequate for technological
reasons, because it represents the homologous population of the molecules with controlled
sequence. Furthermore, it is eheaper and has the high Ievel of purity. Escherichia coli is a
desirable organism for the heterologous expression of proteins owing to its ease of
manipulation, the availability of a variety of cloning and expression vectors, well-understood
genetics and the low cost of the culture (Porter and Larson, 1991 ). In the case of expression of
the metalloproteins the improvement of the purity Ievel of the final product is possible to carry
out by not only using of the specific detergents but also by applying of the affinic resins
(cholate-sefarose) to this proteins during the chromatography purification (Kastner and Neubert,
1991).
The aim of the study is to form LB films of cytochrome P450, both wild type and
recombinant, to study their structure and properties with several experimental techniques and to
compare their behaviour.
In eucariotic mitochondria cytochrome P450scc (CYP11A1) catalyse the key steroidogenic
reaction of cholesterol side chain cleavage to form pregnenolon. The enzyme is active in the
presence of two electron transfer proteins - NADPH-adrenodoxin reductase (ADR) and
adrenodoxin (AD). The first effort was undertaken to prepare Langmuir-Blodgett (LB) films of
P450scc, and their thickness and the amount of adsorbed hemeprotein were evaluated. At
present Langmuir films of proteins are of a particular interest because of the possibility to
receive reproducible, oriented, and well-controlled layers of immobilised macromolecules with
weil defined properties. In spite of the first positive results in P450scc LB films preparation we,
however, still have few knowledge not much about structural and functional properties of
P450scc in thin solid films.

STRUCTURAL CHARACTERIZATION

Isotherms of wild type and recombinant cytochrome P450 monolayers are presented in
Figure 4. Both isotherms were obtained by spreading 10 I of 1.0 mg/ml of protein solution and
compressed with a speed of 3. 5 mm/sec. X axis is expressed in barrier coordinate units, as it is
impossible to calibrate the axis in area per molecule units due to impossibility of calculating the
actual surface concentration of the protein. This problern is general for protein monolayers and
it results from some partial solubility of proteins in the volume of the subphase (Lvov et al.,
1991 ). As it is possible to see in Figure 4, the isotherms are practically identical, indicating that
surface active properties ofthe proteins are more or less similar.

8
 60

e
-.. so
z
!. 40
...:I
~

..."'"'
30
~

Q.
~
u
20
~:I
10
"'
0
0 50 100 150 200 250
barrier coordinate [mm]

Figure 4. -An isotherm ofmonolayers ofwild type (solid line) and recombinant (dashed line) cytochrome P450
at the water surface.

Brewster Microscopy

Brewster angle microscopy ofthe monolayer (Figure 5) revealed homogeneaus distribution

ofthe mater in the layer. This observation indicates the formation ofrather amorphous structure
as no domains were observed. The presence of domains usually means the existence of 2D
crystallites having distingvishable boundaries between them.
Imaging of monolayers of cytochrome P450 at the air/water interface was carried out with
Brewster Angle Microscope (Nanofilm Technologie GmbH, Germany). Monolayers were spreat
at the surface of pure water and compressed with a speed of 30 cm2/min. Images were acquired
when compression was stopped (Paddeu et al, 1997).

Figure 5. Brewster angle microscope image of the monolayer of cytochrome P450. The image size is 600 m ~
400 m. The monolayer was compresse till 20 mN/m.

9
X-ray Scattering

X-ray measurements were carried out with small-angle diffractometer with linear position
sensitive detector (Mogilevski et al., 1984). Cu K radiation ( = 0.154 nm) was used. The
samples were rotated with respect to the incident beam while the intensity in all channels.
Angular resolution of the detector was 0. 0 I o. The curves were acquired in the 2 range of 0.3 -
2.0° (Erokhin et al, 1995).
X-ray reflection curves are presented in Figure 6 for wild type and recombinant proteins
respectively. The curve obtained from the LB film ofwild type protein does not present neither
Bragg reflection, no Kiseg fringes. Such result means that the film is not ordered and there is no
uniformity of the thickness along the sample area. In a case of recombinant protein we see
Kiesseg fringes, whose angular position depends upon the nurober of deposited layers. The
average monolayer thickness, calculated from these data is about 6 nm, corresponding weil both
to the ellipsometric data and to the protein sizes from the Protein Data Bank.

Ellipsometry

Ellipsometric study was performed according to the procedure described in (Tronin et al.,
1995). PCSA null ellipsometer with He-Ne Iaser source (= 632.8 nm) was used for the
measurements. The data were processed according to two-layer model (Tronin et al. , 1994). As
in a case of gravimetric measurements we see the linear increase of the film thickness upon the
nurober of deposited layers (not shown). The average thickness of one monolayer in a case of
wild type protein turned out to be about 4 nm while for the recombinant one about it is 6 nm.
This fact indicates once more that in a case of recombinant protein closely packed monolayer
was built up. In fact, as the protein sizes are identical in both cases, the decreased thickness of
the monolayer in a case of wild type protein can be explained by presence of some empty
regions in the layer. This decreased thickness results from the fact that ellipsometry provides the
averaging through the spot area.

Circular Dichroism Measurements

Circular dichroism (CD) measurements were carried out with spectropolarimeter Jasco J-
710 (Japan). Measurements of CD spectra for samples heated till 95°C were done at heating

1600
1400
~ 1200 - - - - - not treated film
-~·
-
1000 1:
I' --------- thermally treated
·-a soo ,.,
1'. film

§"' 600 ~/'\.

, _.......
... 400 \ ..
200
'~\
..... ~~·~.c..:......
0 ~----~------~----~----~
-- - -~~
--+~- ~+--==~~+=~--~-4
·---
0.06 0. 11 0.16 0.21 0.26 0.31 0.36 0.41
s [1/nm)

Figure 6. Synchrotron X-ray pattem ofLB film containing 20 layers of wild type and recombinant cytochrome
P450.

10
temperature using a Peltier cell (mod. PCT-343, product Jasco, Japan Spectroscopic Co., Ltd.,
Tokyo, Japan). Heating of samples over 100°C was done in a usual Iabaratory oven.
Measurements were carried out at room temperature after cooling the samples. Molar elliptisity
was calculated according to (Nicolini et al., 1993).
The CD spectra recorded for the three different P450 samples (Figure 7) show a very
similar shape with a difference in the "amount" ofthe structure.

A
40000

-40000

wavelength (nm(

• • • • 50°C, I 0 min - · - · - 70°C, I 0 min - - - - 90°C, I 0 min

-20000

wavelength [nm]

- T=25° • • • · T=110°, 10 - - T=150°, 10 - • • T=200°, 10

minuti minuti minuti

c
30000

-20000

wavelength (nm)

- T = 25° • • • · T = 110°, 10 - - T = 150°, 10 - • • T = 200° , 10

minuti minuti minuti

Figure 7. The dependance of the CD spectrum of wild type (A) in solution, B) in LB film) and recombinant (C)
in LB film cytochrome P450 upon temperature.

11
 In order to obtain information about the termostability of the different P450 preparations
we carried out the measurements of the CD signal as a function of the temperature at a fixed
wavelength. In particular we measured the CD value at two different wavelengths: 194 nm
(Figure 7) and 222 nm (Figure 6) in the temperatures range between 25-75°C.
The first order derivative of the CD signal as a function of the temperature gives
information about the melting temperature of the proteins. When the CD signal at 222 nm has
been recorded as a function ofthe temperature we obtained the following melting temperatures:
for wild type Tm=54.1 °C, for native Tm=53.7 oc and for mutant Tm=53.1°C. When the CD
signal at 194 nm has been recorded as a function of the temperature we obtained the following
melting temperatures: for wild type Tm=52.2 °C, for recombinant Tm=51.6°C and for mutant -
Tm=51.8°C. Allthese data show that the thermal stability is very similar in all the three proteins.
CD spectra of LB films of wild type and recombinant protein are similar one to the other
and tothat in the solution (see Figure 7), pointing out that denaturation ofthe protein during the
film deposition cannot be considered as a serious effect. Figure 7 present the variations of the
CD spectra of LB films upon heating for wild type and recombinant proteins respectively. The
behaviour ofthe spectra upon temperature is similar. There is practically no effect ofthe heating
on the secondary structure of proteins, both wild type and recombinant, in LB films up to
150°C, while in the solution the protein becomes to be denatured already at 70°C. Such
behaviour is typical for protein LB films and it was reported for other different types of proteins.
Two factors are responsible for such improved heat stability, namely, molecular close packing
and Jow water content. Previously it was shown, that the molecular close packing plays
dominant roJe in it. Comparative study of LB films of cytochrome P450 wild type and
recombinant revealed the similar surface active properties of the samples. CD spectra have
shown that secondary structure of these proteins is practically identical. Improved thermal
stability is also similar for LB films build up from these proteins. Marked differences for LB
films ofwild type and recombinant protein were observed in surface density and thickness ofthe
deposited Jayer. These differences can be explained by improved purity of recombinant sample.
In fact, impurity can disturb the layer formation, preventing closest packing and diminishing the
surface density and the average monolayer thickness. Decreased purity of the sample can be also
the reason ofpure homogeneity ofthe deposited layers in a case ofwild type protein.

Gravimetrie Measurements

Gravimetrie measurements were carried out by means of home-made gauge with a

sensitivity of 0.57 ng/Hz using quartz oscillators with frequency of 10 MHz. Calibration of the
quartzes balancewas done according to (Facci et al., 1993).0ptical spectra were recorded with
spectrophotometer Jasco 7100 (Japan).

Table 1. Surface density and area per molecule of cytochrome P450 wild type and recombinant
in LB film deposited onto solid substrate.

surface density [ng/mm2] areaper molecule [nm2]

wild type 2.32 40.23
recombinant 3.16 29.53

Results of the gravimetric study of deposited monolayers are presented in Table 1 for wild
type and recombinant protein. Linear dependence of the frequency shift upon the number of
deposited layers indicates the reproducibility and homogeneity of the deposition. Knowing the
molecular weight and dimensions of the protein it is possible to compare the area per molecule
in the film with that for the closely packed system. The comparison of the areas of wild type and
recombinant proteins in deposited layer is presented in Table 1. As it is clear from the table, we

12
have more dense layer in a case of recombinant protein. The area per molecule in this case
(29.53 nm) is of the same order of magnitude as obtained in (Turko et al., 1992) and
corresponds weil to the area per molecule calculated supposing the close packing of molecules
and taking the sizes from the Protein Data Bank. The protein molecule can be estimated as a
block with the following sizes: 5 n~6 n~4 nm, giving, thus, in one cross section the area of
about 30 nm. The value obtained for the wild type protein monolayer is higher, indicating that
closest possible packing was not reached.

FT-IR Spectroscopy

FT-IR spectroscopy and a suitable data processing method proved tobe a very powerfi.il
tool in order to evaluate the structural modifications which occur in proteins when LB films are
obtained in different operating conditions. Infrared spectra were recorded by using a Perkin-
Eimer 1760 FT-IR Spectrophotometer, equipped with a TGS detector and a Perkin-Eimer
mod.7300 computer. The instrumentwas continuously purged with dry air for 15' before data

abs. units

I I I I I I I

1800 cm-1 1400 1359 1209

cm-1

abs. units

1800 cm-1 1400 1359 1209

cm- 1
c 0.03
A

abs. units

1800 cm-1 1400 1359

A
I I I I I I I

cm- 1
1209

Figure 8. FfiR spectra of films obtained by spreading the solution of Cytochrome C in native and denalured
states and LB films ofCytochrome C on silicon wafers in the 1800-1350 cm-1 and 1350-1190 cm-1 regions: A)
solution at pH 7,4 phosphate buffer; B) solution at pH I phosphate buffer (thin line, sample dried at 25°C; thick
line, sample dried at 100°C); C) slowly (A) (thick line) and quickly (B) (thin line) formed LB films.

13
collection and during measurements to eliminate water vapor absorption. The analysis of the
protein aqueous solution was performed by placing 3 I between two disks of BaF2 using a
Teflon spacer 9.3 m thick. Spectra of the buffer were recorded in the same cell and under the
same instrumental conditions as the sample spectra. Difference spectra were generated by an
interactive difference routine to subtract the appropriate solvent spectrum from the spectrum of
each protein solution. Subtraction of water was judged to be appropriate when it yielded an
approximately flat baseline from I900 and I720 cm- 1, avoiding negative side Iobes, and when it
removed the water band near 2130 cm- 1.
The procedure of spectra processing for quantitative analysis by the deconvolution method
has previously been described (Bramanti et al , I996); the Fit Standard Error (FSE) between X-
ray and infrared secondary structure values estimated by our method is 2.5% for -helix, 7.I6%
for structures and 5.I% for other structures (tums and random coils). The qualitative and
quantitative analysis of the conformation of Langmuir-Biodgett (LB) dried films of either
Cytochrome C (Figures 8 and 9, Bramanti et al; I996) or P450 sec (Bramanti et al, I998 in
preparation) on silicon wafers was performed by FT-IR spectroscopy. A deconvolution
procedure was applied to the Amide I band analysis, in order to determine the percentage of the
different secondary structures. Qualitative analysis was performed by exarnining difference
spectra. Films obtained by spreading protein solutions at pH 7.4 and I, dried at 25°C and
I 00°C, on silicon wafers were also examined in order to detect spectral components associated
with denatured protein domains, and to compare them with Cytochromes LB films.

a b

abs. units

4000 3400 2800

cm-1

Figure 9. Fr-IR spectra ofa) Cytochrome C film obtained by spreading the solution at pH 7.4, dried at 25°C, b)
-I
Cytochrome C film obtained by spreading the solution at pH 1, dried at 100°C, c) LB film in the 4000-2800 cm
region.

FT-IR spectroscopy showed that the following important changes characterise LB film
spectra: i) the -helix component is higher (its percentage is 57 and 54% for cytochrome C) than
the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon
substrate (43%), ii) there is an increase in the intensity ofbands attributed to protonated carboxy
group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in
those attributed to deprotonated carboxy groups, iii) the intensity of several bands attributed to
aromatic arninoacids and aliphatic chains increases, and iv) bands due to 0-H stretching
vibrations of crystallisation water are present.
These conformational changes could be induced by protein-protein interaction caused by
the close packing of molecules which occurs during LB film formation; it cannot be excluded
that they may be accompanied by partial changes in the tertiary structure of the protein. A
preferential orientation of protein molecules in LB films can also be suggested. The amide group

14
ofproteins and polypeptides presents characteristic vibrational modes (Amide modes) which are
sensitive to the protein conformation. Amide I (1700-1600 cm- 1 region) is primarily due to the
C=O stretching vibration, Amide II (1600-1480 cm- 1 region) to the coupling ofthe N-H in-plane
bending and C-N stretching modes and Amide III (1350-1190 cm- 1 region) to the C-N Stretching
coupled to the N-H in-plane bending mode. Furthermore, weak vibrations of certain amino-acid
side chains have absorption bands in the 1800-700 cm- 1 region and may make small
contributions to the intensity of characteristic protein Amide bands. The vibrational frequency of
the single components of the Amides is determined by the secondary structure adopted by the
polypeptide chains.

Table 2. Results ofthe frequency deconvolution procedure applied to Amide I ofthe spectra of
aqueous solution, dried films obtained by spreading the protein solution at pH 7.4 and pH 1 on
silicon wafers and LB films ofCytochrome C on silicon wafers. j=N.o gaussian component;_ 1
pj~*= computed peak height; OD=optical density; pj 2 *t computed centrat wavelength (cm );
xj = computed spectral bandwidth at half-height (cm- ).
-I -I
Sampie Oescription j Pjl* 00% Pj2* (cm ) Axj* (cm )
Solution pH 7.4 1 0.31 18 1681 25
2 0.85 49 1657 28
3 0.6 21 1635 27
4 0.23 12 1616 55
Solution pH 1 1 0.15 12 1681 64
2 0.49 39 1658 43
3 0.50 40 1639 52
4 0.11 9 1619 48
film obtained by spreading the protein 1 0.36 22 1684 30
solution at pH 7.4, dried at 25°C 2 0.70 43 1660 28
3 0.18 11 1644 36
4 0.08 5 1637 28
5 0.29 18 1616 90
film obtained by spreading the protein 1 0.36 26 1685 39
solution at pH 1, dried at 25°C 2 0.52 37 1659 31
3 0.36 26 1646 51
4 0.17 12 1616 68
film obtained by spreading the protein I 0.37 22 1684 29
solution at pH 7.4, dried at 100°C 2 0.75 45 1660 27
3 0.2 12 1642 25
4 0.06 4 1637 24
5 0.28 17 1615 85
film obtained by spreading the protein I 0.21 12 1696 38
solution at pH 1, dried at 100°C 2 0.52 31 1660 39
3 0.49 29 1648 69
4 0.35 21 1626 19
5 0.12 7 1611 39
LB film (1120) A 1 0.22 1698 34
2 0.23 17 1683 25
3 0.77 57 1659 34
4 0.09 7 1644 32
5 0.04 3 1631 55
6 0.19 14 1619 83
7 0.02 2 1612 67
8 0.08 1605 80
LB film (1120) B 1 0.21 17 1689 25
2 0.06 5 1682 42
3 0.67 54 1659 35
4 0.13 10 1644 41
5 0.01 1? 1631 31
6 0.16 13 1619 232
7 0.17 1604 82

15
 Figure 8 shows the FT-IR spectra in the 1800-1350 cm" 1 and 1350-1190 cm· 1 regions of
native and denatured Cytochrome dried films, obtained by spreading the protein solution, and
LB films on silicon wafer. Due to the very low value of the intensity of the FT-IR spectrum of
LB films, the highest Amide band (Amide I, 1700-1600 cm- 1 region) was used in order to obtain
quantitative information about the secondary structure composition.
Table 2 summarises all the results for the samples examined, obtained by applying the
deconvolution procedure to the Amide I region ofthe FT-IR spectra. Figure 9 shows the FT-IR
spectra in the Amide I region of the samples examined, compared with the spectra
reconstructed. The results obtained on native protein solution are in agreement with the data
obtained by other techniques.

FUNCTIONAL CHARACTERIZATION

The absorbance spectra of recombinant P450scc before and after the chromatography on
cholate-Sepharose are presented in Figure 10 a and b respectively. It is easy to note the
improvement of the purity Ievel and the quality of preparation ( ratio ~8/A420) of recombinant
sample with respect to wild type. All three types of cytochrome P450scc (wild type, native and
mutant) show the typical maxima of absorbance at 280 nm, 390 nm and 420 nm that correspond
to intact P450scc.
The purity ratios (calculated as the ratio between the absorbance at 390 nm and the
absorbance at 280 nm) have been measured for each preparation and gave the following results:
wild type A,90/~ 80= 0,6, native A390/~80 =0,7, mutant A3'x/~80 =0,7. These results show that
the purity of the recombinant cytochromes is higher respect to the wild type one and depends on
the type of purification.
The absorbance spectra of the proteins (Figure 10) have been recorded before and after
normalizing the sample to the concentration of the high spin fraction of the protein by using the
absorbance value at 390 nm. From these data we can see that native and mutant recombinant
proteins exhibit an identical spectrophotometric behavior. With respect to wild type P450 we
can see an increased peak at 420 nm that becomes more pronounced than the peak at 390 nm.

0.03

0.025

0.02
"'cV
"'...
~
0.015
~
~
< 0.01

0.005

0
350 400 450 500 550 600 650 700
Wavelength (nm)

Figure 10. Typical absorbance spectrum of iso1ated P450scc at neutral pH and room temperature (about 70 % is
in a high-spin form) (thin solid line). Absorbance spectra ofLB films both on quartz and on hydrophobised
quartz (thick solid line). Dashed line- solubilized in 0.3 % sodium cholate and l M NaCI.

16
 This can be explained as more reduced state of these citochromes. CO-reduced differential
spectra (Figure 11) recorded for a period of 150 minutes allow us to obtain the kinetics of CO
binding to P450: this kinetic gives information about potential functionality of the samples. The
CO binding kinetics are reported in Figure 11 before and after normalizing the samples to the
concentration ofthe high-spin fraction ofthe protein (by using the absorvance value at 393 nm).
It is evident that the affinity of mutant and wild tye P450 are very similar while the native
recombinant P450 exhibiths a very lower affinity for CO. This effect can be due to different
hidrofobicity of the mutant and recombinant protein during extraction from inclusion bodies in
the case of heterologous expression into E.coli and by this way more less darnage effect of
detergent used (Emulgene 913).

Spin Transition in LB Film of P450 Cytochromes

It has been known, that physicochemical and enzymatic properties of cytochrome P450
family depend on their spin state. Both high-spin (S=5/2) as welllow-spin (S=l/2) states are two
distinct configurations of five 3d electrons in the heme iron. It has been established, that low-to-
high spin conversion results in heme iron Iiberation from sixth Iigand and it displacement out of
heme plane. Spectrally low to high and high to low spin conversions of cytochrome P450 are
weil determined as changes in position of Soret band which is located at 393 nm (for high-spin
state) or at 416 nm (for low-spin state). To investigate the spin-state equilibrium P450scc was
used often as a model for other P450 species. Spin-state of this hemeprotein is influence by
temperature, pH and depends on binding with substrates and AD Here we present results of our
investigations of P450scc LB films (Figure 11 and Table 3). There is striking fact that in LB
films P450scc, which was before the deposition in high-spin form, exist in low-spin form. This
phenomenon is reversible because after Solubilisation of films P450scc converts its state again to
high-spin state. Moreover we studied electron transfer properties of P450scc in LB films and
found their dependence from the low-spin state of the hemeprotein.
In accordance with the conventional purification procedure isolated P450scc contains one
or two cholesterol molecules in each hemeprotein molecule. At neutral pH and room
temperature about 70% ofthe hemeprotein is in a high-spin form . Typical absorbance spectrum
of such kind of preparation which we used in our experiments is shown in Figure 1. This
spectrum has peaks at 393, 526 and 645 nm that is characteristic of oxidised substrate-bound
high-spin P450scc. After deposition of P450scc LB films both on quartz and on hydrophobised
quartz absorbance spectra of hemeprotein were changed. They had peaks at 416, 53 5, 570 nm
and a shoulder around 360 nm (Figure I 0). There is no peak at 645 nm. This spectrum is usual

0.01
0.008
0.006
8 0.004
~... 0.002
0
"'
..0 0
<_o.oo:t4 475 500
-0.004
-0.006
-0.008
-0.01

Figure II. P450 content measurcd by reduced CO-difference spectrum after deposition-solubilisation treatment
(thick solid linc), of the initial preparation (thin solid line) andin LB films (dashcd line).

17
for low-spin substrate-free hemeprotein. Nevertheless, cholesterol in 50 mM final concentration
did not cause any visible changes in the position of the Soret band of the hemeprotein.
We were interested to desorb the hemeprotein from the supports to understand the
influence ofLB films preparation procedure on the stability ofP450scc: is this low-spin form of
P450 or this is rnixture of P450 and its inactive form, P420. With the aim to diminish
electrostatic and hydrophobic interactions of proteins molecules with each other and with
supports we used 0.3 % of sodium cholate and 1 M NaCI. LB films of the hemeprotein were
adhered rather strong to the supports. As yield from the absorbtion spectra of LB films after 1
hour Solubilisation treatment only about 25 % of immobilised proteins were liberated from the
films. The Soret band of the solubilised hemeprotein has the position at 406 nm that is
intermediate for low- and high-spin states (Figure 10 and Table 3). Indexes of spin-state
equilibrium of initial, LB film deposited and solubilised preparations of P450scc are summarised
in Table 3. As seen from Table 3, P450scc in LB films has spin-state equilibrium index equal to
that of low-spin Tween 20-induced preparation and after the Solubilisation from the films spin-
state equilibrium index is intermediate for low and high-spin states ofhemeprotein.

Table 3. Indexes ofspin-state equilibrium ofP450scc.

Preparation
P450scc initial 1.4 +/- 0.05
P450scc in LB film 0.55 +/- 0.0 I
P450scc after desorbtion from LB films 1.03 +/- 0.0 I
P450scc low-spin (Tween 20-induced form) 0.56 +/- 0.01

After deposition-solubilisation treatment P450 content was measured by reduced CO-

difference spectrum (Figure 11 ). P450 composed about 93 % of the whole hemeprotein content
and was almost equal to P450 content (96 %) in the initial preparation. This fact clearly
confirms that P450scc was stable during the experiment although reduced CO-difference
spectrum ofP450scc in LB films marked presence both P450 and P420 (Gouriev et al; 1996).
As a functional test ofP450scc LB films activity we studied the reduction ofthe CO-bound
hemeprotein in the electron transport chain NADPHJEADRJEADJEP450scc. lt was impossible
to detect the reaction in aerobic conditions. In differential spectra the reduction was seen only in
the presence of glucose oxidase-catalase deoxigenative system by the increase of absorbtion at
420 nm and 450 nm. Hence the rate of P450scc reduction is comparatively slow because in
aerobic conditions soluble molecular oxygen is able to reoxidise hemeprotein with the rate which
exceed P450scc reduction. The results indicate also that electrons can be donated to LB film
incorporated P450scc from the soluble AD and that there is no difficulties in mutual orientation
of reacting molecules. Sodium dithionite is able to reduce P450scc in LB films too. As evident
from absorbance changes in CO-difference spectra (Figure 11) the amount of reduced
hemeprotein is higher for the treatment with this reagent.
1t is known that buoyancy of proteins at the air-water interface and surface tension can
influence on a conformation ofprotein molecules and their functions. To check the influence of
these factors on spin-state equilibrium of P450scc we prepared solid films with random
orientation of the hemeprotein on the support. For this purpose the solution of P450scc was
droped uniformly on the surface of hydrophobised quartz plate. Preparation was placed near the
anhydrous silica gel and dried. As in the case of P450scc LB films the spectrum of the
hemeprotein after evaporation procedure has differences from that of the initial preparation in
the solution. It has two unseparated pikes at 3 93 nm and at 416 nm indicating that spin-state
equilibrium was shifted to the low-spin state. Films with random orientation of hemeprotein
were adhered weakly to the supports. Immediately after immersion in to the 50 mM sodium
phosphate buffer they were solved rapidly and completely. Resolved preparation restored the
high-spin state. Deposition-solubilisation treatment did not influence the stability of the

18
hemeprotein. CO-difference spectrum of resolved P450scc was equal to that of the initial
preparation (data not shown).
There are two factors, temperature and pH, which influence the P450scc spin-state
reversibly and do not cause the Iack of cholesterol from the substrate-binding centre of the
enzyme. P450scc spin state shifts from high to low spin when the temperature is increased and
the inverse transition takes places when P450scc is in mitochondrial membranes. Change in pH
from 6.5 to 8.0 Ieads to the high to low spin transition. As in the case of P450scc LB films
substrate addition at high temperature does not convert the enzyme back to the high-spin state.
However, a temperature decrease reverses the spectral changes completely and a change in pH
from 8.0 to 6.5 increases the high-spin content, although no substrate is added. Conformational
transitions of P450scc molecules upon temperature and pH treatment were reported. We
surmise that during thin films preparation P450scc molecules can change their structure too.
They are able to form new intermolecular bonds. Our data showed that protein-protein
interactions are stronger in oriented layers than in the films with the random orientation of
hemeprotein molecules. Conformational changes Iead to the structural changes in aromatic
amino acids around the heme, in particularly. These changes cause a movement of cholesterol
molecule or at least a movement of its side-chain group in the vicinity of heme group. As the
result ofthis movement heme iron changes its spin-state from high to low. It is possible that this
process is influenced by the Iack of water molecules from the protein surface and really high to
low spin transition is affected by the combination oftwo factors: by protein-protein interactions
in particular mutual orientation and by partial dehydration of protein globule.
It was shown that electron transfer activity of P450scc depends on its spin-state. The most
striking effects ofP450scc low-spin conversion are drastic decreases in its redox potentialandin
its complexability with AD. Oxidised AD (and presumably reduced) interacts poorly with
substrate-free low-spin P450scc (Kd = 600 nM) but much better with cholesterol-bound high-
spin hemeprotein (Kd= 30 nM). The midpoint oxidation-reduction potential of AD is about -
270 mV. High to low spin transition of P450scc Ieads to the decrease in its midpoint potential
from- 305 mV to -410 mV. Therefore electron transfer from reduced AD to low-spin oxidised
P450scc is less favourable. One can see that our approval about low-spin state of P450scc in LB
films coincide weil with its low reduction rate in the native electron transfer chain.
It should be pointed out that we do not know the exact orientation ofP450scc in LB films.
Is this orientation uniform or not ? Because in contrast to monolayers in multilayers (10-50
layers) hemeprotein might be partially disordered. So may be that only apart of surface P450
molecules are able for complexation with AD. The other moment is that because of its
dimensions AD interact mainly with P450scc from upper layer (or layers) and do not penetrate
into the inner hemeproteins sublayers. Perhaps one of the reasons why the amount of reduced
P450scc was increased in the case of sodium dithionite reduction is highest penetrating ability of
this reagent.

THEORY OF ELECTRON TRANSFER

The most advanced area of basic research relating to metalloproteins is the study of
electron transfer between them in aqueous solutions. In such an experiment, the following
reaction is typically studied:

(1)

where A and B can be any of the metalloproteins (typically, physiological partners are chosen),
or one of them is a small electron carrier like ascorbate ion or superoxide ion radical. The
varying conditions include temperature, solution pH and ionic strength. Also, changes are

19
introduced into A or B by means of chemical modifications or (a more recent and advanced
technique) site specific mutagenesis. A special case is A = B, termed electron self exchange.
The aims of such studies are:
identification of the contact sites on A and B through which electron transfer proceeds - this
allows to design mutations in order to optimise either the interprotein contacts in the new
structures like thin films and/or the specificity for a given substrate;
elucidating the electron transfer mechanisms and paths within A and/or B to design single
proteins or complexes of proteins with increased electron transfer rates in the desired
direction or in parallel multiple directions while hampering the undesired (parasite) electron
transfer;
The overall electron transfer process in such systems proceeds as shown by the equation
(I) and is best described as a complex chemical reaction comprising the following stages
(Marcus and Sutin, 1985):
1) collision of two protein molecules with or without subsequent formation of a complex
stabilised by electrostatic or hydrophobic interactions. To distinguish between the latter two
factors, dependencies of the rate of the overall electron transfer process on ionic strength are
usually studied, and attempts are made to theoretically simulate such dependencies (Meyer at al.,
1993; Mauk and Mauk, 1989; Cheddar et al., 1989). Understanding this stage is a key to
modelling protein association into structures with pre-defined properlies such as the thin films
shown later.
2) electron transfer itself in the formed complex or during the collision time (in the latter case,
the electron transfer time should be smaller than the average lifetime of the species called
'encounter complex' formed in the first stage). This stage is determined mostly in multiple
e1ectron transfer pathways occuring in thin protein films.
The dassie theories describing electron transfer within proteins is found in (De Vault,
1984; Marcus and Sutin, 1985) and they relate electron transfer rates to the distance between
the redox centers (in our case, meta! sites), and to two thermodynarnic parameters - DG0, the
driving force of the reaction i.e. the difference in redox midpoint potentials of the two reacting
species (zero in the case of self-exchange) and ls, the reorganisation energy characterizing the
overall difference in coordination of nuclei between the initial and final state of the system in
equation 2:

A- + ß__.:ass=.o::..:cci::::ati::::on-+A -ß-e::.::le=ctr=on.::.:trans=fer~A-B-d=isso=cia::ctio::.:.n-4A + B" (2)

For instance cytochromes P450 feature !arge reorganisation energies which is, of course,
related to electron transfer of their main biological transfer. A variation of 20 A in the distance
between donors and acceptors in protein changes the electron-transfer rate by 10 12-fold i.e.,
basically, electron transfer rate decays exponentially with distance. So, basically, the most
important property for electron transfer is the distance between the redox sites.
However, the body of data on lang range electron transfer in proteins (where the site-to-
site distance is prohibitively !arge) raises the question of whether a protein structure can
influence the rate or path of such transfers, and if yes, what is the mechanism. Answering these
questions requires information on which of the various structural elements composing proteins
support lang range electron transfer. In (Lee et al., 1992), evidence is presented for lang range
electron transfer along the alpha-helix of a synthetic leueine dimer.
In more detailed theories (Beratan et al., 1991; Beratan et al., 1992; Koga et al., 1993;
Liang and Newton, 1992; Liang and Newton, 1993; Onuchic et al., 1992; Tang, 1994; Wheeler,
1990) quantum mechanical calculations are performed to generate electron transfer trajectories
for electron transfer.
These are, however, quite expensive computationally, but the main conclusion that can be
derived from their results confirms that the rate of long-distance electron transfer in proteins

20
rapidly decreases with distance, which is indicative of an electron tunneling process. It was also
found that the distance dependence of electron transfer in native proteins is controlled by the
protein's structural motif. The helix and sheet content of a protein and the tertiary arrangement
of these secondary structural units define the distance dependence of electronic coupling in that
protein. The tunneling pathway model applied in (Beratan et al., 1991) was also successfully
used to model intraprotein-protein electron transfer in ruthenium-modified proteins i.e. the
transfer from the newly designed ruthenium sites on the surface of the protein to the native
meta! site inside the protein (Nocera et al., 1984; Bechtold et al., 1986; Willie et al., 1992).
The analysis ranks the average distance decay constant for electronic coupling in electron
transfer proteins and identifies the amino acids that are coupled to the charge localization site
more strongly or weakly than average for their distance. The conclusion is that pi-electronic
system play a major role in assisting electron transfer so the aromatic side chains are more
preferable for electron transfer than non-aromatic. Also, sulphur atoms can play a major role. In
a recent book (Nicolini, 1996a), we identified which of these pathways dominate the particular
electron-transfer reactions of metalloproteins including seif-exehange and cross-reactions. Also
water molecules appear to play an important role as switches for biological seif-exehange and
cross-exchange biomolecular electron transfer. Note that these water molecules are bound to the
protein surface by hydrogen-band interactions and are completely different in their physical
properties from those of the bulk solvent (Nicolini, 1996a). In all electronic applications the
theory of electron transfer has to be extended to thin solid films in the 3D space to optimize
construction of new materials and nanodevices.

Protein Automata

The design of complex molecular functions is a goal difficult to achieve with the classical
approach of chemistry and supramolecular chemistry. Nature developed a !arge manifold of such
'nanomaterials'. Today, it is not possible to construct such complex materials ab initio, but we
can start out from those molecules that we discover in nature and try to modify their properties
and adapt them towards the demands of technical applications. As shown earlier (for a
comprehensive overview see my recent book on Molecular Bioelectronics - Nicolini, 1996a), the
tools that are necessary to put these ideas into practice are now available. Bacteriorhodopsin is a
possible model for this approach, as motivated by the extraordinary properties of this molecule
and because it is one of the few biopolymers where a sufficiently detailed structure-function
relation is available. Not only may the photochromism of bacteriorhodopsin be of technical
importance, but also its photoelectric properties may be of equal value. However, for optical
applications a wide range of classical materials exist which can be used as references for the
evaluation of whether the biological materials can compete with them. The improvements
attained in the photochemical properties of BR demoostrate the future importance and
effectiveness of gene-technological methods in material science. Modification of the genetic
code facilitates the manipulations of organism that can produce these new 'high-tech' materials
with conventional biotechnological methods. Therefore, the price of such products will be
affordable. An intensive screening for functional biopolymers with technically relevant functions
and their variation and affection will create a new class of 'biomimetic' materials. The
experimental studies done with bacteriorhodopsin have shown that this new approach Ieads to
competitive materials in selected areas.
The cytochromes here introduced, constitute another possible model because of their
multiple pathways for the electron transfer being instead unidirectional in all inorganic materials
such as silicon and gallium arsenide. The main goal in molecular electronics is the search for
molecular assernblies that could be used as circuits which process the information at molecular
scale. The physical nature of these structures implies a great number of interacting molecules, so
the assembly of a molecular circuit is not feasible by using the conventional techniques for
integrated circuits. The picture that every microstructure should interact with its neighbours

21
 only, suggests to describe a molecular circuit by means of a logic-functional model
computationally equivalent to a cellular automaton essentially to a device that assembles itself.
Instances may be found in organic mono- and multilayers made by Langmuir-Blodgett
technique: typical examples are protein (Erokhin and Feigin, 1991) or Iipid (Bianco et al., 1994)
films the surface organisation of which shows domains corresponding to a pseudo-crystalline
phase embedded in a fluid Iipid phase. An interesting property comes from the analysis of lateral
interactions between electric dipoles associated to the hydrophilic heads of Iipids in
homogeneous phase. These dipoles have a non-zero component of the moment parallel to the
film surface, and can be considered as basic elements of a molecular circuit. The surface
distribution of such components shows characteristics similar to a cellular automaton: any dipole
depends above all on closest-neighbour dipoles because the interaction decays as the cube of the
distance, and the time evolution of its orientation is ruled by the same law for all dipoles.
The cellular automata (CA) theory appears tobe a low-cost alternative also for the case of
proteins with large dipole moments, e.g. cytochrome C. The dipole monolayer is considered as a
plane surface, with dipoles bound at their centers, embedded in an isotropic, linear dielectric
material (neglecting any induced polarization ofthe dipoles). All dipoles have the same module
of the moment (all molecules are equal), and the component of the dipole moments normal to
the surface of the film is constant. Consequently the planar component of any dipole moment
has the same module and any dipole can be characterized by three variables: coordinates (x,y) of
the baricenter of the dipole and its angle theta with the positive directions of x-axis. The
electrical interactions among dipoles are expressed by the torsional moment produced on each
dipole. The dipole dynamics is ruled by torsional moment and a viscosity term. Taking into
account only the effects of nearest dipoles in the lattice, introducins a time step to discretize
temporal evolution and allowing only a finite sets of values for the orientation theta of a dipole
we have a fully discrete system. The finite difference equation obtained is the updating law of a
cellular automaton.
In 1948, von Neumann embarked on an ambitious project: to show that phenomena as
complex as life - the survival, reproduction, and evolution of complex forms of organisation -
can be reduced in principle to the dynamics of many identical, very simple primitives capable of
interacting and maintaining their identity. First, von Neumann considered the interaction of
vortices and particles in suspension in some 'primordial soup'. Obviously, such a model was
intractable, thus, following a suggestion by Ulam, he adopted a fully discrete approach: space,
time and even the dynamical variables were defined to be discrete. The resulting cellular
automation theory describes Universe consisting of a homogenous array of cells (Figure 12).
Each cell is endowed with a finite number of states and evolves in discrete time according to a
uniform local transition rule; the rule can be seen as a function whose arguments are the states at
time t of the neighbouring cells (and possibly the state of the considered cell itself) and whose
value is the state of the considered cell at time t+ 1. The rule is uniform in that it is the same all
over the array. Since all the cells 'compute' their new state simultaneously, cellular automata are
often seen as a paradigm of distributed computation. Dyson et al ( 1991) and Lent et al ( 1993)
have given lively accounts of the surprising success of von Neumann enterprise, a self-
reproducing cellular-automaton that anticipated the discovery of the duplicative function of
DNA.
Further uses of cellular automata in biology have been numerous (for a bibliography, see
Toffoli and Margoulus, 1987, 1990). The very name 'Life' for the famous cellular automation
that John Convay invented in 1970 still reflects the biology-motivated origin of cellular
automata. The similarities between cellular-automation behaviour and that of many physical
systems are also quite suggestive. Are these similarities which should be exploited in order to
build a protein-based cell automata from heat- proof, homogeneous and highly stable LB film of
any ofthe metalloprotein here described.
In addition to molecular electronics, the fabrication ofprotein arrays(Figure 12) has many
fundamental and industrial goals. For the industry, uniform coating of glass surface with latexes

22
should reduce light scattering, while providing special optical effects. In a matter of fact,
beautiful colors were produced using 55 nm and 122 nm latexes. Consequently, optics, as weil
as cosmetic industries, are potentially interested in such perfect )arge scale uniform coating.
The universally accepted key factor justifying the use of metalloproteins in electronics is the
time required for triggering an electronic device based on them compared to the one based on
the conventional material. Table 4 below shows that :
they have advantage in the spatial scale - provided the circuit design is planar, the factor is
(100 nm/(0.1-1 nm) 2=10 4 - 106 ;
in the theoretical Iimit, the advantage in speed is 103 which makes the overall advantage in
performance per unit of volume of as much as 107-109 which provides a considerable margin
for material designers.

Figure 12. Von Neumann cellular automaton consisting of a homogenous array of cells.

1t should be noted, however, that the data quoted refer to the fastest known electron transfer
reaction in protein systems. For the more practical cases, including the metalloprotein systems
discussed above, the time is a few magnitudes more, typically, 10-6 to 10- 10 s. Butthis is where
the engineering of electron transfer properties of metalloprotein multilayers engineering comes
in.

Table 4. Orders ofmagnitude oftriggering time and size for the conventional and the single-
electron devices.

Triggering time Device size

Conventional 100 nm
(theoretical Iimit)
Single-electron 0.1-1 nm
(Metalloprotein)
Single-electron lOnm
(Metal granules)

23
APPLICA TIONS

Several applications here summarized are presently being explored.

P450scc-Based Cholesterol Biosensor

Cytochrome P450scc belongs to steroid metabolism enzymes. The reaction catalyzed by

this protein is called side-chain cleavage. Accordingly, steroids not possessing a sidechain are
excluded from the range of possible substrates. The reaction proceeds in three stages, with
hydroxylation at carbon atoms C-20 and C-22 preceding scission of the side-chain at C-20.
Accordingly, the three substances for which P450 is highly specific are cholesterol and the two
reaction's intermediates, 22R-hydroxyholesterol and 20,22R-dihydroxycholesterol (Heyl et al,
1986). Which of the three is preferential is determined by the redox and cytochrome-binding
state of the P450 One feature of cholesterol making it the specific P450scc substrate is the
immediate surroundings of the third position (numbers referre to a figure not shown). For
instance, if the hydroxyl group -OH present in cholesterol at that position is replaced by a !arger
ester group -OOCR, where R is an alkyl radical, this derivative is not processed by P450scc.
However, ifthis hydroxyl group in the 3'd position is replaced by a carbonyl oxygen (=0 instead
of -OH), the activity persists but at a smaller rate.
This substrate is called cholestenone (Sugano et al, 1995). If the hydrogen at the same 3'd
position is oriented differently (3-epicholesterol), P450scc activity also persists(Child and
Kuksis, 1983). Effect of structural modification of cholesterol AlB ring on their side chain
cleavage are also apparent(Morisaki et al, 1980). Another essential feature is the absence of
hydroxyl in the 7th position. A cholesterol with this hydroxyl (7alpha-hydroxycholesterol) is not
processed by a P450scc but by the specific oxidoreductase, not a P450. Finally, there is a steroid
called 22,23-dihydroergosterol which is almost identical to cholesterol, only it is has a double
instead of a single bond between the 7th and 8th carbon atoms. In vivo, it is processed, instead of
P450scc, by P45022ds transforming it into ergosterol (which is also known as the provitamin
02). There is no data in the Iiterature whether P450scc acts on 22,23-dihydroergosterol,
although some biological systems possibly containing P450scc, e.g. rat jejunal brush border
vesicles and erythrocytes, are able to distinguish between the two steroids. For different types of
sidechains, the activity mostly persists but at !arger or smaller rates (Morisaki et al, 1980). It
should be possible to identify the type of steroid side chain by measuring the reaction rate.
However, this is valid only if the above-discussed cholesterol core remains the same in the
analyzed molecules production. Also, there may be non-steroid substrates of P450scc. One
example is methoxychlor, a known organochlorine insecticide. However, it is not a regular
substrate. It is processed by P450scc, but P450scc becomes non-functional after processing the
first molecule of methoxychlor it encounters. This class of substances termed "suicide
substrates". There is a P450 for which methoxychlor is a regular substrate, This enzyme is
Cytochrome P450 IIIA1(Stresser and Kupfer, 1997). To conclude, the available experimental
data and theoretical approaches allow to predict effect of mutations on substrate binding, For
P450scc, however, the range of new substrates to search for is limited to steroids because the
experimental data necessary to calibrate theoretical calculations are mostly available for only
that class of substances. Since 1991, several (less than 10) theoretical calculations were reported
for substrate specificity of P450s, but P450scc was not among them and work is in progress in
my Iabaratory.

Reaction Centers Based Photovoltaic Cells

The recent progress in research indicates that the disentanglement of photosynthesis will
give strong impulses to the application of solar cells. Moreover, as far as the bioconversion of

24
sunlight is concerned, it is known that the photosynthesis starts with a charge separation process
in the photosynthetic Reaction Centers (RC): a photoactive bacterial protein. Therefore, several
experiments have been carried out in order to test the possibility of light to electrical energy
conversion by photochemical cells (or simply photocells) containing such photosynthetic
bacterial proteins (Figure 13). The photocells so far developed utilize different geometries and
different immobilization techniques, therefore it is very hard to classify and compare the
reliability and efficacy in terms of energy conversion of such devices. Nevertheless, the results
suggest that the energy efficiency can at best actually reach 40%-50% percent and it crucially
depends on molecular orientation. Usually, such proteins are immobilized by a thin film
fabrication techniques, such as self assembly, electrical sedimentation, Langmui-Blodgett,
polymer matrix or gel entrapment. Among such techniques, in our opinion the most promising
seems tobe the Langmuir-Blodgett one (see references below). In fact, this technique allow to
organize the protein molecules in an ordered 20 array. There are many publications on films of
RC, including ours. The films were characterized from different points ofview, both structurally
by a combination of new biophysical techniques (see reviews below). Our discovery of an
induced heat-proof and temporal stability by our monolayer engineering had opened a big
possibilities in using the protein in molecular devices development in genera, and of
photovoltaic cell in particular.

Elecmc contacts
Metal electrode

RC LBfilm

ITO
coating
ü light
Transparent
electrode

Photocell structure

Figure 13. Photovoltaic cell based on photosynthetic reaction centers.

In the literature, it was suggested by japaneese authors to make a photodevice, using RC

LB film sandwiched between two electrodes (one of them is transparent). Photovoltage,
registered in this work, was 4-5 mV for the film, containing 44 layers. In our work (the last
being published in Langmuir) we have shown, that it is possible to adjust a tilt of RC molecules
in the layer by controlling the surface pressure. From the other hand, for BR it was shown the
possibility to increase anisotropy by electric field application.We can hope, that applying our
technique of electric field assisted deposition and by optimally controlling the surface pressure
we will be able to increase this number (according to existing estimations, there is only about
12% of prevalent orientation with respect to opposite one; with our technique, we can hope to
increase this anisotropy).
The other possible way of increasing the film anisotropy was suggested by Miyake et al
from MITI-Japan in the LB8 conference (privileged comunication). They biased the substrate
during deposition with respect to the water subphase. He reported, that even in this case the
anisotropy of the film was improved. Thus, the films can be useful for the construction of light-

25
to electron energy converting devices like the photovoltaic cell RC-based here discussed,
working in the range ofvisible-near InfraRed spectrum.

Biocatalytic Devices Based on Cytochromes P450 for Monitoring Drug Metabolism and
Steroid Hormone Synthesis.

Recent research is focused on several cytochrome P450 systems, that are involved in vital
metabolic reactions, including detoxification and biosynthesis of physiologically important
steroid compounds. The chosen proteins aims to the development of novel biocatalytic systems
and analytical devices, capable to perform in vitro the essential steps of xenobiotic metabolism
and steroid hormone synthesis typical for human body and to monitor these reactions through
the application ofbiosensor technology, protein chemistry and recombinant DNA technology. In
order to overcome problems of their instability and improve their reactivity, capability for self-
assembly and controlled orientation on surfaces, recombinant DNA technology and protein
chemistry and modeling is utilized.
More specifically, the development of novel biocatalytic systems is based on the
cytochrome P450 1A2 and P4502B4 responsible for the carcinogen activation and barbiturate
metabolism respectively, and adrenal steroidogenic cytochromes P450SCC, P45011beta,
P45017alpha and their partner proteins (see Bemhardt, Archakov and Skryabin in this volume).
The essential feauture is the construction of semiartificial and artificial monooxygenase systems
with the covalently attached redox donor groups, useful in initiation of catalytic reactions by
nonconventional sources - light, meta! ions or electrochemically. The engineered biocatalytic
devices are supposed to be used also for the development of novel approaches for the high
throughput screening of cytochrome P450 inhibitors to predict the impact of these compounds
on drug and xenobiotic metabolism in humans and to construct nanoreactors, capable to tailor
and modify molecules, important in pharmacology and toxicology.
The metabolism of almest all lipophilic drugs and environmental toxins and carcinogens in
animals and humans is performed by the cytochrome P450 enzymes - a !arge group of heme-
containing proteins which catalyze monooxygenation reactions involving the insertion of
molecular oxygen into the substrate (Gonzales, 1990) Besides that, cytochrome P450 species
are involved also in the biosynthesis of physiologically important compounds, such as steroid
hormones, vitamin D3 and prostaglandins.
These significant roles make P450 cytochromes the target of very intensive research in the
field of toxicology, pharmacology, biotechnology, basic studies correlating the proteins 3D-
structure to its substrate specificity and catalytic activity, mechanism of action, electron transfer
(Archakov and Bachmonova, 1990). Consequently, much of current effort is aimed at the
development of tests, that could be used for the evaluation of exact composition of metabolites,
created in the monooxygenase reactions. However, until now such tests have not received
widespread application, and this is mainly due to the multiplicity of cytochromes P450 at the
molecular Ievel as revealed by recent advances in recombinant DNA technology (Degtyarenko
and Archakov, 1993). With the availability of the individual clones of mammalian cytochromes
P450 it became possible to express them in heterologous systems and to isolate the respective
recombinant enzymes in purified form, without the contamination of closely related multiple
P450 forms. This ability is critical to assess the roJe of individual isozymes in drug metabolism,
carcinogen activation or deactivation and also in the development of novel biocatalysts etc.
(Waterman, 1994) However, the direct application of the heterologously expressed P450
proteins for the above mentioned purposes is limited at present by several factors:
1) The in vivo heterologous cytochrome P450 expression systems may not exactly reflect the
pattems of xenobiotic and drug metabolism in human body due to the differences between the
microorganisms and animal cells in the mechanisms, responsible for the transport and processing
of cytochrome P450 substrates and products;

26
 2) The expression Ievels may vary significantly depending on a particular P450 isoform:
3) The reactivity and stability ofthe heterologusly expressed
cytochromes may not be sufficient enough to enable the long-term preservation of their
properties;
4) the utilization of reconstituted systems requires the use of redox partner proteins and
expensive reductants.
A promising route to overcome the mentioned problems and to develop the required
analytical and monitaring devices based on P450 cytochromes in our opinion will be by
interfacing the cytochrome P450 sturlies with the techniques and approaches developed within
the area of bioelectronics - the new exiting scientific discipline that had emerged recently at the
frontier ofbiotechnology and electronics.
However, before the molecular self-assembling capacities of biomolecules will be explored
either directly as potential biomimetic systems, or as nanoreactors for tailoring individual
biomolecules, several key issues that must be considered (Lava! et al, 1995).
(1) the relationship between the individual building blocks and the nanostructure must be
understood (2) methods must be developed for moditying the molecule in a pre-determined way
that will Iead to a desired structure for particular application (3) templating and
micromanipulation techniques must be developed for refining, or moditying, and stabilizing the
resulting structure. There is also growing belief, that combining protein engineering with the
development ofnew coupling techniques capable ofimmobilizing specific sites on the protein on
the support surface may Iead to new and more effective sensors.
For what concerns the P450 cytochromes, this will require also the detailed understandins
of the mechanisms of inter- and intra-protein electron transfer and dioxygen activation in these
systems.
Since 1978 protein-protein interactions in microsomal P450 systems have been studied by
Archakov et al in Russia. It was found that electrostatic forces play an essential role in
orientating the microsomal P4502B4 and the corresponding reductase for efficient electron
transfer. Since 1991 mitochondrial steroid hydroxylating P450 systems are under incestigation.
Adrenodoxin, the electron donor for these steroid hydroxylases has been expressed in E. coli.
CYP 11 B 1 and CYP 11 B2 from human adrenals were cloned and expressed in cell cultures. The
group works on the expression of these P450s in E. coli. Especially with adrenodoxin many
mutants have been produced and as biochemically as weil as physico-chemicaily characterized.
It could be established that the C-terminal portion of this protein plays an essential role in
electron transfer. Deletion mutant 4-108 was found to be 4-5 times faster in transferring the
elctron to CYP11B1 than the wild type protein. Further mutants with decreased as weil as
increased elecron transfer properties were found.
Moreover, a methodological approach to study the stability of this electron transfer protein
has been developed. Mutants with increased stability were characterized. There is fundamental
experience in the production, expression and characterization of mutants of the mitochondrial
steroid hydroxylase system.
This research is likely to yield significant results in the foilowing areas:
• medical sciences, particularly regarding development of novel approaches for high
throughput screening strategies useful in the evaluation of xenobiotic and drug metabolism in
humans, substitutins the particular form of cytochrome P450 with an "artificial microsome"
and thus modelling in vitro the metabolic pathways of a wide range of compounds,
penetrating humans and animals.
• biotechnology, particularly with respect of optimization of heterologous protein expression
systems and protein purification procedures;
• structural biology, in revealing the basic rules correlating the primary structure (i.e. amino
acid sequence) to the tertiary structure, and thereby function, in an attempt to enhance our
presently limited capability to ad hoc engineer proteins for the desired functional application,

27
• nanotechnology and biosensor technology, gtvmg rise to a novel approach for the
development of precisely controlled biocatalysts and analytical devices, capable to tailor,
modify and detect molecules, important in pharmacology and toxicology.
In respect of the Iang-range economic environment of the project, it should be noted, that
the proposed application of nanofabrication technology and biomolecular engineering for the
synthesis and testing of compounds of pharmaceutical interest is considered to be a promising
approach both to speed up the process of discovery and testing of new drugs and their transfer
from the Iabaratory to the market, with concomitant reduction in costs. So, it is likely, that
successful realization of the project goals will yield results, that would be of considerable
interest for microelectronics, biotechnology and pharmaceutical industry- the rapidly growing
sectors in the world market for the high-tech products.

ACKNOWLEDGEMENTS

The present work was carried with the support of the European Union (Copernicus
Program ERBIC15CT960810) ofthe Italian Ministry ofUniversities and Scientific Technologie
Research (Chapter 2102), the Fondazione Elba and the Polo Nazianale Bioeletrtronica.

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31
MODULATION OF THE ELECTRON TRANSPORT SYSTEM OF OXYGENIC
PHOTOSYNTHESIS

Giorgio Forti, Giovanni Finazzi

Centro di Studio CNR sulla Biologia Cellulare e Molecolare delle Piante.

Dipartimento di Biologia deli'Universita di Milano
Via Celoria 26, Milano, Italy.

INTRODUCTION

Oxygenic photosynthesis of green plants and cyanobacteria is a redox reaction, which

converts the energy of electromagnetic radiation (in the visible region of solar spectrum) into
chemical bond energy, utilizing water as an electron donor, and C02 as the acceptor to
generate carbohydrates and other organic substances, according to the overall equation :

(I)

The photochernical system involved, which is bound to the photosynthetic membranes

(the thylakoids) utilizes light energy to transfer electrons against the electrochemical gradient
from H 20 (Ern= 810 mV at pH 7) to NADP (Em=-0.32V, at pH 7).The electrochernical
work of 1.13 eV is accomplished through the cooperation of two photochernical reactions,
in series. Electron transport from H 20 to NADP is coupled to the synthesis of ATP from
ADP+Pi (inorganic orthophosphate).
The stable products of photosynthetic electron transport ATP and NADPH are then
utilized to activate and reduce C02 to the Ievel of carbohydrates by a multienzyme system
present as a dense protein solution in the stroma of the chloroplasts (or in the cytoplasm of
cyanobacteria) where the thylakoids are embedded. This latter process will not be discussed
in this paper, which is limited to the photochemical events and electron transport producing
NADPH and ATP.
In green plants, including the unicellular green eukariotes, the overall process of
photosynthesis is therefore accomplished within the chloroplast (see fig. 1), through the
cooperation of the events occurring within and on the surface of the thylakoids (light
absorption and excitation energy migration to the reaction centers, primary photochemical
reactions, electron transport and ATP synthesis) and those occurring in the stroma (C0 2
assimilation).

Bwphysics of Electron Transfer and Molecu/ar Bwelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 33
Figure 1. (A) Section ofa chloroplast in the cytoplasm ofa spinach leaf; (B) details ofthe grana within a
chloroplast (C) stacks ofthylakoids and the interconnecting stromal thylakoids. Cw, cell wall; pm,
cytoplasmic membrane; m, mitochondrion; g, granum, consisting ofstacks ofthylakoids; s, starch granule, I
Iyposomes, er endoplasmic reticulum.

34
LIGHT ABSORPTION AND UTILIZATION: THE "PHOTOSYNTHETIC UNIT"

The photosynthetic pigments involved in oxygenic photosynthesis are the chlorophylls

a and b (chla and chlb) in green plants, and chla and the phycobilins (phycocyanin and
phycoerithrin, respectively, in Cyanobacteria and the red algae). The structures of chla, chlb
and the chromophore of phycocyanin are shown in fig.2, and the absorption of the
photosynthetic pigments (dissolved in organic solvents) are compared with the solar
spectrum on the Earth surface in fig.3. It can be seen that the photosynthetic pigments
absorb energy through most of the visible part of the solar spectrum, suggesting the
evolutionary adaptation of the photosynthetic apparatus to the ambient conditions. It must
be considered also that the pigments in the photosynthetic apparatus of plants are bound to
different specific proteins in macromolecular complexes, and these are organized in an
ordered topology in the photosynthetic membranes (the thylakoids). As a consequence of
this, the absorption spectra ofthe pigments "in vivo" are narrowed and slightly red-shifted as
compared to their spectra in organic solvents, and a spectral heterogeneity of the light
absorbing chlorophylls is observed (Jennings et al., 1996). In the case of higher plants,
thanks to the peculiar anatomy of the leaves, the high light scattering of the tissues increases
the light path through the pigment containing cells, and even radiation of wavelengths
between 500 and 600 nm, poorly absorbed by the chlorophylls, is efficiently absorbed and
utilized (Garlaschi et al., 1989).

Struc:ure oi bacteriochloropbyll

Chromophore of phycocyanin

COOHCOOH
I I
CH, CH1 CH 1 CH 3
I I I I
-l...__jiJjfH
M WC CHll l lM :tiCHl
M. I I - M-
O~N ~ N & N 0 N~j ··~r
I I I ry"l Struc:ure oi chlorophyU a

Figure 2. The structures ofbacteriochlorophyll, the chlorophylls a and band the chromophore of
phycocyanin.

35
 600 700
Wavelength (nm)

Figure 3. The spectrum of solar radiation reaching the Earth surface and the absorption spectrum of
photosynthetic pigments.

The photochemical utilization of the light absorbed involves two steps: the migration
of the excitation energy (in the form of the excited singlet state of the eh! molecule) within a
!arge array ofproperly packed pigment molecules (the light absorbing "antenna") and which
eventually brings the excited state to a special chla pair (the "reaction center", RC) where
the singlet is oxidized by an electron acceptor in the primary photochemical reaction. The
oxidized RC chla is then reduced by a primary electron donor, so closing the turnover of the
RC, and the light dependent charge separation is performed. The primary acceptor is then
reoxidized by a chain of electron carriers of progressively higher midpoint potential (Ern),
while the primary donor is reduced by a chain of electron donors. The RC requires that the
primary acceptor and the primary donor are regenerated in the proper redox state
(respectively oxidized and reduced) to perform the next photochemical charge separation. A
scheme ofthe functioning ofa RC is shown in fig.S (see Debus 1992, for a review).
The concept of the differentiated roJe of eh! as light harvesting antenna and RC
performing photochemistry was established by the classical experiments of Emerson and
Arnold (Emerson & Arnold, 1932), who demonstrated that upon illumination of Chlorella

Figure 4. Scheme ofprimary photochemical reaction at a RC (sec text). Al,Dl: primary electron acceptor
and donor, respectively; A2, 02; secondary acceptor and donor, respectively.

36
cells with light flashes of a few l.l.S duration, the maximal 02 yield/flash (in the steady-state)
was obtained only if an adequate dark period (of ca. 20 ms) was allowed between flashes.
They interpreted their observations as indicating that the products of the very fast
photochemical reactions must be utilized by kinetically much slower chemical reactions
("dark reactions") to perform the overall photosynthetic process. They thus defined the
"photosynthetic unit" (PU) as the complex structure including antenna pigments, reaction
Centers, electron Carriersand the enzyme systems required to perform 02 evolution (and C02
assimilation). In Emerson and Arnold's experiments, the size of the PU was of ca. 2400 eh!
molecules/ 0 2 evolved per flash (under conditions of maximum oxygen yield/flash). While
the concept of PU is a basic one to understand photosynthesis, the size of PU has been
found to vary in different plants and different growth conditions: in general, growth under
low light intensity induces a !arger antenna for each RC, and vice-versa.

ENERGY TRANSFER TO THE REACTION CENTERS: "THE PHOTOSYSTEM"

Light absorption by the chls in the Soret region generates the second excited singlet,
which decays very rapidly through thermal equilibration producing the first singlet, also
attained upon light absorption in the red region of the spectrum. Because of the extremely
rapid decay of the second singlet, it is the first singlet which is mostly utilized
photochemically by the reaction center.
The energy absorbed by the antennae chls can "migrate" within the antennae due to the
transfer of the singlet excited state from one molecule to the other and can reach the RCs
where it can be utilized in the primary photochemical reaction. The functional unit consisting
of a reaction center and its antenna proteins is defined the Photosystem (PS). In oxygenic
photosynthetic membranes, two photosystems (PS 1· and PS2) operates in series to perform
electron transfer from H 20 to NADP (see below) The mechanism of excitation energy
transfer can be described by the theory described by Förster (Förster, 1959) which involves
unidirectional transfer through induced resonance. Thus, the velocity of rnigration is
proportional to the integral of the overlapping of the emission spectrum of the donor
molecule with the absorption spectrum of the acceptor molecule. It is inversely proportional
to the 6th power of the distance between donor and acceptor, measured by the distance
between the two oscillating dipoles. lt is also a function of the cosine of the angle between
the two dipoles and the line connecting their centers. The Förster mechanism applies after
thermal equilibration of the excited state. lt still operates efficiently when the distance
between donor and acceptor is in the range of SO to 100 A
The probability for the exciton to be on a molecule having energy E rather than on one
having energy Eo is determined by the Boltzmann' factor, exp((E-E0)/k8 T), where k8 is
Boltzmann's constant. This distribution of energy refers to equilibrium at constant
temperature.
The probability of excitation energy utilization by anyone of the molecular processes
which compete for the same pool of excited states can be defined in terms of the ratio of the
kinetic constant of that process to the sum of the kinetic constants of all the processes
(Butler & Kitajima, 1975). So, the probability of energy transfer is

(2)

where kT is the rate constant for energy transfer, k0 for thermal decay, and kF for
fluorescence emission. In the same way, the fluorescence yield is

(3)

where kp is the rate constant for the primary photochemical reaction (which includes in this

37
case kT). PS 1 is not tluorescent at physiological temperature; its tluorescence can be
observed at low temperatures. The chl tluorescence measured at room temperature is
therefore emitted mostly by PS2. The term p express the concentration of open centers,
where the primary acceptor Q is oxidized, as is the case in dark adapted chloroplasts, and
the tluorescence is at its minimal Ievel, F0 . Upon illumination, Q is progressively reduced. In
the presence of an inhibitor of its reoxidation by the intersystem chain, Q becomes totally
reduced, i.e. p = 0, and tluorescence reaches its maximum Ievel, Fm. In the presence of very
strong light Fm is approached even in the absence of the inhibitor, though not attained. The
difference Fm-Fo= Fv is the "variable tluorescence". The increase of tluorescence upon
illumination is a measure of the progress of Q reduction, and is indicated as the tluorescence
induction curve; the area above the tluorescence induction is a measure of Q available as the
electron acceptor (Malkin and Kok, 1966; Murata et al., 1966). The ratio FviFm is therefore
a useful indicator of PS2 photochemical activity. Independently of the redox state of Q, the
tluorescence may decrease if a quencher is formed in the antenna or at the RC; the quencher
will dissipate thermally the absorbed energy, a situation indicated by the increase of ko in
equation 3. Both tluorescence and photochemistry ofPS2 will be decreased by the rise ofko
(see equation 3).
The very important observation by Joliot and Joliot (Joliot & Joliot, 1964) that the rise
oftluorescence from Foto Fm is sigmoidal, has been interpreted to indicate that cooperativity
exists between PS2 units. The tluorescence rise curves could be represented by the equation

Fv = (1-a)(J-p)I(J-a(l-p)) (4)
where a represents the probability for exciton migration from a PS2 unit having the RC
"closed" (Q fully reduced) to a neighbor unit with open RC. The value of alfa was estimated
by fitting the tluorescence rise curves to the model, and was found to be in the range of 0.4
to 0.6. The velocity ofPS2 turnover could accordingly be described by the equation:

v = Xoß p/(1-a (1-p)) (5)

where ß is the optical cross section ofPS2 and Xo a parameter related to the product ofl, the
photon tlux absorbed and the quantum yield of photochemistry (Paillotin, 1976). This
equation was used to calculate the velocity of NADP reduction by isolated thylakoids, and
was found to describe accurately the measured velocity (Jennings & Zucchelli, 1986).

. . . . . . . .... . . . ... . .. . . . ...!~~

-Time .......................................................... ..
t
Lq.ton

Figure !I.Kinetics of fluorescence induction in dark adapted tylakoids in the presence of an inhibitor of Qa
reoxidation. The excitation light intensity was of lOOJ.Lmol m·2 sec·1 of 440 nm radiatiom. Emission was
detected at 685 nm. Other explanation and symbols: see text. From Hipkins and Baker, 1991.

38
 Using equation (5), equation (4) is easily integrated to give the time dependence ofthe
fluorescence rise:

F(t) = F.n(t)(Fm-Fo)+Fo (6)

where:
F () -I [a·F".(t)+I-a-F•• (t)] -
t - n
a·F".(t) 0 ·t
+-- z (7)
•• a·F•• (t)+I-a (a·F•• (t)+I-a)·(I-a) I-a

The cooperativity among several PS2 units is an obvious advantage in terms of the
efficiency of PS5 photochemistry inasmuch as it allows the utilization of photans absorbed
by PS5 units having closed centers.

THE EMERSON EFFECT AND THE BILL SCHEME OF PHOTOSYNTHETIC

ELECTRON TRANSPORT

The quantum yield of photosynthesis (defined as the ratio electron transported/hv

absorbed) or its reciprocal quantum requirement, is not constant all along the absorption
spectrum of the photosynthetic pigments: the classical observations of Emerson and
collaborators (Emerson, I957) established that at the far-red end of the chl absorption
spectrum the quantum yield dropsrather abruptly (above 685 nm; the so-called "red drop"),
and that the superimposition of a beam of lower wavelength produces a superadditive effect
(the "Emerson enhancement"). Such enhancement was also observed when the long and
shorter Iambda lights were separated by a dark interval of a few seconds (Myers & French,
I960), an observation which rules out the possibility of a cooperation between excited states
produced by the absorption of different oscillators, and indicate that the enhancement effect
must involve more stable chemical intermediates.
Emerson's observations are easily interpreted in the frame of the "Z scheme" of Hili
(Hili & Bendall, I960) (see an up-to-date version ofit in fig.6): two photochemical reactions
are operating in series, and the electron acceptor ofone ofthem (reaction 2) is reoxidized by
the product of the other photoreaction (reaction I), through a chain of electron carriers, in
sequence according to the electrochemical gradient. The two photochemical reactions are
catalyzed by two different RCs, each one collecting excitation energy on its antenna
consisting of ca. 300 chl molecules, organized in several chl-protein complexes (review by
Jansson, 1994). The Emerson's photosynthetic unit, defined by experiments in vivo where
photosynthesis was measured as 02 evolution, consists of PS2, PS 1, the electron transport
chain connecting them and the enzyme system performing C02 assimilation. The absorption
spectrum of PS2, which oxidizes water, is largely overlapping but not identical with that of
PSI, which reduces ferredoxin, then NADP and finally C02. PSI absorption extends into the
far-red up to 720-730 nm, where PS2 has negligible absorption. These spectral differences
account for the Emerson enhancement effect. While light of A.>ca. 690 nm is very poorly
absorbed by PS2 and therefore has very little efficiency for 02 evolution, it activates PS 1
thereby reducing ferredoxin and reoxidizing all the electron carriers ofthe intersystem chain.
As a consequence, the addition of shorter wavelength radiation activating PS2 will produce
a cooperative etfect (superadditive electron transport from H20 to ferredoxin). Such effect
has a spectrum with peaks at the wavelengths where PS2 absorption exceeds PS I
absorption.
This can be observed in vivo (Emerson, 1957; Canaani and Malkin, I984) as weil as
with isolated thylakoids (Govindjee et al., 1964).

39
 -
cn
0
>
_e
w

Figure 6. Scheme ofthe electron Iransport pathway in oxygenic photosynthesis. For explanations see text.
On the left, the Ernpotentials ofthe electron carriers are represented. ASC, ascorbate; MDA, the radical
monodehydroascorbate produced in the 1-electron oxidation of ascorbate.

The basic criterion for establishing the Z scheme of electron transport (the Hili
scheme) has been the observation that certain electron carriers, such as cytochrome f
(Duysens et al., 1961) and plastoquinone (see review by Witt, 1979) are oxidized by PS I
activation and reduced by PS2 activation. This property has been used to establish whether a
newly discovered electron carrier belongs to the intersystem electron transport chain.
At the reducing side ofPS2, a molecule ofpheophytin (pheo) is reversibly reduced by
the RC chla, named P680 after the peak wavelength of its bleaching upon oxidation (Kiimov
et al. 1980, Witt 1990, Debus, 1992). This reaction is performed in few ps; ca. 400-600 ps
are then needed to transfer the electron from pheo· to the D2 protein bound plastoquinone
molecule, Qa (Schatz et al., 1987). Qa is a one electron acceptor, so its reduction is a one
quantum-one electron process (reviews by Debus, 1992; and by Renger, 1993). The next
carrier is a plastoquinone (Qb) molecule reversibly bound to the D 1 protein of the RC (the
same polypeptide which binds P680). Qb accepts two electrons produced by two successive
photochemical events, and when fully reduced it is protonated and PQH2 dissociates from
the RC, because the binding constant to D 1 of the protonated species PQH 2 is much smaller
than that of PQ. The Qa-Qb electron transfer is the step where the transition from a one
electron to two electron transfer occurs. PQH2 rapidly diffuses into the Iipid phase of the
membrane, and constitutes a pool common to many PS2-PS 1 units (Cramer and Knaff,
1989, but see Joliot et al. , 1992 and Lavergne et al., 1992, for an alternative hypothesis). lt
is reoxidized by the complex cytochrome f-cytochrome b6-Rieske iron-sulfur center. The

40
oxidation of plastoquinol is the rate Iimiting step of photosynthetic electron transport (it has
an half-time of ca.10 ms), releases protons into the Iumen and is controlled by the H+
concentration inside the internal (lumenal) space of the thylakoids. The electrons can be
recycled across the membrane by the two heme groups of cyt.bs63 (so named after the peak
of its a band), a process which re-reduces PQ and as a result transfers more protons from
the stromal to the lumenal side of the membrane (Mitchell, 1975; Crofts et al 1983 ),
contributing to the creation of the proton electrochemical potential which is the source of
energy used for the synthesis of ATP coupled to electron transport (see below). Cytf
(Em=365 mV, see fig.6) is reoxidized by the copper protein plastocyanin (PC, Em=380
mV), which is present in solution in the thylakoid Iumen and can bind to PSI(Haenel et al.
1980), where it is reoxidized by the photoxidized RC in about 15-20 IJS. The RC eh! a of
PS I, (P700, after the absorption peak of its bleaching upon oxidation) is photooxidized in
the ps time scale (Shuvalov et al., 1986; review by Goldheck and Bryant 1991). The primary
acceptor is a chla molecule, which becomes reduced to the anion radical; this is reoxidized
by a molecule of phylloquinone. Three iron-sulfur centers are membrane bound
intermediates before the reduction of the iron-sulfur protein ferredoxin (Fd), which is in
solution in the chloroplast stroma (Forti & Grubas, 1985). Fd forms a onelone complex with
the thylakoid bound flavoprotein ferredoxin-NADP reductase (FNR) (Foust et al., 1969),
which simultaneously binds NADP. The electron transfer to bound NADP occurs probably
in two steps through the intermediate anionic radical ofFAD, the prosthetic group ofFNR.
On the oxidizing side of PS2, the primary donor to PS2 is the tyr residue 161 of the
Dl protein (see reviews by Witt, 1990; Debus, 1992 and Renger, 1993). Its one electron
oxidation occurs in the ns time scale; tyr 161 is then reduced by the Mn containing water
oxidation complex. It has been shown that when single turnover, saturating flashes are fired
on dark adapted green cells or isolated thylakoids, 0 2 evolution has a periodicity of 4,
starting however at the third flash (Koket al., 1970). These Observations were interpreted to
indicate that each turnover of PS2 is required to advance from a state, Sn, to Sn+l: S4
oxidizes 2 molecules of H 20, thus returning to S0 • In the dark, an equilibrium must exist
between S, and So, with a ratio of ca. 4 S dSo, to account for the observation that ca. 4 tim es
more 0 2 is produced by the third flash than by the 4th. The periodicity of 0 2 production is
damped with increasing number of flashes, and is completely lost usually after 20-25 flashes.
This is due to the failure to utilize the flash (misses) and double hits (advances from Sn to
Sn+2 during the flash) statistically distributed among the !arge number of PS2 units. The "S
state" indicates the number of positive charges accumulated in the Mn enzyme: when 4
positive charges are accumulated (state S4, requiring 4 turnovers of PS2 starting from So),
two H20 molecules are oxidized and one 0 2 molecule is produced. These established facts
indicate that dioxygen and not atomic 0 is the product ofwater oxidation in photosynthesis,
and that 4 quanta are required by PS2 to perform the reaction; this means that not less than
8 quanta are required for the overall process which requires an equal number of
photochemical events in PS 1.
As stated before, the time range for the reduction of the RC species in the 2 PS is
rather different (ns for PS2 against IJS for PSI). On the contrary, the time range for
oxidation of their excited states is rather similar, suggesting that the RC species Iasts for
Ionger time in the case ofPSI. lt is known that RC is an efficient sink on excitation energy
(see, e.g. Goldheck and Bryant, 1981 for a review). This different quenching properties of
the two reaction centers could, at least partially, explain why PS2 is fluorescent at room
temperature, while PS 1 is not.

PHOTOPHOSPHORYLATION

The charge separation of the photochemical reactions of PS2 and PS I produces an

electric potential difference across the thylakoids, negative on the outer surface (Witt, 1979)

41
because the electron acceptors are located close to the stromal side, whilst the donors are
close to the lumenal side of the membranes. Furthermore, the proton-producing reactions of
electron transport release the Ir into the lumenal water space (which is a continuum within
each chloroplast), whilst the proton binding reactions (the reduction of Qb) take up protons
from the stromal side. This is due to the topology of the electron carriers as they are
organized in the architecture of the thylakoids. The result of such sovramolecular
organization is that the photochemical reactions and the following electron transport are
coupled to the formation of an electrochemical potential of protons across the membranes,
which are intrinsically very impermeable to H+. Such potential is utilized by the membrane
bound enzyme ATP synthase for the synthesis of ATP from ADP and inorganic phosphate
(Pi), according to the chemiosmotic theory ofMitchell (Mitchell, 1977).
The electrochemical potential of protons can be described by the equation

(8)

(where \jl is the electric potential; the other symbols have their usual meaning).
A\j/ is formed in the ps time scale both at PS2 and PS 1 RCs, and the two PSs
contribute to the same extent to it (Witt, 1979, 1996). Alf/ values above 200mV are
observed a few ns after the beginning of illumination (Witt, 1979). A slower formation of
membrane potential is due to the electron recycling by the cyt brf complex (Joliot and
Delosme, 1974). However, the electric potential in the thylakoids decays rather rapidly due
to the inward diffusion .of anions (Witt, 1979), mainly er which is transported through a
specific channel ( Schönknecht et al., 1988). In the steady state, A lf/ is usually in the range of
10-30 mV, while most ofthe AGH is accounted for by ApH.
The synthesis of ATP is defined by the reaction catalyzed by the ATP synthase:
ADP+Pi~ATP+H20+ H+. This reaction is endergonic, and the value of its AGo is =7.6
KcaVmole, at pH 7.4. Such an unfavorable thermodynamic situation is overcome in the
thylakoids (as weil as in mitochondria) by the fact that ATP synthase is asymmetrically
located across the membrane, and couples the synthesis of ATP to the translocation of
protons from the lumenal to the stromal side. The overall reaction is therefore:

(9)

The loss of the proton electrochemical potential is coupled in this reaction to the
increase of the chemical potential of ATP synthesis (Mitchell, 1977), and ATP synthesis
requires that AGATP+AGI <0. The opposite reaction, i.e. ATP hydrolysis, occurs when
AGATP > AGI. In this case, protons are translocated into the Iumen. Equilibrium is attained
when the ATP/ADP+P; ratio is suchthat the thermodynamic potential oftbis system is equal
to the proton electrochemical potential.
The coupling of ATP synthesis to photosynthetic electron transport is linked to the
impermeability of thylakoids to protons, which makes possible the formation and stability of
AG.i, and to the peculiar properties ofthe ATP synthase ofthylakoids. This enzyme is made
of two components: one (CF0) is a 4-subunits (4 polypeptides) strongly lipophilic moiety
crossing the membrane, the other (CF 1) consists offive different polypeptides (a, ß, y, o, e)
and protrudes out of the stromal surface of the thylakoids (Boekema et al., 1988). The
active enzyme is made of 3-(a), 3(ß), 1(y), 1(o), 1(e) subunits (Boekema et al., 1988). CFo
has the function of proton transfer from the Iumen, where it becomes protonated at the low
pH value, to the surface ofthe membrane where it is linked to CF 1. CF 1 binds ADP at the

42
catalytic site only when a threshold value of ~GH is attained; in the presence of Pi, ATP is
forrned and released into the outer space (the stroma in intact chloroplasts) tagether with the
protons transported across the membrane by CFo. ATP is thus made available for C02
assimilation in the stroma.
One important feature of the ATPase-ATPsynthase of thylakoids is that its activation
requires the conversion of an inactive form into the active one. This activation is endergonie
and requires a ~GH value higher than that required for ATP synthesis. The activation is
accompanied by the release from the enzyme of a ADP molecule tightly bound to an
allosteric site (Junesch and Graber, 1985). The activation reaction is rapidly reversible
(Junesch and Graber, 1985; Fromme and Graber, 1990) when ~GH decreases, so that the
enzyme is inactive when ~GH is below the threshold required for ATP synthesis. This feature
therefore prevents ATP hydrolysis which would otherwise occur. However, the enzyme can
be stabilized in its active form upon reduction of an -S-S- bridge (by thiol compounds).
Under these conditions, i.e. the enzyme stabilized in its active form, ATP is hydrolyzed at a
high rate. The hydrolysis is the complete reversal of the ATP synthesis reaction, including
the proton translocation across the membrane, in this case into the Iumen. The hydrolysis of
ATP generates therefore ~pH (Junesch and Graber, 1985;Fromme and Graber, 1990). ATP
hydro Iysis can occur in vivo by this mechanism, and the regulation of the redox state of the -
S-S-/-SH of the enzyme is performed in the chloroplasts by the ferredoxin-thioredoxin
reductase-thioredoxin system (see review by Ort and Oxborough, 1992).

MECHANISMS OF REGULATION AT THE LEVEL OF LIGHT ABSORPTION

AND PHOTOCHEMICAL CONVERSION

A - Light Absorption Balance of the Two Photosystems

Any departure from the equal distribution of photans to the two photosystems
decreases the quantum yield of photosynthesis, because the electron transport system
requires the two photochemical reactions in series to transfer one electron from H20 to
NADP. To achieve equal distribution of the absorbed energy to PS2 and PS 1 they should
have equal absorption cross section throughout the spectrum, or their difference of
absorption should be in some way compensated. It is known that this is not the case. The
distribution ofthe pigment protein complexes is unbalanced in favor ofPS2 antenna, which
receives the excitation energy from ca. 60% of the pigment molecules, versus 40%
belanging to PS 1 antenna (Thornber et al., 1987).
A regulation of the size of the antennae has been reported by Bonaventura and Myers
(Bonaventura and Myers, 1969). They observed that when Chlorella cells performing
steady-state photosynthesis are suddenly transferred to an illumination regime unbalanced in
favor ofPS2 (A.=650 nm), two types offluorescence changes occur: a very rapid (in the ms
time scale) increase due to the over-reduction of Qa, followed by a slow decrease requiring
several minutes (5 to 10). They described this slow transition as the "state 1 (high
fluorescence)-state 2 (low fluorescence)" transition. The state 1 was restored upon
illumination with a 710 nm beam (absorbed mainly by PSI) in several rninutes. These
reversible state 1-state 2 transitions were seen as an adaptation of the relative size of the
antennae of the two photosystems to the prevailing illurnination conditions.
The biochemical mechanism ofthe state 1-state 2 transition is understood as due to the
activity of a thylakoid bound protein kinase which is activated when the PQ pool is over-
reduced, and phosphorylates a threonine residue close to the N-terrninal of LHC II (the
major chl a-b protein complex which belongs to the antenna ofPS2), exposed on the stroma
side of the membranes (see review by Allen, 1992). Upon phosphorylation of LHCII, a
decrease (15-25%) of PS2 fluorescence and photochemistry is observed, and a

43
corresponding increase of PSI photochemistry (Fofti and Fusi, I990). The decrease of
fluorescence concerns Fo and Fm to the same extent (Allen, I992), and their ratio does not
change. A fraction of the phosphorylated LHC II has been shown to migrate from the grana
paftitions to the stroma-exposed membranes, where PSI is concentrated (see Allen, I992;
also Lavergne et Briantais, I996). As a consequence of the increased ratio of PS I to PS2
photochemical activity, PQH2 is reoxidized and the kinase becomes inactive. A thylakoid
bound phosphatase dephosphorylates LHC II, which migrates back to the grana paftitions
where it is reintegrated into the PS2 antenna. The time course of LHC II phosphorylation-
dephosphorylation and the accompanied changes of PS2 fluorescence and photochemical
activity are in fair agreement with the kinetics of state I-state 2 transitions in vivo.

B-Protection against Excessive ßlumination

The primary photochemical reactions of photosynthesis have rate constants exceeding

by several orders of magnitude those of the electron transpoft reactions and of the enzymatic
reactions of C02 assimilation. As a consequence of this fact, high concentrations of chl
excited states in the antennae give rise to the generation of chl triplet in relevant
concentration. The latter, reacting with 02, Ieads to the formation of different chemical
species harmful to the photosynthetic apparatus. Among these are the formation of 02
singlet, 02- and OH radical, which are species very reactive with proteins, Iipids and the
pigment molecules themselves, leading to inactivation of the system and eventually to
bleaching of the pigments. The protective function of carotenoids has been clearly
recognized (Demming-Adams, I990), and the general pathway for protection is through the
thermal dissipation of the energy absorbed in excess of the kinetic capacity of the electron
transpoft system.
Of course, thermal dissipation of the energy absorbed is useful when the excited states
in the antennae are present in excess of the amount that can be utilized photochemically,
while it would be deleterious to the efficiency of the system if energy were dissipated in
competition with photochemical utilization. In fact, the onset of thermal dissipation as a
function of incident light intensity has been the object of contrasting repofts. In sunflower
leaves it was observed only at high light intensity (Demming-Adams, 1990), whilst rather
low intensity produced thermal dissipation in pea leaves (Genty et al., 1990). A mechanism
for turning on and off the thermal dissipation has been found to be dependent upon the value
of A<iH across the thylakoid membrane (Wright and Crofts, 1970; Krause et al., I982). Such
dissipation competes with photochemical utilization of energy as weil as with fluorescence
emission (see equation 3); for this reason, it has been defined "high energy quenching" of
fluorescence, qE. The phenomenology and mechanisms of qE are widely discussed but still
controversial (see, e.g. Crofts and Yerkes, 1994; Hofton, 1996; Briantais and Lavergne,
I996 for discussion). It will only be mentioned here that qE can be easily distinguished
experimentally from the photochemical quenching (qP) of fluorescence, which is due to the
presence of the electron acceptor Qa. In its presence, photochemistry competes successfully
with fluorescence, because kp >> kr (see equation 3). When dark adapted leaves (or isolated
chloroplasts) are ·exposed to a shoft light flash of high intensity (oversaturating with respect
to electron transpoft rate), Qa becomes fully reduced and the Fm Ievel of fluorescence is
attained transiently. Continuous illumination at rather high intensity causes the decrease of
fluorescence, both Fm and F0 . The recovery of the Fm Ievel occurs in ca. I min in the dark
(the time required for dissipation of ApH), or it can be observed in isolated chloroplasts in a
few ms upon dissipation of AGH by an uncoupler. The qE is due to the formation of
quenchers in the antennae ofPS2 (Lavergne and Briantais, I996), which efficiently dissipate
the energy absorbed in excess of the kinetic capacity of electron transpoft, so protecting the
photosynthetic apparatus from inactivation. However, the fraction of excitation energy

44
which is transferred to the reaction centers has been reported as being utilized with
unimpaired efficiency for photochemistry (Genty et al., 1989; Lavergne and Briantais, 1996).
So, the estimate of Fm·-FJFm• (where Fm· and F. are, respectively, Fm in the quenched
state and the steady-state fluorescence) has been claimed to be a measure of the quantum
efficiency of the absorbed photons. The mechanism of qE is still unknown; however, in
intact leaves it has been observed that fluorescence quenching is correlated with the de-
epoxidation ofthe carotenoid violaxanthin to yield zeaxanthin (Demmig-Adams, 1990). This
process, however, is not an absolute requirement for qE (Demmig-Adams, 1991). The roJe
of carotenoids seems to be to amplity the quenching phenomena induced by the L1GH,
through direct energy transfer from the first excited singlet of the chls to the carotenoids,
and thermal relaxation ofthe latter to the ground state (Phillip et al. 1996).

REGULATIONS OF PHOTOSYNTHESIS AT THE LEVEL OF ELECTRON

TRANSFER PHENOMENA

A - The "Photosynthetic Control"

1t is weil known that the low lumenal pH consequent to proton uptake into the Iumen
coupled to electron transport inhibits the electron transport itself Such inhibition is
suppressed by uncouplers which annihilate L1GH across the membrane, or is strongly
decreased by ADP plus P;, through the onset of ATP synthesis, which decreases the L1GH.
This phenomenon has been called "photosynthetic control", in analogy with the same
phenomenon of the "respiratory control" observed in respiratory electron transport. lt was
demonstrated that the reoxidation ofPQH2 by the cytochrome bJ complex (known tobe the
rate limiting step of photosynthetic electron transport) is sensitive to high proton
concentration in the Iumen (Rumberg and Siggel, 1969, Hurt and Hauska, 1981).
This regulatory phenomenon has been discussed and temptatively explained in
thermodynamic terms, saying that when the lumenal pH becomes very acidic the oxidation of
plastoquinol by cytochrome f, coupled to proton release into the Iumen, approaches
equilibrium. F or these reason the forward reaction would be slowed down. Though
theoretically sound, such explanation could hardly apply to the real conditions of PQH2
oxidation in the thylakoids, because the L1Em ( - 370 mV) is such that the system is anyway
far from equilibrium.
Therefore, this explanation is difficult to rationalize when the magnitude of the L1pH
and the redox potential of the electron transfer cofactors (Hauska et al., 1983) are
considered, unless some additional hypotheses on the thermodynarnics at the quinol
oxidation site are formulated (see, e.g. Cramer et al. 1996 for a discussion). Moreover,
slowing down of the initial rate of PQH2 oxidation by protons was recently reported in
experiments performed under single turnover flash illumination in living algae, under strict
anaerobiosis (Joliot and Joliot, 1994, Finazzi et al., 1997). Under these conditions a large
L1GH is already established in darkness (Joliot and Joliot 1994), and the PQ pool is
completely reduced (Bennoun, 1982). Of course, undere these conditions, this slowing down
cannot be explained by a simple mass effect.
An alternative hypothesis has been made, that the lumenal proton concentration
regulate the catalytic activity of the electron carriers of the cytochrome bJ complex. To
explain the pK of- 6 that is usually found in measurements of the electron transport rate as
a function of pH, the presence of a protonable residue in the vicinity of the Rieske iron sulfur
cluster has been proposed (review by Rieb, 1984). This residue would modulate the
efficiency of electron transfer either through a direct participation to the reaction (Bendall,
1982), or through the catalysis of quinol oxidation. This latter possibility can be rationalized,

45
proposing a simple kinetic model (Finazzi and Rappaport, unpublished), where two states of
the cytochrome br/ are considered according to their protonation: a protonated state where
the rate constant of quinol oxidation, kp, is slow and an unprotonated one where the rate
constant, ku, is faster. Since the half time of PQH2 oxidation is modulated by the lumenal
pH, the equilibration between these two states must be fast when compared to the electron
transfer rate, i.e. faster then 10 ms (see above). In this model the kinetic parameters can be
directly estimated from the pH dependence curve, since ku would be the rate constant
calculated at neutral pH, and kp at acid pH. Fitting pH titration data according to this model
is satisfactory, suggesting that the pH might affect the structure of the quinol oxidizing site
in cytochrome br/ complex.

B - Photosystem 2 Turnover

The turnover of the PS2 reaction center can be regulated at two Ievels: (a) the
photochemical reaction itself, or the reactions on its oxidizing and/or reducing side; (b) the
transfer of excitation energy from the antenna.
lt has been reported that PS2 photochemical activity is inhibited by protons produced
during the activity itself, and that the inhibition is reversed by lipophilic uncouplers and by
the presence of ADP+Pi (Finazzi et al., 1992; 1995). These observations indicate that
membrane localized protons produced during H20 oxidation inhibit PS2 activity and that
such protons are available to the ATP synthase for ATP synthesis coupled to ~GJ
utilization. This autoregulation ofPS2 through the protons produced by its activity seems to
concern the reaction centerrather than the antenna, because it does not affect the Ievel ofFo
nor of Fm but only the velocity of fluorescence rise from F0 to Fm, i.e. the rate of PS2
primary photochemistry. The mechanism of inhibition of PS2 by protons is not known but
probably involves the efficiency of stabilization of the electron coming from the pheophytin
to Qa.
It thus appears that both the proton-producing reactions of photosynthetic electron
transport, H20 oxidation and PQH2 oxidation, are regulated by the protons that they
produce, either localized within the membrane or released into the Iumen. Moreover, both
regulations can hardly be explain by the thermodynamic equilibrium when the electron
transfer cofactors and the coupled proton translocation into the Iumen are working against
high value of ~GJ. It is therefore attractive to think about a similar mechanism of regulation
of the electron transport rate by protons that may operate through a regulation of the
tumover rate of the electron carriers, involving the conformational change of some protein
component. The "pockets" where quinones are reduced in PS2 and oxidized in the
cytochrome br/ complex, indeed, are predicted tobe rather similar (see Degli Esposti et al.,
1993 forafurther discussion on this topic).

C - The Switch of Electron Acceptorsat the Reducing Side of the Photosystem 1

It has been found that other electron acceptors, alternative to Fd, can be reduced at the
reducing side of PS I: one of them is 0 2, in the so called Mehler reaction, after the name of
its discoverer. Univalent reduction of 0 2 generates the anion radical 02. The enzyme
superoxide dismutase (SOD), present both in thylakoid bound form and in solution in the
chloroplast stroma, disproportionates 02· to yield 0 2 and hydrogen peroxide. The overall
stoichiometry of such electron transport system is the uptake of one O:z/4 electrons
transported across the chain. This electron transport pathway is the same as that reducing
NADP up to the Fd step, and is coupled to ATP synthesis (Forti & Jagendorf, 1961). The
univalent reduction of 02 at the reducing side of PS 1 is a slow reaction (ca. 15 to 20 times
slower than NADP reduction). However, the H20 2 formed in the process involving SOD
reacts rapidly with ascorbate (which is always present in rather high concentrations in the

46
chloroplast stroma) through the catalysis of ascorbate peroxidase (Miyake and Asada,
1992), producing the radical monodehydroascorbate, MOA (Miyake and Asada, 1992). The
latter is also produced by the direct reaction of ascorbate with 0 2-. MOA is an efficient
electron acceptor from PS 1 (Forti and Ehrenheim, 1993): electron transport from H20 to
MOA occurs at a rate of about 50% the rate ofNADP reduction and competes with NADP
for electrons at the reducing side of PS 1 (Forti and Ehrenheim, 1993). The coupling of this
electron transport system to ATP formation occurs with the same stoichiometry as in the
case of NADP reduction (Forti and Elli, 1995), as would be expected because the same
electron transport system and the same photochemical reactions are involved.
Ascorbate has a dual function in this system: (a) as a scavenger ofthe harmful oxygen
species (02- is a very reactive substance, which inactivates many enzymes and structures of
the photosynthetic apparatus ), and (b) as a catalyst of electron transport coupled to ATP
formation.
Reduced ferredoxin (and possibly other reductants generated by PSI) may be
reoxidized by the intersystem electron carriers; cyt.b 6 and PQ are the most likely candidates
for this function. In this way, a cyclic electron transport araund PS 1 is set on, dependent
only on PS 1 photochemical reaction and therefore activated also by light absorption in the
far-red end of eh! spectrum. This process is coupled to ATP formation in isolated thylakoids
(Arnon, 1977) (it is called cyclic photophosphorylation) and was demonstrated to occur in
vivo under conditions where the electron flow from the reducing side of PS2 is inhibited at
the Ievel of Qa- by a specific inhibitor (Forti and Parisi, 1963), but not under physiological
conditions (Tanner and Kandler, 1969).
The stoichiometry ofprotons translocated across the membrane/ATP synthesised (see
equation 3) is still under debate, though 4 seems tobe likely. On the other hand, the ratio of
H+ translocated inside the lumen/electron transported from H20 to NADP is also uncertain,
though most experimental results indicate that it should be close to 2 (Witt, 1979). If these
ratios are correct, they would be in agreement with the stoichiometry of ATP/NADPH =1
found in most experiments over the last 30 years. Only a few reports of values between 1
and 1.3 can be found in the literature, and they need to be corrected for low Ievels of the
simultaneously occurring Mehler reaction and/or cyclic electron transport araund PS 1. Such
corrections are rather uncertain, because of the experimental difficulty of estimating very
Iow Ievels of the rates of the interfering reactions, with the accuracy necessary to correct the
ratio ATP/NADPH observed, not to mention the theoretical difficulty of attributing a
stoichiometry to ATP formation in cyclic electron transport. The problern of the
ATP/NADPH ratio is obviously important to understand the quantum requirement of
photosynthesis, as the requirement of 3 ATP/2 NADPH for the assimilation of one C02 is
weil established. This implies that the extra ATP needed must be supplied by utilizing more
photons. In fact, a !arge number of measurements of quantum requirement in vivo or in
intact chloroplasts assimilating co2 reported in the Iiterature indicate that not less than 10
quanta/C02 are required. This would be compatible with the utilization of 8 quanta to
perform the reduction of2 NADP and the coupled synthesis of2 ATP, and 2 more quanta to
produce the extra ATP required.
Phosphorylation coupled to the electron transport triggered by the Mehler reaction
seems to be the most likely mechanism to generate ATP in the chloroplasts stroma (where
C02 assimilation occurs) at the rate required for steady state photosynthesis to proceed. The
Mehler reaction is known to activate ascorbate oxidation, the production of
monodehydroascorbate and the fast electron transport with MOA as acceptor for PS 1
(Miyake and Asada, 1992; Forti and Ehrenheim, 1993) which is coupled to ATP synthesis
(see above). Furthermore, it occurs at rates compatible with the observed rates of overall
photosynthesis (Forti and Ehrenheim, 1993), whilst the rates of cyclic phosphorylation
observed in isolated thylakoids are rather low (Arnon, 1977), and their measure in vivo

47
under physiological conditions is not feasible. Moreover, the functioning of cyclic electron
transport around PS 1 in cyanobacteria has been challenged (Myers, 1987). The real
physiological relevance of photophosphorylation coupled to cyclic electron transport around
PS 1 is therefore doubtful.
The switch at the reducing side of PS 1 from NADP reduction to Oz and MDA
reduction is easily understood in terms of the depletion of NADP when NADPH cannot be
reoxidized because of the Iack of ATP which prevents the formation of 1,3 bis-
phosphoglycerate (the electron acceptor for NADPH in the Calvin cycle). As soon as the
Mehler reaction plus MDA reduction generate ATP, NADPH is reoxidized and electron
transport is switched back to NADP.

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50
ELECTRON TRANSFER IN MITOCHONDRIAL STEROID HYDROXYLA TING
CYTOCHROME P450 SYSTEMS: ROLE OF ADRENODOXIN

Rita Bernhardt

Universität des Saarlandes, Fachrichtung 12.4- Biochemie,

P.O. Box 15 11 50, D-66041 Saarbrücken, Germany

INTRODUCTION

Cytochrome P450 systems catalyze the following reaction:

RH + o2 --------------> ROH + H2 0

They are involved in the metabolism of various exogeneaus as weil as endogeneaus

substrates such as drugs, xenobiotics and steroid hormones. The most important reactions
are summarized in Fig.1. The reactions catalyzed by cytochromes P450 are diverse and
include hydroxylation, N-, 0- and S-dealkylation, sulfoxidation, epoxidation, deamination,
desulfuration, dehalogenation, peroxidation, and N-oxide reduction. Their substrates
include - besides steroids, drugs and xenobiotics - fatty acids, prostaglandins, as weil as a
multitude of foreign compounds such as anesthetics, organic solvents, ethanol, alkylaryl
hydrocarbon products, pesticides, and carcinogens (for review see: Bernhardt 1996).
It is obvious that this diversity of substrates and catalyzed reactions cannot be
managed by a few different isoforms only. When the first classification of the family
members was performed in 1991, 154 different P450 genes have been described grouped
into 27 gene families. Ten of these families, including 18 subfamilies were found in
mammals (Nebert et al. 1991 ). The report also recommended a new nomenclature of the
P450s to minimize confusion introduced by the use of different names by different groups
for the same enzyme. CYP is used to characterize the respective protein as a hemoprotein.
The first arabic number defines the gene family, the following Ietter the subfamily and the
second number the individual enzyme, e.g. CYPIA1 for cytochrome P450IA1 (previously
P450c). Members of the same gene family are defined as usually having :S 40% sequence
identity to a P450 protein from any other family. This definition was made arbitrary, but
has turned out to be very useful. Mammalian sequences within the same subfamily are
always >55% identical. In 1993 the number ofP450 genes already grew to 221 existing
in 36 gene families (Nelson et al. 1993).

Biophysics of Electron Transfer and Molecular Bwelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 51
 Biotransformation
of drugs

I
Biosynthesis of Metabolism of
steroid hormones xenobiotics

/
Biosynthesis of
fatty acids,
prostaglandins,
-
"" C t h p
y oc rome 450
- Bioactivation of
potential carcinogens

!
vitamin D / ""-

Transformation of
herbicides and
~Synthesis of
phytoallexins
insecticides
Degradation of
terpenes, alkanes
a.s.o.

Figure 1. Reactions catalyzed by cytochrome P450 systems.

The last published update reported 481 P450 genes and 22 pseudogenes in 85 eukaryotic
(including vertebrates, invertebrates, fungi, and plants) and 20 prokaryotic species (Nelson
et al. 1996). For more information on these families and newly discovered P450 genes see:
http://dmelson. utmem. edu/nelsonhomepage.html.
In the adrenal gland essential steroid hormones such as glucocorticoids,
mineralocorticoids and androgens are produced by a series of P450 enzymes, which
catalyse a multistep process providing the organism with the effective hormones (Fig. 2).
The initial steps, the side-chain cleavage reaction performed on cholesterol as a precursor
yielding pregnenolone (catalyzed by P450scc, CYP11A1) and successive dehydrogenation
at the 3ß-position to form progesterone, are identical in the three pathways. For cortisol
biosynthesis progesterone is 17a-hydroxylated to 17a-hydroxyprogesterone, which
contrasts with the aldosterone synthesis pathway, where, due to a Iack of 17a-
hydroxylase/17-20-lyase expression in the zona glomerulosa, no 17a-hydroxylation
occurs. Both pathways then proceed with 21-hydroxylation to 11-deoxycortisol or 11-
deoxycorticosterone, respectively. 11-deoxycortisol in glucocorticoid synthesis is 11 ß-
hydroxylated to yield cortisol, whereas 11-deoxcorticosterone in mineralocorticoid
synthesis, in addition, is 18-hydroxylated and 18-oxidized to form aldosterone, with
corticosterone and 18-hydroxycorticosterone as further metabo1ic intermediates (Miller
and Tyrrell, 1995; Bemhardt, 1996). In humans, rat and mouse the final steps in cortisol
and aldosterone production, precisely 11 ß-hydroxylation in the zona fasciculata/reticularis
and 11 ß-hydroxylation, 18-hydroxylation and 18-oxidation in the zona glomerulosa, are
performed by two distinct enzymes, namely 11ß-hydroxylase (P450 11 p, CYP11B 1) and
a1dosterone synthase (P450aldo, CYP11B2) (Momet et al., 1989; Matsukawa et al., 1990;
Domalik et al., 1991). P450 11 p enzymes of other species like that in bovine (Wada et
al., 1985), porcine (Yanagibashi et al., 1986) and frog (Nonaka et al., 1995) were shown
to catalyze the synthesis of gluco- as weil as mineralocorticoids.
The mitochondrial P450s (P450scc, P450 11 p and P450aldo) obtain the necessary for the
oxygen activation and steroid hydroxylation electrons by an NADPH-dependent redox
system consisting of a flavoprotein, adrenodoxin reductase (AdR), and an iron-sulfur
protein, adrenodoxin (Adx) (Fig. 3).

52
 I Mitochondria [ Endoplasmic reticulum I
CHOLESTEROL

Testes
Adrenal+ (CYP11A1)
Ovary
CYP1. 17a-HYDROXY- CYP1' DEHYDROEPIANDRO-
PREGNENOLONE PREGNENOLONE
Adrenal PREGNENOLONE Adrenal STERONE
Testes I, Testes I
+( 3pHSD ) Ovary 3ßHSD )
t( Ovary • ( 3ßHSD )

CYP17..
PROGESTERONE CYP 1• 17a-HYDROXY- ANDROSTENEDIONE
Adrenal PROGESTERONE Adrenal
I Testes Testes
t( CYP21 ) ovary Ovary

11-DEOXYCORTICO- 11-DEOXYCORTICO-
STERONE Adrenal I ( CYP21 )
STERONE

Adrenal I CYP11 82
t CYP1181

CORTICOSTERONE 11-DEOXYCORTISOL

Adrenal + ( CYP11 82)

18-HYDROXY-
CORTICOSTERONE 11-DEOXYCORTISOL •-+-------------_.
Adrenal + ( CYP11 82) Adrenal + ( CYP11 81 )

ALDOSTERONE CORTISOL

Figure 2. Biosynthesis ofsteroid hormones. 3ßHSD, 3ßhydroxy-ß5 -steroid dehydrogenase-ß5 -isomerase

VI
w
 Mitochondrial steroid hydroxylase
Figure 3. Model ofthe organization ofthe mitochondrial steroid hydroxylase systems.

Three different models have been suggested: the first is the "shuttle" model, where
adrenodoxin sequentially forms binary complexes with the reductase and the cytochrome
P450, thereby transferring one electron per interaction cycle. In a second model a ternary
complex of adrenodoxin reductase, adrenodoxin and CYP 11 A 1 is formed and the two
electrons are transferred by two one-electron transfer steps within thls complex. Finally,
the necessity oftwo molecules ofadrenodoxin for one electron transfer has been proposed
(cf. Bernhardt 1996). Recognition and interaction of adrenodoxin with its redox partners
is mainly based on electrostatic interactions of negatively charged amino acids on the
surface of adrenodoxin (Coghlan and Vickery 1991) and positively charged amino acids of
the reductase (Geren et al. 1984) and the cytochrome P450 (Wada and Waterman 1992).
The mechanism of electron transfer in these systems is not very weil understood yet and
needs further investigation.

RESULTS AND DISCUSSION

To study the roJe of distinct protein domains and arnino acids of adrenodoxin in
interaction with adrenodoxin reductase, CYP II AI and CYP 11 BI as weil as in electron
transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis, expressed
in Escherichia coli, and their structural and functional properties have been characterized in
detail.

Expression of Adrenodoxin in Escherichia coli

Two expression systems for adrenodoxin have been developed (Uhlmann et al., 1992).
Whlle the first system (Fig. 4) used the Ieader sequence of the outer membrane protein A
(OmpA) to direct the newly synthesized protein into the periplasm, fusion of a start codon to
the mature sequence of Adx Iead to direct expression of the protein into the cytoplasm of E.
coli. Interestingly, the [2Fe-2S] duster has been assembled in both expression systems as has
been shown by EPR spectroscopy (Uhlmann et al., 1992). The expression yield was,
however, about an order of magnitude hlgher when using the direct expression system into

54
the cytoplasm (Uhlmann et al., 1992; Erdmann et al., 1994). Normally, about 800-1800
nmol Adx/1 E. coli culture (about 10-25 mg/1) were synthesized. This amount is enough to
turn the bacterial cells into brownish coloured ones, due to the absorption of the [2Fe-2S]
duster in the visible region (Ämax at 415 and 455 nm}, and to measure the EPR spectra of
Adx and its mutants using the bacterial cells, without the need of protein purification
(Uhlmann et al., 1992}.

Expression of native adrenodoxin

import through
mitochondrial membrane cleavage of the mature Adx
pre-Adx presequence - (matrix)
(post-translational)

Expression of adrenodoxin in Escherichia coli

OmpA Ieader cleavage of

sequence '-.... export OmpA Ieader mature Adx
OmpA/Adx ----'---+ periplasm (periplasm)
sequence of /
mature Adx

Start codon
(ATG)/mature
Adx
direct expression
cytoplasm
-
Figure 4. Expressionsystems for adrenodoxin.
mature Adx
(cytoplasm)

Role of Tyr 82 for the Function of Adx

Adx contains no tryptophan residue in its primary structure and only a single tyrosine
residue in position 82. This Tyr82 has been suggested on the basis of chemical modification
studies to be involved either in the interaction with AdR or in the electron transfer
(Taniguchi and Kimura, 1975, 1976}. Three mutants have been produced to check this
proposal: Y82F, where tyrosine has been replaced by another aromatic residue,
phenylalanine, Y82S, where the hydroxyl group (although an aliphatic instead of an aromatic
one) has been mainatined, and Y82L, where tyrosine was replaced by another hydrophobic
residue, Ieu eine. After rep1acement of Tyr82 by other amino acids, the consequences of this
change on the structure of the mutants compared with that of the wild type protein have to
be investigated in detail. Unehangerl absorption, CD and electron spin resonance spectra as
weil as redox potentia1s indicated that the environment of the [2Fe-2S] duster was not
affected by the mutations (Beckert et al., 1994). Furthermore, 1H-NMR studies revealed that
the overall structure of adrenodoxin was not changed by replacement of Tyr82 (Beckert et
al., 1995). Thus, replacement of Tyr82 by phenylalanine, serine, and leueine did not cause
any remarkable structural changes of the protein. To check whether the rep1acement
influences the functional properties of Adx, the kinetics of cytochrome c reduction and
substrate conversion with CYP11A1 and CYP11B1 has been studied.
The Ymax values in cytochrome c reduction, CYP11A1-dependent formation of
pregnenenolone from cholesterol and CYP 11 B 1-dependent 11 ß-hydroxylation of 11-
deoxycorticosterone were also not influenced when using Y82F, Y82S or Y82L instead of

55
wild type protein. Since the second electron transfer is rate-limiting in most P450-dependent
reactions what holds true for the mitochondrial steroid hydroxylases (cf. Bernhardt 1996),
this means that the single tyrosine residue in Adx is not involved in the intra- or
intermolecular electron transfer (Beckert et al., 1994; Bernhardt et al., 1995).
Moreover, replacement of tyrosine 82 did not affect AdR binding as shown by
unchanged cytochrome c activity (Fig. 5A). Although this reaction does not occur
physiologically, it is a widely used model for the electron transfer from reduced AdR
reductase to Adx since the flavin-to-iron electron transfer appears to be the rate-limiting step
in cytochrome c reduction (Lambeth and Kamin, 1979). There are, however, slight changes
in Km values when measuring the enzymatic activities with cytochromes 11 A 1 and 11 B 1 as
electron acceptors (Fig. 5B,C). These results suggest that Tyr82 is not directly involved in
the interaction domain with AdR, but either directly or indirectly affects binding of the
cytochromes P450, although to a different degree (Beckert et al., 1994).
When looking at the three-dimensional structure of Adx, which very recently has been
obtained for the truncated mutant Adx4-108 in collaboration with the group of Udo
Heinemann/Berlin (Müller et al., 1998), it can be seen that, in fact, Tyr82 is localized close
to the putative binding site of Adx redox partners (Fig. 6), comprising a highly negatively
charged region containing residues 72-79 (Coghlan and Vickery, 1991 ). It seems
conceivable that replacement of Tyr82 by other residues either can directly affect binding of
CYP 11 A 1 or CYP 11 B 1 or can change slightly the position of the interacting domain thus
influencing the interaction with the cytochromes P450 more indirectly. A clear answer can
only be obtained after crystallization of the elctron transfer complexes.

C-Tenninally Truncated Mutants of Adx and Their Influence on Electron Transfer

First experiments with Adx revealed that upon isolation of this protein from adrenal
mitochondria a form, consisting of 114 amino acids was obtained and shown to be active as
electron transfer protein (Tanaka et al., 1973). Later, a 14-amino acid C-terminal extension
peptide was found in the nucleotide sequence of Adx cDNA, so that the mature Adx
contains 128 amino acids (Okamura et al., 1985). Isolation of this protein from adrenals,
however, Iead to multiple truncated forms of Adx, possessing different sizes of the C-
termini, varying in lenght from 114 (Tanaka et al., 1973), 121, 124, and 125 amino acid
residues (Hiwatashi et al., 1986) up to 127 amino acids (Sakihama et al., 1988).
Overexpression of Adx in E. coli produced full-length Adx (Uhlmann et al., 1994),
which, however, was prone to proteolysis to some extent. To obtain Adx with high
proteolytic stability and yet maintained functional properties, the role of the C-terminal
region of Adx was studied by analyzing truncated mutants Adx 4-114 and 4-108, where
amino acids 1-3 and 115-128 or 109-128, respectively, had been removed. Removal of
amino acids 1-3 appeared to be beneficial, since native and wild type adrenodoxin showed,
in addition to C-terminal, also some N-terminal proteolysis. The mutants were shown to be
of the expected composition, but contained an additional methionine at the first position
resulting from an uncleaved start codon (Uhlmann et al. 1994). The absorption spectra of all
mutants studied were identical to that of the wild type. However, EPR, CD and redox
potential measurments of mutants 4-114 and 4-108 revealed that the structures of these
mutants differ slightly from that of wild type adrenodoxin. EPR spectra of wild type Adx are
characterized by two g-values: g_l_ = 1.94 and gll = 2.03 (Uhlmann et al., 1992). The mutants
showed signals, where the position of gl_ was identical to that of native Adx, but broadened,
while g II was shifted to a smaller value. The CD signals of mutants 4-114 and 4-108 in all
three wavelenght ranges measured (absorption ofthe peptide region, aromatic residues, and
iron-sulfur duster) were increased. In addition, the redox potentials of these mutants were
considerably lower than that of wild type.

56
%Km
700 ~----------------------------------------------------,

6oo cytochrome c

500

400

300

200

100

0
wr Y82F Y82S Y82L 4-128 4-114 4-108 T54S T54A H56Q H56T H56R

%K m

700

600 CYP11A1

500

400

300

200

100

0
wr Y82F Y82S Y82L 4-128 4-108 T54S T54A H56Q H56T H56R

Figure 5. Kinetic constants of adrenodoxin mutants.

Interaction of adrenodoxin mutants with adrenodoxin reductase was assayed following

the reduction of cytochrome c at 550 nm. In order to characterize the electron transfer
properties of the adrenodoxin mutants with the terminal electron acceptors cytochrome c
(A), CYPIIAI (B) and CYPIIBI (C), reduced cyochrome c was analyzed directly and the
products of the respective hydroxylation reaction, pregnenolone and corticosterone, were
analyzed by HPLC. The Km and Vmax values and their standard deviations (not shown here)

57
 %Km
700 r--------------------------------------------------------.

soo CYP11 81

500

400

300

200

100

0
WT Y82F Y82S Y82L 4-128 4-1 14 4-1 08 T54S T54A H56Q H56T H56R
Figure 5. (continued)

were calculated from 5-6 independent experiments and are relative to the adrenodoxin
concentration.
Adx, being -342 ± 5 mV for Adx4-114 and -344 ± 5 mV for Adx4-108 compared with -274
± 5 mV for the wild type Adx.
Deletion of residues 115-128 or 109-128 did not essentially affect the interaction with
the electron donor adrenodoxin reductase as shown by nearly unchanged (besides mutant
Adx 4-1 08) cytochrome c reduction activity (Fig. SA). In contrast, interaction with the
electron acceptors, CYPI1A1 and CYP11B1, was influenced. In CYP11AI-dependent
cholesterol conversion, mutants 4-108 and 4-114 exhibited 3fold and Sfold decreased Km
values (Fig. SB), respectively, while the binding affinity for CYP11Al raised nearly 3fold
and 2fold, respectively (Uhlmann et al. 1994). The Vmax values did not change upon
deletion ofthe C-terminal region. When measuring the CYP11Bl-dependent conversion of
deoxycorticosterone to corticosterone, mutants 4-108 and 4-114 also showed decreased K",
values (6fold and 3fold, respectively (Fig. SC). In this case, however, also the Vmax values
increased, being 5. 5 nmol product/min/nmol CYP 11 B 1 for wild type Adx, 11 .8 nmol
product/min/nmol CYP11Bl for mutant 4-114, and 19.7 nmol product/min/nmol CYP1181
for mutant Adx 4-108 (Table 1). lt could be clearly demonstrated that this increase in
product formation correlates with an increase of the electron transfer rate of the first
reduction step, which is experimentally accessable (Table 1). Furthermore, the data suggest
that the electron transfer coupled interaction of adrenodoxin with CYP 11 AI and CYP 11 8 1
is determined at least in part by different features of the cytochromes. This observation is
supported by site-directed mutagenesis studies of amino acid residues Tyr82 (see above),
His56, and Thr54.
Taken together, the truncated mutants are shown to possess equal or even better
properties as electron transfer protein than the wild type protein, while exhibiting increased
proteolytic stability.
In fact, diffracting crystals could be obtained using mutant Adx4- l 08. In collaboration
with the group of Udo Heinemann!Berlin the three-dimensional structure of this protein has
been deduced with 1.85 Aresolution (Müller et al., 1998).

58
 Figure 6. Three-dimensional structure ofadrenodoxin. The 30-structure has been obtained at 1.85A
resolution by Müller et al. (submitted). The backhone structure is shown. In addition, residues, which are
described in this paper have been depicted.

Using this knowledge, further experiments can now be designed to understand the
molecular basis for the increased electron transfer ability ofthe truncated mutant Adx 4-108.
Calculation of the coupling of all amino acids of Adx to the iron-sulfur duster using the
PATHW AY II program developed by Beratan and coworkers ( 1990, 1991, 1992) revealed
that the best pathways leading from the duster to the surface of the protein are less than 20
A away from residue 108 of Adx (Fig. 7). Thus, a possible explanation could be that the
additional 20 amino acids could "cover" the interaction between Adx and CYP 11 B 1, but not
CYPI1A1 dose to this pathway (Rottmann et al., unpublished results). Further sturlies using
this program and mutants of Adx are necessary to understand the structural background of
the increased electron transfer rate of mutant Adx4-108 to CYP 11 B 1 and the mechanism of
electron transfer in cytochrome P450 systems in general.

Table 1. Kinetic parameters ofthe CYP11B1-dependent reaction oftruncated

adrenodoxin mutants ( data taken from Uhlmann et al. 1994).

Adrenodoxin Ymax kapp

nmo1 corticosterone s·l
~roduced/min/nmo1 CYP11B1
Wild type 5.5 ±0.6 0.31 ±0.04
Adx 4-128 5.6 ±0.4 0.39 ±0.05
Adx 4-114 11.8 ± 1.0 0.92 ±0.10
Adx 4-108 19.7 + 1.8 1.40 + 0.07

59
Mutants near the Coordinating Cys55

The region around the coordinating cysteine residues of adrenodoxin is rather

conserved within the family of the [2Fe-2S] type ferredoxins. A histidine residue can be
found in most of the vertebrate type ferredoxins in the position next to Cys55 of Adx (Fig.
8). In the position corresponding to Thr54 of Adx, threonine or serine has been conserved in
all [2Fe-2S] ferredoxins. From this observation it was tempting to Iook, what effect a
replacement of these conserved residues by other amino acids would cause and, especially,
to find out, whether these amino acids are able to modulate the electron transfer properties
of Adx. lt could be demonstrated that Thr54 replacement by serine did not change the
interaction with AdR, while replacement by alanine Iead to an about 3fold decrease of the
Km value (Fig. SA). The efficiency of cytochome c reduction was not changed for both
mutants. There was also no change in the efficiency of the electron transfer to CYP11Al.
The Km values for both mutants are, however, decreased 3-Sfold in the CYP11 Al-dependent
cholesterol side-chain cleavage reaction (Fig. SB). Interestingly, the Km value for the
CYP11B 1-dependent formation of corticosterone was only changed 2-3fold, while the Vmax
value for this reaction increased about 2fold (Fig. SC). Again, the increase in the efficiency
of product formation correlates with an increase in the reduction rate of CYP 1 I B 1
(Uhlmann and Bernhardt, 1995).
Furthermore, mutants T54S and T54A exhibit decreased redox potentials, which correlate
with the increase ofthe electron transferrate to CYP11B1 (Uhlmann and Bernhardt, 1995).
Thus, the residue in position 54 seems to be involved in modulation of the redox potential
of Adx and this way the electron transfer rate to CYP 11 B 1, but not to CYP 11 AI.

Figure 7. Electron transfer pathways from the iron-sulfur cluster to the most coupled region of adrenodoxin.
The coupling was calculated using PATIIW AY II (Onuchic and Beratan, 1990).

60
 Histidine is conserved in the position corresponding to His56 in Adx only in vertebrate
type adrenodoxins (besides terpredoxin)(Fig. 8). lts position in the three-dimensional
structure of Adx (Fig. 9), however, points at a prominent roJe in electron transfer, since it is
localized between the interaction helix and the cluster containing core region (Müller et al.,
1998). In fact, replacement of His56 by glutamine, threonine or arginine drastically changes
the interaction with the redox partners of Adx (Fig. 5, Table 2). However, the efficiency of
product formation was not decreased, indicating that the rate-Iimiting step was not
influenced by the amino acid replacement (Beckert et al., 1995; Beckert and Bernhardt,
1997). Interestingly, the rate of the first electron transfer to CYP 11 Al slightly decreased for
mutant H56R compared with that for the wild type protein, while it was increased for
mutants H56T and H56Q, reflecting a correlation of the redox potential with the logarithm
ofthe rate constant ofthe first electron transfer in the system with CYPllAl (Beckert and
Bernhardt, 1997). No such correlation has been observed when studying the first electron
transfer from Adx to CYPIIBI (Table 2), indicating that in this case other properties, e.g.
hydrophobic interactions, are more important. Taken together, this data suggests that either
the first and second electron transfer are differentially regulated (implying different
requirements for the adrenodoxin P450 electron transfer complex) orthat another step (e.g.
product release) and not introduction of the second electron is rate-Iimiting in these systems.
43 * * 61
Adxl bovin FGACEGTLACSTCHLIFED
Adx_pig FGACEGTLACSTCHLIFED
Adx_sheep FGACEGTLACSTCHLIFEQ
Adx human FGACEGTLACSTCHLIFED
Adx mouse FGACEGTLACSTCHLIFED
Adx rat FGACEGTLACSTCHLIFED
Adx chick FGACEGTLACSTCHLIFED
Adxh drome EGACEASLACTTCHVYVQH
Putx_psepu VGDCGGSASCATCHVYVNE
Fer ecoli EHACEKSCACTTCHCIVRE
Terp_psesp VAECGGSCVCATCRIEIED
Fer caucr DADCGGACACATCHVYVDE
Fer6 rhoca DADCGGACACSTCHAYVDP
Thcc_phoso VAECGGQAMCATCHVYVES
Fer haein HHACDGSCACTTCHVIVRE
Ferl_anasp F-SCHS-GSCSSCVGKVVE
Ferl anava F-SCQS-GSCSSCVAKVVE
Fer_spipl Y-SCRA-GACSTCAGTITS
Ferl_spiol Y-SCRA-GSCSSCAGKLKT
Ferl nosmu Y-SCRA-GACSTCAGKIVS
Ferl_pea Y-SCRA-GSCSSCAGKVVG
Ferl_phyam Y-SCRA-GSCSSCTGKVTA
Figure 8 Alignment of the amino acid sequences of representative vertebrate, plant and bacterial ferredoxins
around three cysteine ligands ofthe [2Fe-2S] cluster (positions 46, 52, and 55 of adrenodoxin). Mutation
targets T54 and H56 of adrenodoxin are indicated (*).

Table 2. Stopped flow kinetic and redox parameters ofHis 56 mutants of adrenodoxin
(data taken from Beckert and Bernhardt, 1997).

Adrenodoxin Redox potential k.,pp(CYPllAl) k,.pp(CYPllBl)

mV s·I s·I
WT -274 0.9 0.68
H56Q -302 1.8 0.56
H56T -340 2.0 0.61
H56R -339 0.6 0.43

61
Figure 9. Three-dimensional structure around residue His56 of adrenodoxin (structural data from Müller et
al. , 1998).

1t is worth mentioning that replacement of His56 by glutarnine, threonine or arginine

also changes the stability of the polypeptide chain. Using a method, which decreases the
destruction of the iron-sulfur duster upon heating, calculation of thermodynamic data from
scanning calorimetric studies became possible (Burova et al., 1995). lt was shown that the
transition temperatures ofmutants H56Q, H56T, and H56R were decreased by 5.0, 2.7, and
2.1 °C, respectively. Moreover, mutant H56R produced substantial local changes in the
region around positions 56 and 82, as indicated by reduced heat capacity change and
fluorescence measurements (Burova et al. I996).

Role of ProlOS

Since deletion of residues I 09-I28 of Adx Iead to an increase in the rate of the first
electron transfer and the efficiency of corticosterone formation catalyzed by CYPIIB1, it
was temping to check the influence of the residue in position 108 on the electron transfer
properties of Adx. Mutant Adx 4-I07, lacking the single in Adx contained proline residue,
Pro I 08, did not show EPR signals indicating that Pro 108 plays an essential role for the
folding and/or assembly ofthe /2Fe-2S/ duster. In-vitra reconstitution ofthis mutant gave a
fully active protein. The kinetic parameters for the interaction with the redox partners were
similar to those obtained for mutant Adx 4-108, indicating that Pro 108 does not influence
the electron transfer properties of Adx. However, mutant Adx 4-107 showed a higher
sensitivity to urea denaturation and had a significantly lower denaturation temperature,
44.8°C compared with 51.7 oc for Adx 4-I08 (Uhlmann et al., 1997), suggesting that
Pro I 08 plays an important role in stabilizing this protein.

62
 ILE12

Leu57

HIS 56

Figure 10. Three-dimensional structure around residue ProlOS ofadrenodoxin (structural data from Müller
et al., 1998).

This conclusion could be confirmed by studying mutants, where Pro I 08 was replaced by
alanine, serine, Iysine or tryptophane. The redox potentials of these mutants were similar to
that of Adx 4-1 08P and the kinetic parameters for mutants Adx 4-1 08A and Adx 4-1 08S in
the cytochrome c and CYP II A 1-dependent assays were not significantly changed, showing
that there is no effect on the electron transfer properties of these mutants compared with
Adx 4-1 08P (Grinberg and Bernhardt, submitted). However, when using mutants Adx 4-
108W and Adx 4-1 08K, a decrease in the Vmaxvalues, accompanied by an increase in the Km
value for the CYP11Al-dependent reaction was observed. This effect could be due to the
charge of the Iysine residue and steric hindrance of the bulky tryptophane residue upon
protein-protein interaction. Thus, it can be concluded that Pro I 08 does not have a direct
influence on the electron transfer properties of Adx. Its essential rote in protein stabilization
could be demonstrated studying the thermodynamic parameters of protein unfolding. The
transition temperatures were lowered by 5.4 -14.1 oc compared with Adx 4-108P (Grinberg
and Bernhardt, submitted). When looking at the three-dimensional structure of Adx 4-108 it
can be seen that Pro I 08 makes important contacts with Arg 14 and His 56, thus bringing
together different regions ofthe protein (Fig. 10).

SUMMARY

Cytochromes P450 need electrons for oxygen activation and following substrate
conversion, which they get from NAD(P)H either via a FAD and FMN-containing reductase

63
(microsomal P450s) or an iron-sulfur protein and a FAD-containing reductase (bacterial and
mitochondrial P450s). In most P450 systems the electron transfer comprises the rate-Iimiting
step for the product formation. Interestingly, the rate of electron transfer varies by a factor
of 100-1000 among different P450 systems. The reason for this observation is still obscure.
In our laboratory, studies on the mechanism of electron transfer in mitochondrial steroid
hydroxylating P450 systems are being performed.
In the bovine adrenal cortex, the mitochondrial cytochrome P450scc (CYP11A1) catalyzes
the side-chain cleavage of cholesterol to produce pregnenolone, whereas the P450
dependent 11ß-hydroxylase (P45011B1) is responsible for the formation of corticosterone
and cortisol as weil as aldosterone. Both proteins receive the necessary electrons for oxygen
activation via an electron supporting system, consisting of adrenodoxin, the adrenal /2Fe-2S/
protein, and adrenodoxin reductase, a flavoprotein. In order to study the roJe of distinct
protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase,
CYP 11 A 1 and CYP 11 B 1 as weil as in electron transfer, mutants of adrenodoxin have been
prepared by site-directed mutagenesis, overexpressed in Escherichia coli, and their
structural and functional properties have been characterized in detail. Different, but partially
overlapping binding sites for these three proteins have been found using mutants with
replacements ofThr-54, His-56, Tyr-82 and the deletion mutants Adx 4-114 and Adx 4-108.
Using this series of mutant proteins owing distinctive structural parameters such as redox
potential, microenvironment of the iron-sulfur duster, electrostatic properties, and
conformational stability, the contribution of the electronic and conformational states of
adrenodoxin to the driving forces of the complex formation and electron transfer has been
investigated. The apparent rate constants for CYP11A1 reduction were generally
proportional to the adrenodoxin redox potential under conditions in which protein-protein
interactions were not affected by steric constraints or charges. In contrast, no such general
correlation has been observed with CYP11B 1, where -in addition to the redox potential-
hydrophobic effects seem to be of great importance. Interestingly, the deletion mutants Adx
4-114 and Adx 4-108 show increased rates of the electron transfer to CYP 11B I and
increased Vmax values upon CYP 11 B 1-dependent corticosterone formation, but not upon
interaction with adrenodoxin reductase or CYP11Al. The reason for this acceleration of
steroid hydroxylation (corticosterone formation from 11-deoxycorticosterone) is not
understood yet and needs further investigation.
The results of our study are being further discussed taking into account the three-
dimensional structure of adrenodoxin, which has very recently been resolved in colaboration
with U. Heinemann!Max Delbrück Center ofMolecular Medicine, Berlin-Buch.

ACKNOWLEDGMENTS

This work is supported by a grant from the EC Copernicus IC15-CT96-0810 as weil as

by a grant from the Deutsche Forschungsgemeinschaft, Be 1343/1-3.
I would like to thank Dr. Volker Rüdiger, Stephanie Bechtel, Mathias Rottmann and Karin
Müller for graphical work and Dr. Frank Hannemann and Asya Grinberg for critical reading
ofthe manuscript. The help ofMrs. Gabriele Schon in typing is greatly appriciated.

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Wada, A., and Waterman, M.R., 1992, Identification by site-directed mutagenesis oftwo Iysine residues in
cholesterol side chain cleavage Cytochrome P450 that are essential for adreonodoxin binding, J. Bio/.
Chem. 267(32):22877.
Yanagibashi, K., Haniu, M., Shively, J.E., Shen, W.H., and Hall, P., 1986, The synthesis ofaldosterone by
the adrenal cortex. Two zones (fasciculata and glomerulosa) posses one enzyme for 1lbeta-, 18-
hydroxylation, and aldehyde synthesis, J. Bioi.Chem. 261(8):3556.

66
PREPARATION, STRUCTURAL CHARACTERIZATION AND FUNCTIONAL
COUPLING OF TETHERED MEMBRANES TO SOLID SUBSTRATES

Wolfgang Knoll"'b,c), Natascha Bunjes•,c>, Morgan Denyer•,d) Claudia Heibel•>,

Mieko Matsuzawab>, Renate Naumann•>, Andreas Offenhäusser•,b>, Jürgen
Rühe•>, Eva-Katrin Schmidt•>, Axel Sinner•>, Christoph Sprößler•>

•> Max-Planck-Institut fur Polymerforschung, Ackermannweg 10, 55128

Mainz, Germany
b> Frontier Research Program, The Institute of Physical and Chemical
Research (RIKEN), Wako, Saitama 351-01, Japan
c> Center on Polymer Interfaces and Macromolecular Assemblies,
Department of Chemical Engineering, Stanford University, Stanford, CA
94305-5025, USA
d) University of Glasgow, The Centre for Cell Engineering, Glasgow G12
8QQ, UK
•> Merck KGaA, Frankfurter Straße 250, 64271 Darmstadt, Germany

INTRODUCTION

One ofthe remaining major challenges of current scientific "hot topics" at the boundary
between physics, chemistry, biology, medicine, materials science, and mechanical and
electrical engineering is the interface between the living world of biomolecules, cells and
tissues and the technical world of implants, sensor surfaces or signal transducers in neuro-
electronic circuits. The understanding, design, fabrication, control and modification of these
"bio-interfaces" will be in the center of many scientific activities aiming at compatibilizing
the two spheres in an effort to not only induce a passive mutual toleration, but rather
generate an interactive network of components originating from living organisms and, e.g.,
microelectronic devices (Nicolini, 1996).
Our efforts in this particularly ambitious multidisciplinary field of bio-electronic signal
transduction is schematically depicted in Figure 1. Basically, 3 different categories of
interfacial bio-architectures are being investigated in our groups. The first one (cf. the top
left cartoon in Fig. 1) involves the controlled build-up of complex multilayer assernblies
(Knoll et al, 1997) based on "classical" physico-chemical preparation schemes involving,
e.g., Langmuir monolayers assembled from amphiphiles at the water/ air interface (Reiteret
al, 1993; Blankenburg et al, 1989), their deposition onto solid substrates by the
Langmuir/Biodgett/Kuhn technique (Kuhn et al, 1972; Petty, 1996; Schmitt et al 1991 ), the

Biophysics of Electron Transfer and Molecu/ar Bioelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 67
self-assembly process of thiols and disulfides to Au surfaces (Ulman, 1996; Häussling et al,
1991; Spinke et al, 1993) or silane derivatives onto oxides(Ulman, 1996; Chen and Frank,
1989) or the alternate polyelectrolyte deposition by electrostatically controlled adsorption
from solution (Decher and Hong, 1991; Knoll, 1996). Typically, non-specific interactions are
used in the assembly process as weil as highly selective and specific bio-recognition
reactions, matrix materials are combined with (bio-) functional units in an attempt to
generate supramolecular architectures with a certain tailor-made property and functionality
profile. Typical examples of such functional layers are electron transfer units assembled in an
oriented way by means of a highly organized mediator layer at an electrode in contact with
an aqueous solution (Feng et al, 1995). This whole range of research activities, however,
will not be discussed further in this review.
Instead, we will concentrate on interfacial architectures that involve, in one way or
another, membranes tethered to a solid substrate via buffer layers of different chemical
nature and composition (Sackmann, 1996). The investigated membranes are either artificial
bimolecular Iipid layers (Beyer et al, 1996a; Advincula et al, submitted; Bunjes et al
submitted), functionalized by the incorporation of proteins in a reconstitution step(Bunjes et
al submitted; Naumann et al, 1995, 1997) (cf upper middle cartoon in Fig. 1), or biological
membranes of cells(Fromherz et al, 1991; Offenhäusser et al, 1997) (upper right in Fig. 1)
or cell assernblies (Gross et al, 1977; Sprößler et al, submitted). Both areas will be discussed
in some detail.
As to the employed substrates, we fully concentrate here on electronic devices, ignoring
optical configurations, e.g., in bio-sensors based on surface plasmon (Zizlsperger et al
submitted) or integrated optics formats (Weisser et al, submitted). In particular, we will
discuss several examples for the electrochemical characterization of tethered membranes
coupled to Au-electrodes (lower right of Fig. 1) and we will introduce first results on the
functional coupling of cells with the gate electrodes of a field effect transistor (FET) array
(cf Iower left ofFig. 1).

TETHERED ARTIFICIAL LIPID BILA YERS

Basically, two different strategies can be taken in the attempt to assemble complex
supported membrane architectures (cf Fig. 2). One is a sequentiallayer-by-layer deposition:

/
Bio-electronic

""'
Signal Transduction

/
electrode

FET

Figure 1. Schematic of our Bioelectronic Interface Program aiming at coupling various supramolecular
architectures to electronic devices.

68
the substrate is functionalized, e.g., by a self-assembled monolayer (SAM) for (noble) metal
(Steinern et al, 1996) or oxide surfaces or by plasma treatment in the case of an inert
polymer support (van Os et al 1997), to generate the reactive sites that can be used in the
next step to covalently couple a polymer cushion that acts as the tether layer (Advincula et
al, submitted; Beyer et al, 1996b). Finally, a Iipid monolayer is built, either in a Langmuir
transfer process of a pre-organized film, or by self-ordering from solution, and (partially)
coupled via reactive components ("anchor" groups) to the polymer support (Beyer et al,
1996a; Advincula et al, submitted).

(a)

uh,rhwAn,hmhmtJ
Surface Polymer Lipid Monolayer
Fu nctionalization Attachment Deposition

(b)

Figure 2. (a) sequential build-up of polymer-supported Iipid layers: ehemisorption of linear or branched
polymers to funetionalized self-assembled monolayers followed by deposition of Iipid layers. (b) polymer
supported Iipid layers through end group attaehment of teleehelies or multifunctional copolymers.

The second approach is a one-step process involving either so-called telechelics, i.e.,
oligomeric tether units with a Iipid moiety on one end and a substrate-specific reactive group
at the other (Heibel, 1996), or multifunctional polymers, with co-monomer units that mirnie
Iipid molecules, other ones that eventually play the roJe of the polymer cushion, and a few
reactive monomers that allow for a covalent, and hence stable coupling to the substrate
(Spinke et al, 1992). Typically, the exposure of the solid support to a solution of these
multifunctional elements Ieads to adsorption and binding and eventually, through a
spontaneous microphase separation!self-organization process to the desired supported
monolayer architectures (Erdelen et al, 1994).
The final tethered bilayer is then obtained by either a simple vesicle fusion step (Tamm
and McConnell, 1985; Stelzle et al, 1993; Dusch! et al, 1994; Groves et al, 1997, 1995;
Lingler et al, 1997) or by a Langmuir-Schäfer transfer of a second monolayer, again firstly
prepared at the water/air interface. The advantage ofthe vesicle fusion process is the ease of
protein incorporation into supported membranes because established protocols for the
isolation, purification and reconstitution of functional units from bio-membranes into
liposomallipid bilayers can be employed (see below) (Bunjes et al submitted).

69
 It is evident that the preparation of such complex hybrid architectures combining, e.g.,
elements of a random disordered polymer coil with the liquid-crystalline (smectic) structural
motive of a Iipid layer, can only be successful (and does not remain wishful thinking based
on an intriguing cartoon) if a comprehensive characterization gives experimental evidence of

'-------Teflon Cuvette
' - -- - - - -Heatina Element

lethered Bilayer

(a) (b)
1
R R (c)

0~------------~
time t E
0 /deg
Figure 3. Combined surface plasmon optical/ electrochemical set-up used for simultaneaus structural and
functional characterization of tethered membranes. The right panel gives schemalies of (a) angular scans of
surface plasmon spectroscopy; (b) the corresponding kinetic mode, i.e., recording the reflected intensity at a
fixed angle ofincidence as a function oftime; (c) cyclic voltammetry as one example for electrochemical
investigations.

70
the actually achieved (internal) structure, order, and orientation of the various functional
components, their dynamic features, and their (synergistic) functional properties. This
requires a whole battery ofhighly sensitive and surface-selective analytical tools.
Many of the results discussed in the following were based on an experimental set-up
that combines features of a surface plasmon spectrometer (SPS) (Aust et al, 1994) with
those of an electrochemical cell (Kienle et al, 1997). This is schematically depicted in Fig. 3.
For the optical characterization of the interfacial architectures a Iaser beam is coupled in a
Kretschmann prism set-up to the thin Au layer in contact with the electrolyte. Scanning the
angle of incidence while monitoring the reflected intensity allows for the determination of
the angular position of the resonant excitation of a surface plasmon mode (cf. Fig. 3(a)).
This resonance angle, determined by the energy-momentum matehing condition between
incident photons and excited surface plasmons, depends on details of the optical properties
of the interfacial architecture: E.g., any change in the layer thickness by an adsorption or
desorption process from solution results in a shift of the angular position of the resonance
(cf. Fig. 3(a)) which can be sensitively monitored in the corresponding reflectivity (ATR)
scans. A Fresnel fit routine then yields the thickness change provided the refractive index of
the Iayer is known (or given by a reasonable assumption). Kinetic information of interfacial
processes can be obtained if the reflected intensity is recorded at a fixed angle of incidence
(Knoll, 1997). If this angle is chosen such as to correspond to a reflected intensity on either
one ofthe steep slopes ofthe PSP resonance (cf. Fig. 3(a)) the shift ofthe whole curve as a
function of time can be used to obtain diffusion constants or rate constants of interfacial
binding events (Fig. 3(b)). The simultaneaus recording of electrochemical parameters, e.g.,
electron transfer data from cyclovoltametry (cf. Fig. 3(c)) or capacitance and conductance
data from impedance spectroscopy gives valuable information as to the correct conclusion
from the structural analysis and, of course, about the functional performance of the
interfacial architecture.
These data, have to be complemented by many other ones from additional experimental
techniques. E.g., optical thickness values need to be compared to structural information
obtained from X-ray (XR) and neutron reflectometry (NR) measurements. While the former
method typically is limited to dry samples in air (see below) NR can also be performed easily
in an evanescent total internal reflection mode with the sample in contact to an aqueous
medium (Schmidt et al, 1992). Moreover, the possibility to selectively replace in the various
sub-layers protons by deuterons allows for a very powerful modification of the relative
scattering contrasts which yield valuable additional information supporting (or questioning)
the respective structural models.
Other experimental methods that we routinely employ include i) XPS (ESCA) and
Auger spectroscopy to identifY atomic or molecular species at the interface (Heibel, 1996;
Beyer et al, 1997), ii) SIMS (Hagenhoff et al, 1993) and other mass spectrometries, e.g.,
heavy ion induced (HIID) (Schmidt et al, 1991a) or spontaneaus desorption (SO) time-of-
flight mass-spectroscopy, (Schmidt et al, 1991b), in order to identifY whole molecules or
molecular fragments of deposited layers, iii) FTIR and, to some extent, surface plasmon
field-enhanced Raman spectroscopies to identifY molecular species and orientation of highly
organized layers (Nemetz and Knoll, 1996), iv) specific forms of NMR, e.g., 29 Si-NMR, to
determine the degree of surface reaction (Heibel, 1996; Rühe, 1994), v) scanning probe
microscopies in an attempt to determine structures and structural defects at the submicron
and molecular Ievel (Weisenhorn et al, 1992; Hara et al, 1996; Tamada et al, 1997), vi)
fluorescence techniques (including spectroscopy (Liebermann and Knoll, in preparation),
microscopy and FRAP (Beyer et al, 1996a), fluorescence recovery ~er p_hotobleaching)
and, of course, vii) other optical techniques like ellipsometry in external (Hickel et al, 1990)
or total internal reflection geometry (Habicht et al, submitted).

71
 (1)

(2}

(3)

Figure 4. A sequential approach to polymer-supported Iipid monolayers: (I) NHS-ester silanes are attached
to the surfaces, in subsequent reaction steps poly(ethylene-imine) or poly(ethyloxazoline-stat-ethyleneimine)
polymer (2) and Iipids (3) are bound to the monolayers.

72
POLYMER-SUPPORTED LIPID MEMBRANES ON SI02 SUPPORTS

The first example that we summarize concerns the build-up of polymer-tethered Iipid
bilayers based on the sequential deposition approach. Some details ofthe employed chemical
principles as weil as some preparation steps are given in Fig 4. The Si02 substrate (either a
glass slide, an oxidized wafer, or a thin layer of Si02 evaporated onto a Au or Ag substrate
to allow for the surface plasmon spectroscopic characterization of the various reaction steps
(see Fig. 5 below), was first coated in a self-assembly process by a dimethyl-mono-chloro
silane derivative with a N-hydroxy-succineimide reactive ester group (NHS) at the alkyl
chain end. This monolayer formation, followed also by IR, XPS and 29Si-MAS NMR
investigations, could be identified and quantified by the shift in the SPS resonance curve
relative to the SiOx-coated Ag substrate. This can be seen in Fig. 5. Assuming a refractive
index ofn=l.5 a layer thickness of IIA is obtained from the Fresnel fit calculation.
The next preparation step was the adsorption and covalent coupling of the polymer as
the cushion for the Iipid layer. Various systems were used with a strong focus on
poly(ethyloxazolines) (PEOx) prepared by cationic ring opening polymerization (Heibel et
al, 1996), its copolymers with ethyleneimine, prepared by partial hydrolysis of PEOx, linear
poly(ethyleneimine) (LPEI) obtained by complete hydro Iysis of PEOx, and, for comparison,
commercially available branched poly(ethyleneimine) BPEI. The time course and the final
layer thickness could be monitored by SPS. The covalent linkage in methanol under Argon
of some of the amino-groups of the PEOx-co-PEI systems which is catalyzed by an excess
ofpyridine was proven by extended soxhlet extraction ofthe coupled polymer layer for 14h
in absolute CH2Ch. The final layer thickness which could be controlled by the concentration
of the polymer solution from which the adsorption proceeded could be derived again from
SPS. The polymer layer given in Fig. 5 was assembled for Jh from 0.5M solution at T=50°C
and had a thickness of d=36A.
The last step on the way to the final architecture was the assembly of a Iipid monolayer
onto the polymer support. As a test system we choose the reactive ester of NHS and
myristic acid. Its assembly for 12h at T=50°C from CH2Cb solution under Argon with
excess of pyridine resulted (again after soxhlet extraction 14h, abs. CH2Ch) in a layer
increase by 18A, in qualitative agreement with the value of a Iipid monolayer.

0.8
0::
~ 0.6
·:;:
.ü
QJ
~ 0.4
0::

0.2

48
O.Ol..-....13-8--4.L..0--4-'-2--....J4W4UL...!L..C.4IL6_ _ _j
9/ deg

Figure 5. Surface plasmon resonance measurements ofsurface-attached multilayers with increasing

complexity (a) reference substrate (glass/ Ag/ SiOx-coating), (b) 1.1 nm thick NHS-silane SAM, (c) NHS-
silane layer + 3.6 nm PEOX; (c) NHS-silane-SAM/ PEOX/ 1.6 nm Iipid monolayer. Symbolsare
experimental data points, full curves are Fresnel fits.

73
 One important structural/functional feature of these polymer-supported monolayers is
their ability to take up water (cf. Fig. 6(a)). One way of testing this is the SPS-optical
determination of the layer thickness increase as a function of increasing relative humidity.
The experiments were done in a closed chamber in which the interfacial layers could be
exposed to relative humidities controlled by saturated solutions of different salts ( cf. Fig.
6(b)). The monolayer/polymer system described above showed a significant change in
reflectivity which indicated a swelling with increasing humidity which was, however, fully
reversible (see Fig. 6(b)). For comparison we display in Fig. 6(c) the quantitative analysis of
such swelling studies for a system that was prepared by mere electrostatic coupling of an
arachidie acid monolayer to physisorbed BPEI. As expected, the water up-take is most
pronounced at high relative humidities.

(a)

(b)
0.12

·f 0.10
tl<V
c;::
~ 0.08

0.06

i i t i t relative
7% 23% 43% 70% 98% 0% humidity

(c) 5
E
.E 4
Q
c: ~I
n; 3 I

0
e-
8 2
0 - 0 - /0
,I
.!:
1
/--c---
0 ...
--
_!>/-
:r:
0
40 60 80 100
relative humidity I%

Figure 6. The swelling behavior of polymer-supported monolayers in different relative humidities: (a)
Schematic ofthe water up-take by the polymer; (b) sequence ofreflectivity measurements at constant angle
of incidence taken at different relative humidities for a monolayer of partially hydrolyzed PEOX (degree of
hydrolysis 40%) attached to a SiO. -NHS monolayer at25°C. Onto this layer a monolayer ofDMPC/ CI4-
NHS had been transferred by the LBK-technique; (c) Quantitativeanalysis ofswelling experiments
performed with the system Ag/ physisorbed BPEI/ arachidie acid monolayer.

74
 0
II>
.!!!.
NE
.=
:c::II>
0
u 0 .1
:c::
Ci

15 20 25 30 35 40
Temperature I ac
Figure 7. Diffusion coefficients obtained by FRAP measurements for a dextrane-supported DMPC bilayer
containing 30 mole% cholesterol in water at different temperatures.

Another structural/functional parameter concerns the fluidity of the supported

membranes. Many incorporated functional units require a liquid-like membrane environment.
One conceptional goal of our research on interfacial supported membrane architectures,
therefore, is the preparation of fluid Iipid bilayers. The required physical decoupling of the
polar headgroups of the Iipids from the substrate surface is one of the main tasks of the
polymer cushion. The experimental technique to measure the lateral diffusional mobility of
Iipid molecules in a membrane Ieaflet is FRAP. We could show that for a number of
combinations lipid/polymer lateral diffusion constants could be found also for tethered
membranes as they are characteristic for a fluid membrane, e.g., in Iiposomes or vesicles
(Beyer et al, 1996a). The example presented in Fig. 7 was obtained from a dextran-
supported DMPC-bilayer mixed with 30 mole% of cholesterol. This important biomembrane
constituent is known to reduce the fluidity in the liquid-crystalline phase and to enhance the
lateral mobility in the gel-phase. In addition, sharp phase transitions of pure phospholipid
bilayer membranes are smeared out. Correspondingly, we find at high temperatures a
diffusion coefficient that is representative of a very efficient lateral diffusivity of ~I J..Lm2 /sec
which decreases upon a decrease of the sample temperature by more than an order of
magnitude.
AJI systems and a broad range of other chemical realizations of the polymer supported
bilayer concept are now available and ready to be coupled to the oxide surface of
microelectronic devices. The sequential build-up offers also the possibility to functionalize
other substrate materials, an extension which only requires a chemical modification of the
first anchor layer in order to account for the different reactive sites on the other support.

THIOPEPTIDE-SUPPORTED MEMBRANES ON AU ELECTRODES

The next example of the tethered membrane concept is based on a peptide sequence
used to support a Iipid bilayer. The principles ofthe coupling concept are displayed in Fig. 8.
Firstly, an amino acid sequence is assembled from solution and covalently attached to the Au
substrate via the cystein end group. The resulting layer formation can be followed by SPS
and by X-ray reflectometry (Bunjes et al, submitted).
Fig. 9 displays the surface plasmon resonance curves of the bare Au and after coupling
ofthe peptide layer, respectively. From the Fresnel fits one obtains a peptide layer thickness

75
 +

- 0
H'N"'VO. .0 ....II
+ H ..P. "yo~
0 0 l 0
0~

Figure 8. Schematic representation ofthe thiopeptide monolayer chemisorbed on the gold support and the
in-situ coupling ofterminal COOH groups with DMPE.

20 25 30

9 / deg

Figure 9. SPS reflectivity scans taken from (a) the bare Au-substrate, (b) after chemisorption of the peptide
layer, and (c) after the covalent coupling of a DMPE layer.

76
of d=I2 A. Forthis particular sample we tested a contrast variation approach in SPS which
allows one to differentiate between thickness and refractive index. By performing SPS
experiments with the thin layer in contact with different media (i.e., with media of different
refractive index, e.g., in air, water, buffer, etc.) a separate determination of n and d is
possible within the Iimits of a "box model" for the layer. The refractive index for the peptide
tether thus derived was n= 1.41.
The structural data obtained from the optical sturlies were confirmed by XR. The
corresponding measurements are displayed in Fig. IO. Plotted are the reflectivities as a
function of the momentum transfer q, which corresponds to the reflection angle. The top
most curve recorded from the bare Au substrate which was a thin film of d=45 nm thickness
(and thus identical to the ones used in SPS) evaporated onto a glass slide shows above the
critical angle for total internal reflection at Qc~0.75 nm· 1 intensity oscillations, so-called
Kiessig fringes that originate from the interference of partial X-ray waves reflected from the
airlAu and Au/substrate interface, respectively. The experimental data can be weil described
by a Fresnel fit that gives, in particular, the Au layer thickness, its electron density and two
roughness parameters, characterizing the air/Au and Au/substrate interface. The next curve
was measured after the peptide layer was assembled. Despite being an extremely thin organic
monolayer coating its influence on the interference pattern is clearly visible as a substantial
angular (phase-) shift of the sequence of constructive and destructive interferences. The
corresponding Fresnel fit gives a thickness of the peptide layer of d=lSA. in excellent
agreement with the optical data.
The next preparation step intended the covalent coupling of Iipid molecules to the
(activated) COOH-endgroups ofthe peptide layer (cf. Fig. 8). The reaction was carried out
in DMF at room temperature and the resulting layer again characterized by SPS and XR,
both performed again in air, i.e., after rinsing and drying. Fig. 9 also displays the reflectivity
curve obtained after the second coupling step. The Fresnel fit yields a totallayer thickness of
d=26A. which points to only a partial coverage of the peptide layer by Iipid molecules.
In this context it is interesting to note that the XR data displayed also in Fig. I 0 gave a
total layer thickness of d=35A., the experimental data, however, could be fitted only by
introducing a remarkable roughness at the Iipid/air interface. Considering the differences in
the lateral averaging for SPS and XR, respectively, this result is reasonable in that the optical
technique gives a mean thickness, averaging coated areas and free peptide domains, whereas
XR "sees" the holes as a rough surface topography!
The differences ofthe layer thicknesses obtained by the two techniques are summarized
in Table I and compared with theoretical expectations based on structural model
assumptions (Bunjes et al, submitted).
Up to this point of the preparation procedure the optical sturlies can be complemented
by XR performed in air. For this type of measurements a strong X-ray generator, e.g.,
equipped with a rotating anode is sufficient, a synchrotron X-ray source is not required. For

Table 1. Thickness values as determined by surface plasmon spectroscopy (SPS) and X-ray
reflectometry (XR).

d/nm
SPS XR
Thiopeptid - Layer 1.2 1.5
Thiopeptid - Iipid monolayer 2.6 3.5
Thiopeptid - Iipid doublelayer 5.1
same with ATPase
I per Liposome 8.1
10 per Liposome 8.8
20 per Liposome 7.3

77
 10°

10"2 a)

10""
b)
=:
~
-~ c)
-=as 10"6
c<II
=:
10"8

J Q·IO

1o·'2

0.5 1.0 1.5 2.0

·I
q / nm

Figure 10. XR spectra (a) of the bare substrate which is a 45 nm thick Au layer evaporated onto the glass
support giving rise to the observed Kiessig fringes; (b) after the chemisorption of the peptide layer, and (c)
after the covalent coupling of the Iipid layer. The sample architectures were identical to the ones used for the
SPS investigations (cf. Fig. 9).

9
10 ATPase
8 l ATPase

7 20 ATPase

6
§
......
"d 5

2
0 2 4 6 8 10 12 14 16 18
tl h
Figure 11. Real time recordings (reflectivity vs. time) ofthe fusion ofthe thiopeptide Iipid monolayer with
Iiposomes with incorporated ATPase CFoF1 the concentration ofwhich with respect to the Iipid was 8 mg
ml"1, whi1e the protein concentration varied from 23 to 461 flg ml"1 CFoF1 corresponding to an average
number of enzymes of 1, 10 and 20 CFoF1 per Iiposome, respectively.

78
 u
-60
;\

1\
-50

<(
-40
,l~ 0 50
-- 100
JYI\ .
jt 1
::J.. -30 c (ATP) I mmol L· 1
.._

-20

-10

10
0 -0 .5
U/V
Figure 12. Square wave voltammetry (corrected for the blank curve) of a peptide-supported Iipid double
layer with incorporated EF0F 1 in the presence of !(--), 6 (===), (-·-·-), 75 (-- -), and 89 mmol L" 1 ATP
( ....... ).Insert: Peak height as function ofthe concentration of ATP without (-- -) and with EFoF1 (-).

the next steps that have to be performed in contact with an aqueous phase typically only NR
in a total internal reflection geometry would be applicable and has been used to characterize
supported membranes. However, the need to use a neutron reactor Iimits the practicability.
The optical techniques, on the other hand, still operate under water and give valuable further
structural data, e.g., on the bilayer formation.
The vesicle fusion can be followed on-line, as it is demonstrated in Fig. 11. After the
Au-substrate functionalized with the peptide-supported monolayers has been mounted in the
SPS cell filled with buffer, vesicles loaded with ATPase from E. coli and chloroplasts were
injected and their fusion onto the tethered monolayers was followed in the kinetic mode of
SPS. One can see that the complete fusion is a rather slow process (and requires that the
fusing Iipid membranes are in a fluid state). Interesting, however, is the fact that the final
thickness increase seems to be largely independent of the degree of protein reconstitution in
the fusing vesicles: For preparations with a molar ratio between 1 and 20 proteins (on
average) per vesicle the thickness of the supported membrane at the end of the fusion is
almost constant- and very !arge (cf Table 1). A tentative explanation is an enrichment ofthe
protein in the tethered membrane, an interpretation that is supported by the finding of a very
active proton translocation across these membranes.
These systems that were very weil characterized structurally could then be investigated
functionally by electrochemical methods, because they were assembled directly onto suitable
Au electrodes and already in contact with electrolyte. It was therefore Straightforward to use
chronoamperometry to test for the pumping activity ofthe incorporated proteins.
The results are displayed in Fig. 12. Upon sweeping the potential from +0.3V to -0.8V
one finds a cathodic wave at ca. -0.7V if ATP is present in the external aqueous medium.
The peak intensity increases with increasing ATP concentration and then saturates (cf. the
inset in Fig. 12). In the absence ofthe protein, i.e., for supported matrix bilayers only, ATP
has no effect (also shown in the inset of Fig. 12). The underlying mechanism is the active
translocation ofH+ across the membrane by the protein using ATP as the energy source. The
protons accumulating in the cleft between membrane and electrode, are reduced by an
electron transfer step from the electrode, and this latter step is monitared during the
experiment.

79
 Figure 13. Photograph and schematic cross section ofthe encapsulated chip, for details see text. The
Japanese 'I 1 coin gives a scale.

We should point out that these membranes are far from being perfect and electrically
tight. Rather, they exhibit specific resistivities of R-1 Ok0cm 2 (at capacities in the order of
C=4J.!F/cm2) only. Nevertheless, the transient character of this very specific proton
translocation event and its selective recording during a voltage sweep allows for a functional
test of these architectures which is rather unique. The mechanistic picture of the involved
elementary translocation steps is further confirmed by the fact that dicyclohexylcarbodiimid
(DCCD), a known decoupler ofthe respiratory chain in biomembranes, reduces the observed
cathodic wave, and only 3.5mM DCCD in the aqueous compartment is enough to
completely suppress the electron transfer process: now it is easier for the protons to diffuse
back across the leaky membrane than to pick up an electron from the electrode surface.
The presented results on tethered Iipid membranes are very first steps only, but,
nevertheless, in a very promising direction. If the feasibility of the structural and functional
coupling of artificial membranes to solid electric/electronic transducer surfaces is further
confirmed, v~ry exciting prospects can be envisaged for the practical use of such complex
interfacial architectures in novel sensor applications (Cornell et al, 1997) or for drug
screening, e.g., when combined with genetically modified functional units. From a

80
fundamental point of view, these supported membranes may prove to be a model system for
many basic biophysical and biochemical studies offering a mechanical long term stability by
far superior over other much more fragile systems like the black Iipid membranes (BLMs)
(Knoll, 1994).

WHOLE CELL COUPLING TO MICROELECTRONIC DEVICES

Compared to supported artificial Iipid bilayer membranes cells in contact with a solid
support are considerably more complex systems. As far as the bio-engineering of the
interactive cell-device coupling is concerned this has consequences mostly with respect to
two important features: the far more demanding requirements for the multi-component mix
of the buffer in contact with the cell and the different concepts that need to be realized and
optimized for the control ofthe cell/substrate interaction.
The first problem, i.e., physiological buffer components in contact with a
microelectronic element is mostly solved by the proper design concept for the device. Si-
based process technology and device formats turned out to be very weil suited for being
combined with the environmental requirements of a living and developing cell, e.g., a neuron
(Fromherz et al, 199 1; Weis and Fromherz, 1997; Offenhäusser et al, 1997a).
Our design concept and its current realization is shown in Fig. 13. ( a) displays a
photograph of the chip mounted on a standard chip-carrier and with the mini petri dish
attached to it. (b) gives a schematic cross section showing the chip, the bond wires, the inner
silicone ring sealed to the chip and the chip-carrier by means of a silicone rubber inside a
glass container (Offenhäusser et al, J997b ).
The chip itself is designed so as to integrate 16 FETs within an area of 800x800 J..Lm2
with a shared source contact and individual drain contacts for separate but simultaneaus
read-out of the FET signals. Fig. 14 shows micrographs of the active chip surface in
different magnifications. The lower picture shows a single gate. The effective gate
dimensions were varied between IJ..Lm and 9J..Lm in length and I ÜJ..Lm and 28J..Lm in width. The
gate oxide thickness, one of the most important performance parameters was varied between
4nm and 30nm. The data acquisition electronics allows for the parallel monitaring of 4 FET
signals and is fully computer controlled.

Figure 14. The FET-array in different magniftcations. The arca ofthe 4 x 4 transistor array is 800 x 800
11111' . The gate dimension in thc lowcst frame is 8 x 18 11111 2

81
 A more difficult challenge to meet concerns the cell/substrate interface. Depending on
the cell type individual solutions will be required. E.g., the "classical" way to culture neurons
from the mammalian central nervous tissues would be seeding the cells together with glia
cells. The latter would secret the extracellular matrix, adhesion proteins which physisorb to
the substrate and to which the neurons would easily adhere. However, this would mean a
substantial separation distance between neuron and gate surface and hence would cause a
largely reduced electronic coupling of any capacitive or conductive changes across the cell
membrane to the FET. Fora tight coupling, therefore, glia-cell- and serum-free media have
to be used for the in vitro culturing of neurons.
The control of cell adhesion then has to be achieved by means of a suitable surface
modification by chemical means. This includes the preparation of device coatings that are
cell-repellent as weil as domains where the surface functionalization attracts cell and
stimulates them to adhere and to develop to their full functionality .
An example for the passivation of a selected area on the chip towards cell adhesion is
given in Fig. 15. The coating process was based on a so-called grafting-from approach for
the preparation of polymer brushes, i.e., densely packed end-grafted linear polymer chains
(Rühe, 1994). This concept involves the coating of the substrate by a monolayer of radical
initiator molecules, in our case modified by monochloro-silane endgroups so as to allow for
the covalent attachment to the gate oxide. If such a molecule is activated by heat or, for our
purpose, by light the decomposition of the initiator generat es a radical covalently attached to
the substrate, which in the presence of suitable monomer units triggers the formation of an
end-grafted macromolecular chain by polymerization from the surface. If the initiator
monolayer is exposed to light through a mask a pattern of the polymer coating can be
generated. In the example presented in Figure 15 a 20 nm thick polystyrene film was
prepared using an electron microscopy copper grid as a mask. The randomly seeded PC 12
cells only adhere to the areas that were not covered by the thin PS film. Moreover, for the
developing cells it can be seen that also their dendrite-like protrusions seem to avoid the PS
covered regions.
The complemental behavior, i.e., the promotion of cell adhesion to certain areas by an
appropriate surface functionalization can also be achieved. Our approach is based on the

Figure 15. PC-12-cells grown on a FET-ehipafter random seeding. The device surface was partially covered
with a 20 nm thick polystyrene film grown via the "grafting-from" techilique by activating the initiator SAM
deposited to the Si02 surface with UV light through a mask leading to ca. 80 x 80 ~m' PS pads separated by
ca. 20 ~m wide uncoated stripes. Cells adhere only to the non-passivated stripes whereas PS acts as a cell
repellent.

82
 observation that only certain "active domains" of the cell adhesion protein laminin are
recognized by the cells and trigger their settling. So we developed the strategy to use a short
peptide sequence of 20 amino acids that mirnie this binding domain of the u chain of the
protein and couple it to the substrate surface (Matsuzawa et al, 1996). The corresponding
reaction scheme is shown in Fig. 16. Firstly, the silanol groups ofthe glassy surface are used
to covalently couple an aminosilane compound. These amino groups are reacted with one
end of a heterobifunctional crosslinker the other end of which is then used for the final
coup1ing ofthe peptide. We could show that this way neuronal cells can be cultured in-situ
and triggered to develop after a few days in vitro a regu1ar morphological pattern with a
resting potential negative enough to also allow them to fire action potentials.
A particular aspect of our strategy is the possibility to generate a laterally patterned
functionality on the substrate, i.e., to decorate only certain areas with the adhesion
promoting peptide while others remain inert (or are modified by the repellent PS layer). This
can be achieved at different Ievels of this sequential reaction scheme, and basically all the
protocols developed for the patterning of silane self-assembled monolayers can be applied
(Matsuzawa et al, 1997). In the example shown in Fig. 17 a regular pattern of 401J.m wide
stripes separated by 401J.m gaps of bare glass were functionalized by the peptide. Random
seeding of neurons resulted after 4 days in vitro in a selective settling of the cells to the
functional areas only and also their neuritic processes were found to selectively grow along
this preferred surface coating.
When taken together, the positive Stimulation of cell attachment by the peptide
coupling and the negative repellent function of the PS coating should enable one to generate
a surface pattern that follows the lateral arrangement of the FET array on the chip. One of
the various goals of this research program, e.g., the experimental realization of a neuronal
network coupled to external electrodes monitaring the individual activities of the neurons
connected to each other in a controlled way by synapses is schematically depicted in Figure
18. Using similar masks as the ones employed for the chip fabrication it will be possible to
functionalize by Iithographie procedures only the gate areas and some connecting surface
channels with the peptide while completely passivating the rest of the chip surface. The
employed processing steps, by no way, are limited to the relatively small number of FETs

(a) (b)

OH OH OH OH OH OH OH OH OH
I I I I I I I I I
~ ~ & ~ ~ & & & &
Aminosilane Film
Formation

(d)
(c)

r-crcrcrcrcrcrcrcr
XXXXX s s s s s s s s s
XXXXXXXXX
er Cystein-labeled
NH NH NH NH NH NH NH NH NH
~~~~~~~~~ NH NH NH NH NH NH NH NH NH
~ & ~ a a a a a a ~~~ ~ ~ ~ ~~~
sH Peptide a a a & ~ a a & a
.
. Gouplmg
p,epflde )

Figure 16. Scheme for the covalent coupling of a cystein-labeled peptide chain to a Si02 surface, e.g., the
gate electrode of a FET.

83
 -
4 0 J1m

Figure 17. Neuron adhesion and dendritic outgrowth pattern on a glass substrate partially functionalized
with a peptide sequence mimicking a binding domain of the adhesion protein laminine. After random
seeding of the fetal rat-hippocampal cells to the substrate only the peptide-coated stripes of 40 ~-tm width are
populated after a few days in vitro.

synapse

Figure 18. Schematic of a neuro-electronic device consisting of an assembly by neuron cells coupled to
microelectronic devices and communicating to each other through synapses.

integrated in our chip so far. It thus will be possible to experimentally follow the transition
from the interplay of a few neurons to the chaotic response of a )arge number of information
processing units.

RECORDINGS FROM A SYNCHRONIZED CELL MONO LA VER COUPLED TO

THEFETCHIP

The last example discussed concerns the synchronized firing of action potentials of an
electrically and mechanically coupled monolayer of rat cardiac myocytes cultured on the
FET array (Sprößler et al, submitted). The cells were dissected from 3 days old rat hearts
and seeded randomly across the bottom of the mini petri dish. After ca 4 days in vitro a
single cell layer developed and started to synchronously beat and fire action potentials in a
correlated way. The electrical (and mechanical) synchronization between the single cells
originates from gap junctions that guarantee the transfer of the excitation from one cell to its
neighbors. This is schematically depicted in Fig. 19(a}, lower part. A micropipette impaled

84
into one of the cells allows for the recording of its action potential. An example is given in
Fig. 19(a) upper part. The inward sodium current is the initial event triggering a fast rising
action potential. The calcium current carries the plateau phase of the action potential and the
potassium current repolarises the cell.
The coupling ofthe celllayer to the chip surface allowed us to monitor the cell activity
by the gate electrodes of the FET array. Two types of signals could be found (cf Fig.
19(b)): a low amplitude signal shown in the right part ofFig. 19(b) with typically 300 to 500
J.!V peak-to-peak amplitude and a giant signal shown in the left part with a different shape
but a maximum amplitude ofca. 25mV. As we could show by simulations with an equivalent
circuit the smaller signal can be explained using an extended point-contact model. The first,
fast rising part of the extracellular signal is mainly originated by the convolution of the
intracellular voltage VM(t) with the high-pass filter element consisting ofthe capacitance of
the membrane CM and the seal resistance R1 of the junction. The second part of the signal
comes mostly from the contribution of current flowing across the membrane in the contact
area due to active ion channels. The difference between the !arge signals and the normal
signals is due to either a leaky membrane or an unusually high seal resistance. This means
that the membrane resistance RM is in the range ofthe seal resistance R1 ofthe junction.
While the electrical activity of an assembly of communicating cells like the
cardiomyocytes could be probed by a maximum of 4 electrodes at most (because of
limitations in space needed for the micromanipulators) the external electrode concept with

glass
microelectrode

0.5 sec
Figure 19. Schematic ofthe experimental set-up used for electrical recording from rat cardiac myocytes.
Measurements of action potentials from a cell layer afler 4 days in vitro were performed simultaneously with
an intracellular microelectrode (upper trace) impaled into the beating cell grown on a FET (lower traces).
Two types of signals can be observed that originale from differences in the contribution of active membrane
elements (Matsuzawa et al, 1997).

85
 channel # 16

Sms 10

recor
sites

beating-
center
Figure 20. 4 different FET recordings from a cardiac myocyte monolayer synchronized in its mechanical
beating and electric firing pattem by a pace·maker cell. The different location ofthe FETs on the chip, lower
part, relative to the beating center (X) results in a different time delay of the corresponding action potential
recordings (upper traces).

an array of FETs allows for a multiplicity in the monitaring of cell potentials that will give
eventually new insights into the experimental validation of existing 2-dimensional network
model of cardiac cells, the schemes of cell communication, and the mechanisms of learning
and memory. As a first step into this direction we present in Fig. 20 the results obtained
from the heart muscle cell layer by the simultaneaus recording of potential changes in 4
single cells sitting on different FETs of our sensor array. The spatial arrangement of the
transistors that were monitared is schematically depicted in Fig. 20(a). The corresponding
signals recorded in parallel are shown in Fig. 20(b). Clearly seen is the time delay between
the individual action potentials. This effect is due to the fact that during the cell development
eventually one cell, the one with the highest frequency of spontaneaus firing, takes the role
of a pacemaker, dominating and synchronizing all other cells. The known spatial
arrangement of the recording detectors and the temporal sequence of the simultaneously
recorded action potentials allows us to exactly localize the pacemaker cell (indicated by X in
Fig. 20(a)) and to determine the velocity by which the excitation wave propagates across the
cell monolayer, in our system with 0.2m/sec. This recording scheme can be easily extended
to all 16 FETs on our chip and, of course, more transistors can be integrated into the device.

CONCLUSIONS

lnteractive biocompatibility, i.e., the communicative coupling of technical components

and functional elements of living organisms remains a challenge for modern research on
biointerfaces. Tethered membranes, either in the format of a supported artificial bimolecular
Iipid layer functionalized by the reconstitution of receptors, ionophores (carriers, pores) etc.,

86
or as bio-membranes of living cells or tissues will play a central roJe in this field. On the one
hand side the question is as to how much complexity of a natural membrane is required for a
specific technological application, e.g., in biosensors or in various formats of bio-/neuro-
electronic devices. Can we disassemble a cell, select certain functional units (in their
wildtype or in a genetically modified version) and reassemble and reconstitute an artificial
membrane that mirnies certain recognition or transport properties of the biosystem, but is in
close contact with a transducer element and is more stable and robust, an essential
requirement for technological applications. For certain sensor formats, e.g., this certainly
will be sufficient to optimize the functional needs for the membrane part. The stability of
such tethered membrane systems might be even further enhanced by the incorporation of
polymerizable Iipids which after cross-linking to a macro-Iipid (network) enhances the
mechanical robustness of the whole device. However, for many other envisaged
configurations for the coupling of biological elements with microelectronic devices we will
have to work with whole cells, we will need to couple the complex multicomponent
architecture of a biomembrane in an interactive way to a technical support. As we have seen
other concepts that are needed to provide the required biocompatibility. Moreover, for a
single cell coupled to a device, the question needs to be answered as to how stable and vital
a biological sub-system can be, once it is disconnected from its life-supporting environment.
What we gain, e.g., in sensor formats, are the secondary amplification cascades that
follow a single molecular recognition event. It is weil conceivable that only by these
mechanisms, optimized by evolution, we will be able to reach the sensitivity needed to detect
the low Ievels of certain analytes in body liquids, for environmental analysis, or in food
processing. The combination of the tethered membranes concept with the principles of
microsystems technology will eventually also allow us to develop formats for massive
parallel sensing. Lithographie approaches for lateral patterning substrates are easily
transferable to the fabrication needs of compartimented membrane device configurations, for
artificial supported membranes ("membrane corrals") as weil as for the parallel read-out of
the response of many individual selectively sensing cells. This next generation of sensor
arrays will be much more powerful, faster in response, and a Iot cheaper.

ACKNOWLEDGMENTS

This work profited from intense discussions with many colleagues. We are particularly
grateful to the advice given by 0. Beyer, C. Duschl, C.W. Frank, M. Hara, R. Johnston, E.
Jonczyk, S. Maus, C. Naumann, H. Ringsdorf, E. Sackmann, F.J. Schmitt, K. Tamada, and
H. Vogel. Excellent technical support was given by Y. Hasegawa. Parts of this work were
financially supported by the Bundesministerium fur Bildung, Wissenschaft, Forschung und
Technologie (BMBF) (Proj.-Nr. 0310852 and 0310895) the Deutsche
Forschungsgemeinschaft (DFG Of 22/2-l ).

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89
TARGETED EXPRESSION OF MAMMALIAN CYTOCHROMES P450SCC AND
P4502B4 IN YEAST SACCHAROMYCES CEREVISIAE.

Michael A. Eldarov, Vadim E. Sidorovich, Galina E. Pozmogova, and

Konstantim G. Skryabin

Center "Bioengineering" Russian Academy of Sciences, Moscow, Russia

INTRODUCTION

Cytochrome P450-dependent electron transfer systems represent a diverse and

important class of metalloproteins that show significant promise as a source of new materials
for bioelectronics, design of new biosensors and multistage enzyme processing systems (for
discussion see Nicolini et al., 1995; Pashkevitch et al., 1996).
P450 enzymes are responsible for a wide variety of important biological functions,
including the biosynthesis of all steroid hormones and the metabolism of almost all lipophylic
drugs and environmental taxins and carcinogens (Gonzales, 1990; Guengerich, 1992). The
recent progress in understanding the structure-function relationships in these enzymes,
heterologous expression and purification of P450s have already resulted in the construction
of several electrochemical systems based on recombinant P450 enzymes incorporated in Iipid
films with applicability for drug discovery studies, biocatalysis and synthesis of of
hydroxylated compounds (Estabrook et al., 1996; Zhang et al., 1997).
The construction of bioelectronic devices based on P450 enzymes requires elevated
amounts of these proteins in highly purified form, and this is only possible through the
application of recombinant DNA technology.
Recently we have reported the high Ievel expression of mammalian mitochondrial P450
enzyme- P450SCC (or P45011A1) bothin bacteria and yeast (Pashkevitch et al., 1996).
This protein is part of mitochondrial mini-electron transfer chain, including also adrenodoxin
and adrenodoxin reductase as redox partners, that are all tagether responsible for the initial
and rate limiting step in the synthesis of steroid hormones - cholesterol side-
chain cleavage (Miller, 1988). The construction of bioelectronic devices based on P450SCC
may require simultaneaus production of its protein partners in heterologous hast. Here we
describe the construction of systems for co-expression of P450SCC, ADX and ADR in N-
terminally modified form in yeast, showing that functional heterologous electron transfer
chain can be reconstructed in yeast when these proteins are retargeted to novel intracellular
compartments. We also show, that the constructed expression systems can be used for high-
level production of another P450-cytochrome - microsomal rabbit cytochrome P4502B4.
The data is presented and discussed in view of the current progress in the heterologous
expression of P450 cytochromes and their redox-partners.

Biophysics of Electron Transfer and Molecu/ar Bioelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 91
HETEROLOGOUS EXPRESSION OF P450 CYTOCHROMES: EXPRESSION
SYSTEMSAND STRATEGIES.

Production of mammalian cytochromes P450 in heterologous systems has become an

area of major interest for a variety of reasons and is being applied to study the role of
individual isozymes in drug metabolism, carcinogen activation or deactivation, to study
structure-activity relationships, in the generation of cell lines capable of synthesizing steroid
hormones from cholesterol, andin the development ofbiocatalysts etc. (Waterman, 1994).
Several heterologous expression systems for P450 enzymes have been developed
during last decade, including bacteria, yeast, mammalian and insect cell lines, and plants.
Despite of significant progress in this field, successful expression of these proteins remains
largely empirical at present.
P450 cytochromes, as weil as other metalloproteins, exert their in vivo function in
specialized intracellular compartments, and their biogenesis inside the cell often involves
specific targeting and processing steps. This notion, obviously, should be accurately consider
in designing expression strategy. The latter should be based on thorough inspection of such
properties, as protein subcellular localization, requirements for post-translational
modifications, possible interaction of heterologously overexpressed protein with the cellular
metabolic machinery.
For different proteins different approaches for organella-targeted gene expression can
be utilized to improve their production Ievel in heterologous host, and this requires
manipulation with their signal sequences.
The direct expression of unmodified recombinant human cytochrome P450 enzymes
(P450s) in Escherichia coli has proved to be extremely difficult. Sturlies on optimization of
expression of both microsomal and mitochondrial P450s in different systems clearly show,
that the most critical region, that should be manipulated for successful expression is that
around the start codon for translation. In contrast to soluble bacterial cytochromes, of which
the best studied are P450cAM and P450sM-3 (Lu et al., 1994; Ravichandran et al., 1993) both
microsomal and mitochondrial eukaryotic P450s contain N-terminal extensions, that are
responsible for their targeting to the appropriate subcellular compartments (Black, 1992).
The insertion of microsomal P450s into the endoplasmic reticulum membrane (ER) is
directed by hydrophobic N-terminal signal-anchor sequence that also functions as a stop-
transfer signal, leaving the rest ofthe P450 protein on the cytoplasmic side ofthe membrane.
A mechanism highly similar to this signal-recognition particle dependent insertion of proteins
in the ER is likely to be responsible for localization of P450s expressed in E.coli to the
bacterial inner membrane (Saier et al., 1989). However, the correct folding of P450
polypeptide into holoenzyme shows striking dependence upon the type of expression vector
and recipient strain used (Waterman, 1994). The most promising results were obtained with
the pCWori+ vector and with modified N-terminal sequence from bovine cytochrome
P45017(-alpha), where silent and neutral changes were made to enrich the AT-content and
reduce the probability of hairpin formation in this region of mRNA (Fisher et al., 1992). By
this approach expression Ievels of as high as 400 nmollliter of culture can be achieved (>25
mgll).
Recently a more general strategy aimed at production of unmodified P450s in high
Ievels in E. coli was suggested, based on NH2-terminal translational fusions to bacterial
Ieader sequences (Pritchard et al., 1997). The two Ieader constructs, based on pe!B and
ompA sequences, when fused to CYP3A4, CYP2A6, CYP2E1 all resulted in the production
of spectrally active, functional proteins, and marked improvement in the recovery of active
P450 in bacterial membrane fractions, when compared with the conventional (17alpha-)
approach.

92
Construction of Optomized P450 Enzyme Systems

From the studies with individual mammalian cytochromes P450 produced in different
environments, it became evident, that for the creation of systems with increased reactivity
and stability, it is necessary to express simultaneously both the P450 enzymes and its redox
partners. Several approaches have been used to improve the system, and they can be roughly
classified in two categories: generation of in vivo systems for stable and regulated CO-
expression of P450 with oxido-reduction proteins (a) and construction of enzyme fusions
(b).
In vivo systems. Reported variability in the expression of different mammalian
cytochromes P450 in S.cerevisiae (Oeda et al., 1985; Cullin and Pompon, 1988) could be
attributed to the fact that the S.cerevisiae P450 reductase activity is limiting (Murakami et
al., 1990), and also that yeast reductase protein Yred (coded by the CPR1 gene) is only 35%
homologaus to the mammalian enzyme and may not couple efficiently with mammalian
enzyme.
Co-expression in yeast ofmammalian P450 with the yeast reductase was reported using
two expression cassetes on a single plasmid and resulted in several-fold enhancement of the
P450- dependent activity as compared with the expression of P450 alone (Murakami et al.,
1990). Besides reductase, mammalian cytochrome b5 is also required for the optimal
monooxygenase activity of particular P450 isoforms (White and Coon, 1980). This
potentiation roJe have been utilized in the development of diploid yeast strains, combining of
chromosomal alleles, specifying the regulated overexpression of Yred and/or human b5
(Pompon et al., 1996). In the engineered strains the overproduction of Yred and/or b5
increased significantly the turnover numbers of the heterologously expressed human P450
isoforms.
Several P450 species are involved in the biosynthesis of physiologically important
compounds, such as steroid hormones, vitamin D3 and prostaglandins. For example,
cortisol, one of the key intermediates in the synthesis of steroid hormones, is biosynthesized
from cholesterol through five enzymatic reactions, four ofwhich are P450-catalyzed (Miller,
1988). Therefore, these steroidogenic P450s are applicable to practical bioconversion
processes for steroid hormone synthesis if the respective enzymes can be overproduced and
their activity and stability be improved.
The initial and rate-limiting step of steroid hormone biosynthesis in various
steroidogenic tissues is the side cleavage reaction of cholesterol to yield pregnenolone which
is catalyzed by mitochonrial cholesterol side chain cleavage cytochrome P450 (P450SCC).
As mentioned above, this mitochondrial P450 is part of the electron transfer system in
eukaryotic cells that also includes adrenodoxin (ADX) and adrenodoxin reductase (ADR)
(reviewed by Usanov et al., 1990). ADX and ADR are synthesized on cytoplasmic
ribosomes as large precursors and then transported into the mitochondrial matrix and inner
membrane respectively, where they are post-translationally processed into mature forms. To
address the feasibility of the yeast system for the expression of proteins of mitochondrial
P450-monooxygenase complex the influence of the organella-targeting sequences on the
assembly of the mammalian mitochondrial electron-transfer chains in yeast were examined in
Saccharomyces cerevisiae (Akiyoshi-Shibata-M et al., 1991). Elimination ofthe mammalian
mitochondrial N-terminal signal from the ADX and ADR cDNA genes led to the Iocation of
the expressed mature proteins (mat-ADX and mat-ADR) to the cytoplasm of the
recombinant yeast strains. By use ofthe in vitro reconstituted system it was shown, that mat-
ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450SCC.
These finding enabled the same authors to proceed with the creation of a novel functional
electron transport chain between the cytoplasm and microsomes in yeast by simultaneously
expressing mat-ADX, mat-ADR and a modified rat cytochrome P450c27, whose
mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of

93
 bovine cytochrome P450c17 (Sakaki et al., 1992). The modified P450c27 hemoprotein was
correctly Jocalized on yeast microsomes and exhibited the P450c27-dependent
monooxygenase activity by addition of bovine ADX and ADR. So, it happens that the
destination ofP450c27 to either mitochondria or microsomes in yeast depends solely on the
amino-terminal targeting signal.
Protein fusion systems. The naturally occurring soluble CYp102 (B.megaterium
cytochrome BM-3), and nitric oxide synthase provide examples of P450-dependent catalysis
performed by a single polypeptide chain, comprised of P450 and reductase parts
(Degtyarenko and Archakov, 1993). The discovery of these self-sufficient enzymes had
stimulated the efforts aimed at the construction of several genetically engineered fused
proteins between P450 and reductase (Murakami et al., 1987; Sakaki et al., 1990, 1994).
These artificial fused enzymes were found to be superior in their turnover numbers and
stability in yeast microsomes. These efforts resulted in the following rules for construction of
efficient fused enzymes. Firstly, the P450-part should be placed at the amino terminus of the
fused enzyme. Secondly, the amino-terminal hydrophobic sequence of NADPH-P450
reductase, which serves as a membrane anchor to the Iipid bilayer, should be removed.
Finally, the length and sequence of the hinge region between the P450 and reductase parts
should be optimized (Shet et al., 1994). P450-reductase fusion proteins can be rather easily
purified (Parikh et al., 1997) and at least one particularly active fusion between P450cl7 and
soluble form of rat reductase was already successfully employed for the construction of
bioelectrochemical device, capable of prolonged synthesis of hydroxylated steroids
(Estabrook et al., 1996) .
Interestingly, the conservation of redox donors in P450 systems is not absolute, and in
some cases the heterologous electron donors can replace the homologaus ones in mediating
electron transfer to P450s. For instance, it was shown (Sakaki et al., 1996), that
microsomally-targeted mitochondrial P450c27, expressed in yeast, can accept electron form
the yeast NADPH-P450 reductase, whether the latter was co-expressed either separetely, or
within the fusion protein with mic-P450c27.
Contrary to this reported success in converting the mitochondrial P450c27 to
microsomal form, the similar approach failed when it was applied for the retargeting of
another mitochondrial P450 enzyme - P450SCC (P45011Al) to microsomes (Biack et al.,
1994). It was observed, that such protein, as weil as its different fusions with ADX and
ADR were not functional outside mitochondria of COS-1 cells. So, it was concluded that the
mitochondrial environment is strictly required for functional activity of P450SCC in
eukaryotic cells. However, from these findings it was not apparent, whether P450SCC can
be active in the ER of eukaryotic cells if soluble ADX and ADR are provided. The possible
difference in the Iipid and protein composition between mammalian and yeast ER should not
also be neglected.
This notion has prompted us to undertake the current research aimed at the
investigation of the requirements for functional expression of the mitochondrial cytochrome
P45011Al outside the mitochondria in yeast ER and to simultaneously test our expression
system for the production of a normal microsomal mammalian P450 enzyme - cytochrome
P4502B4.

RECONSTRUCTION OF MAMMALIAN MITOCHONDRIAL CYTOCHROME

P450-DEPENDENT ELECTRON TRANSFER CHAIN IN YEAST.

In order to investigate the requirements for functional expression of the mitochondrial

P450s outside the yeast mitochondria we have created the following gene constructs: i.
mature form of P450 11 A gene lacking N-terminal signal sequence P450 11 AI; ii. chimeric
form of P45011Al cDNA bearing N-terminal synthetic sequence (cl7 signal sequence) for

94
 ER targeting and anchoring instead the mitochondrial sequence (c I7P450 II AI); iii. mature
form ofcDNA of ADR and iv. matureform ofcDNA of ADX. All above mentioned cDNAs
were inserted into the high-copy number YEpES30-50 expression vectors (Sidorovich et al.,
I996) for targeted expression in ER and cytosol of the yeast cell (Figure I). The expression
vectors were co-transformed into the yeast strain t334 (Hovland et al., I989) yielding two
recombinant strains expressing one particular form ofP450IIAI and both electron transport
proteins -ADR and ADX.

YEpSCC4

MURAJ lb u orul YEpADR2

~~mADX · ~ b u orul YEpADX2

Figure I. Schematic structure of the expression cassettes used for cytochrome P450 11 A 1 -dependent enzyme
system Co-expression. All cDNAs were expressedunder the strong inducible S.cerevisiae GALJ promoter
(PaALJ!· Transcription terminationwas mediated by the presence ofthe terminator from the S.cerevisiae
PGKJ gene(tPGKII· URA3, LEU2, TRPJ-yeast selective markers; 2).! ori-yeast Saccharomyces cerevisiae's
origin of replication; mADR, mADX, mSCC-coding sequences formatureform ofNADPH-adrenodoxin
reductase, adrenodoxin and cytochrome P45011AI , respectively; cl7- denotes the first 18 codons for N-
terminal signal sequence for ER membrane retention. The names ofthe corresponding plasmids are given to
the right.

1 2 3 4

c
D

Figure 2. Subcellular fractionation ofthe cytochrome P45011Al-dependent enzyme system expressed in the
yeast S.cerevisiae. Recombinant cells carrying the multicopy plasmids YEpADR2, YEpADX2, YEpSCC3
and YEpADR2, YEpADX2, YEpSCC4, respectively were converted to spheroplasts, lysed and subjected to
sequential differential centrifugation. Equivalent amounts of subcellular extracts were separated on an SDS-
polyacrylamide gel, followed by electroblotting to a nitrocellulose filter. NADPH adrenodoxin reductase
(panel A), adrenodoxin (panel B}, matureform of cytochrome P45011 Al (mP45011AI) (panel C) and N-
terminally modified form of cytochrome P450 II AI (c 17P450 II AI) (panel D) were detected with a rabbit
polyclonal anti- NADPH adrenodoxin reductase antibody (panel A), a rabbit polyclonal anti- adrenodoxin
antibody (panel B) and a rabbit polyclonal anti- cytochrome P45011Al antibody (panel C and 0),
respectively. Aperoxidase -conjugated secondary antibody and 3,3'-diaminobenzidine served for
visualization of bands. The presence of these proteins was assayed in the supernatant (S II, lane 2) fractions
ofa 11,000 x g.. centrifugation, as weil as the pellet (PIOO, lane 3) and supernatant (SIOO, lane 4) fractions
ofa 100,000 x g•• centrifugation ofthe SII fractions. NADPH adrenodoxin reductase (panel A},
adrenodoxin (panel 8) and cytochrome P450 II AI (mP45011Al) (panel C and D) purified from bovine
adrenal cortex milochondria were used as control proteins (lane I).

Subcellular fractions were prepared from transformed yeast cells by differential

centrifugation (Akioshi-Shibata et al., 1991 ). Sampies were subjected to Western blot

95
analysis using polyclonal antiborlies against the purified P450 II AI, ADR and ADX. The
P450 II A 1 protein was detected as a single band in the P 100 fraction of the recombinant
yeast strain t334[YEpSCC3; YEpADR2; YEpADX2] at an apparent molecular weight of 54
kDa co-migrating with the purified bovine protein (Figure 2, panel C). A PI 00 fraction of
the strain t334[YEpSCC4; YEpADR2; YEpADX2] producing N-terminally modified
P450 II AI (c 17P450 II AI) harbored two bands, one of 54 kDa co-migrating with the
purified bovine P45011Al and an additional band with a slightly higher molecular weight
which could correspond to an N-terminally modified form of P45011 Al (Figure 2, panel D).
Whether targeted to ER or to the cytosol, expression products of c 17P450 11 A 1 and
mP45011Al produced ER membrane associated P45011Al proteins only.
Recombinant ADR and ADX possessed the same electrophoretic mobility as the
respective proteins purified from adrenal cortex mitochondria. Mature form of the
recombinant ADR was found both in ER membrane and cytosol fractions (Figure 2, panel
A). The recombinant ADX was identified only in the cytosol fraction ofthe strains (Figure 2,
panel B).
The alkaline treatment of ER membrane fractions of both recombinant strains was
performed with 100 mM Na2 C03 , pH 11.5 (Pernecky et al., 1993). Confirming its tight
membrane integration, c 17P450 II AI was not removable by alkaline treatment (Figure 3,
panel D). Surprisingly, mP45011Al was found to be embedded into the ER membrane
(Figure 3, panel C). In contrast to this findings, mADR was extracted from the membrane by
alkaline treatment only partly (Figure 3, panel A).

1
.
\
2
-
(
3
..
4
.
.
~
5

J
A t.& ... .......

c
Figure 3. Characterization ofthe association ofNADPH-adrenodoxin reductase. matureform cytochrome
P450 !lAI and N-terminally modified cytochrome P450IIAI expressed in the yeast S. cerevisiae with the
P IOO subcellular fraction .. The lysates werc of recombinant yeast strains were scquentially centrifuged at
11,000 x g., and 100,000 g., and PIOO fractions of respective strains were then treated with the following
reagents prior to centrifugation at I00,000 x g., to generate supernatant (S) a nd pellet (P) fractions: lane 1-
NADPH-adrenodoxin reductase and cytochrome P45011AI purified from bovinc adrenal cortex
mitochondria, 15 ng each; lanes 2 and 3-50 mM PBS, pH 7.4. Sand P fractions, rcspectively: lancs 4 and 5
- 100 mM Na2 COJ, pH 11.5. Sand P fractions, rcspectivcly.

nm

Figure 4. Fe2+ vs Fe2+ CO difference spectra of the PIOO

subcellular fraction prepared from the yeast S. cerevisiae
t334 strain transformed wwith YEpADR2, YEpADX2,
YEpSCC3 plasmids.

96
 The yield of expression was estimated by means of reduced CO difference spectrum of
the microsomal fractions of the recombinant strains. Spectrum was not detected in the
microsomal fraction bearing cl7P45011Al protein (not shown). In contrast absorption
maximums at 420 and 450 nm were detected in the membrane fraction consisting
mP45011AI protein (Figure 4). The yield of expressionwas estimated at 1.2 nmol/liter (10
pmol/mg ofmicrosomal protein).
Cytochrome c reductase activity was determined with the subcellular fractions prepared
from the recombinant strains (Table I). Cytochrome c reductase activity in subcellular
fractions ofthe control strain t334[YEpES30; YEpES40; YEpES50] was also measured and
subtracted as endogenaus background. Both recombinant mADR and mADX were found
active for transfer of electrons to cytochrome c in an in vitro assay.

Table 1. NADPH-adrenodoxin reductase and adrenodoxin activity in microsomes from

transformed yeast cells.

Microsomes 1 Reductase activity 2

(nmol cyt. c reduced/min mg protein)
t334[YEpSCC3; YepADR2] 5.8
t334[YEpSCC3; YepADX2] 3.6

1P45011Al content was always in the range of 10-25 pmol/mg microsomal protein.
2 The reductase activity measured as described (Akiyoshi-Shibata et al., 1991) shows the mean values ofat
least two independent determinations. The standard deviation was less than I 0%.

The hydroxylase activities of yeast microsomes expressing mP45011AI and

ci7P45011A1 proteins supplemented either mADR and mADX expressed in cytosol of the
yeast cell or ADR and ADX purified from bovine adrenal cortex were compared. For these
studies non-radioactive cholesterol dissolved in 100 mM HEPES, pH7.4, 0.5% Tween 21
was added into the reaction mixture. The reaction mixture contained 4 mg microsomal
protein, 1.0 unit glucose-6-phosphate dehydrogenase, 2mM glucose-6-phosphate and either
8 mg cytosol protein fraction of the recombinant yeast strain or 1nmol ADR and 10 nmol
ADX in 0.5 ml 100 mM HEPES, pH7.4, 1 mM EDTA, I mM DTT. The tubes were
vortexed and pre-warmed at 28°C. Incubations were started by addition of NADPH to 0.5
mM. Incubations were stopped by addition of equal volumes of both acetone and n-hexane
in 20 minutes. Proteins were separated by addition of ammonium sulphate. Tubes were
vigorously vortexed. Organic phases were separated and dried under vacuum. The
trimethylsilyl ether derivatives of steroids were prepared. A Hewlett-Packard HP-5988A gas
chromatograph coupled with a mass spectrometer was used for identification of steroids. GC
was carried out on a fused-silica capillary column (12 m, 0.2 mm internal diameter) coated
with methylsiloxane SE-30. Oven temperature was programmed from 80°C to 280°C with a
10°C/min increment. The column was coupled to the mass spectrometer ionization chamber.
The source temperature was set at 200°C and the energy ofbombarding electrons at 70eV.
GC/MS analysis of steroids demonstrated that mP45011Al microsomes supplemented with

Figure 5. Schematic structure of the expression cassette for cytochrome P450284 expression in the yeast
S.cerevisiae. Cytochrome P450284 cDNA was expressedunder the strong inducible S.cerevisiae GALJ
promoter <PaALI!· Transcription terminationwas mediated by the presence ofthe terminator from the
S.cerevisiae PGKJ gene(IPGKli· TRPJ-yeast selective marker; 2~ ori-yeast Saccharomyces cerevisiae's origin
of replication; P450284(ö2-27)-coding sequences for rabbit cytochrome P450284 with deleted amino acids
2-27; c17- denotes the first 18 codons for N-terminal signal sequence for ER membrane retention.

97
purified bovine ADR and ADX were able to convert about 1% of cholesterol to
pregnenolone for 20 min. Whereas the same microsomes supplemented with the cytosol
protein of the strain expressed ADR and ADX produced Ievel of pregnenolone that was
below the Iimit of detection for the analysis. Neither c17P45011A1 microsomes
supplemented with purified ADR and ADX nor recombinant ones produced detectable Ievels
of pregnenolone.

HIGH YIELD EXPRESSION OF CYTOCHROME P450 2B4 IN YEAST

Among the large cytochrome P450 family (EC.1.14.14.1.) the species of the 28
subfamily compise major phenobarbital-inducible isoforms serving in the bioactivation and/or
detoxification of a wide variety of compounds of pharmacological and toxicological
importance . There are at least three highly related cytochrome P45028 types, termed 80,
81 and 82 (Gasser et al, 1988). Cytochrome P450284 catalyses with high rate N-
demethylation of benzphetamine and 16alfa-hydroxylation of testosterone. These activities
increase in the microsomes from phenobarbital induced rabbits (White and Coon, 1980).
The attempts to express the unmodified, as weil as N-terminally truncated forms of
P450284 in E.coli in sizable amounts failed (Pernecky et al., 1993). It was reported that
unmodified P450284 protein can be expressed in 334 yeast with yields up to 10 pmoles per
mg of microsomal protein (Kedzie et al., 1991), or as a C-terminal fusion protein with
glutathione-S-transferase in E.coli (Pernecky and Coon, 1996).
We have used the gene coding for the N-terminally truncated P450284(~2-27) gene
(Pernecky et al., 1993) and have inserted it into the expression vector YEpES50 (Sidorovich
et al., 1996) in frame with the synthetic signal sequence c 17 (Figure 5). The resulting plasmid
(YEpc17/284(~2-27)/3'UTR) was transformed into the recipient yeast strain t334 (Hovland
et al., 1991 ). Subcellular fractions were prepared from transformed yeast cells by differential
centrifugation (as described in the caption for figure 2). Sampies were submitted to Western
b1ot analysis using polyclonal antiserum against the purified P450284.
The P450c17/284(~2-27) proteinwas detected as a singleband in the P100 fraction of
the recombinant yeast strain with an apparent molecular weight of 53 kDa, comigrating with
the purified rabbit liver protein (not shown).
To assay for insertion into the membrane of P450c17/284(~2-27), the microsomal
fraction of the yeast strain t334[Yepc17/284(~2-27)/3'UTR] was extracted with alkaline
solution (pH 11 .5)- the condition commonly used to discriminate between integral and
peripheral localization of membrane proteins. It appeared, that the recombinant protein
P450c17/284 in which the N-terminal amino acids 2-27 were replaced by synthetic 18 amino
acids long signal sequence was tightly associated with the microsomal membranes (Figure
6). These results showed that the used artificial N-terminal signal sequence is sufficient for
membrane insertion ofthe modified P450284.

Figure 6. Probing the interaction of cytochrome P450c 17 /284(~2-27)

1 2 3 4
with the P100 subcellu1ar fraction . The cell 1ysate of recombinant
yeast strain was treated with the reagents as described below prior to
centrifugation at 100,000 x &.v to generate supematant (S) and (P)
fractions: lane 1-cytochrome P450 2B4 purified from rabbit liver
microsomes, 30 ng; lanes 2 and 3 -50 mM PBS, pH 7 .4, S and P
fractions, respectively; lanes 4 and 5- 100 mM Na2C03 , pH 11.5, S
and P fractions, respectively.

98
 The expression rate was estimated by measurement of reduced CO difterence spectrum
ofthe PIOO membrane fraction ofthe recombinant yeast strain. Absorptionmaximum at 450
nm was detected in the membrane fraction consisting P450c 17/2B4(1l2-27) protein (Figure
7). No spectrum was recorded from the control microsomal fraction derived from the yeast
strain transformed with the expression vector YEpES50. The Ievel of expression was
estimated at 25 nmoles/liter (0.48 nmol/mg of microsomal protein). This certifies the
efficient folding ofP450cl7/2B4(1l2-27) enzymein the ER membrane ofthe yeast cell. The
recombinant cytcohrome possessed the catalytic properties similar to that of native P4502B4
towards 7-pentoxyresorufin (not shown).

DISCUSSION

We have shown, that mammalian mitochondrial steroid-hydroxylating P450 dependent

system can be functionally reconstructed in yeast by co-expressing soluble ADR and ADX
and mature P4501IAI form. The P450IIAI protein without any apparent signal sequence
was tightly localised to the yeast ER membrane, as evidenced from the subcellular
fractionation studies and probing its membrane interactions with high salt, alcaline and
detergent washes. The replacement of mitochondrial targeting sequence of P450 ll Al with
that of microsomal one of P450c 17 prevented its assembly in the holo-form and resulted in
the site-specific cleavage of the expressed protein, presumably at the boundary between the
signal sequence and mature part.
This observation may be rather important for further development of yeast strains,
capable of producing valuable steroid compounds using heterologous expression of

+O .OlS

-O.OlS

nm 400 450 soo

Figure 7. Fe2+ vs. Fe2 +CO difference spectra ofthe PIOO subcellular fraction prepared from the yeast
S.cerevisiae t3 34[Yepcl7/2B4(A2-27)/3 'UTR] strain.

99
 mammalian steroidogenic P450 cytochromes. The potential of metabolic engineering of
yeast strains towards production of steroid compounds have been recently demonstrated
(Dumas el al., 1996).
However, further developments in this field have to consider several important issues,
like complex genetic and physiological control of steroid uptake by yeast (Shinabarger et al.,
1989), absence in yeast of specialised mechanisms for steroid transport in mitochondria of
steroidogenic tissues (Kim et al., 1997).
1t is thus envisaged that retargeting of the key P450 enzymes in functional form from
mitochondria to ER may help to overcome these natural limitations of yeast systems for
expression of mammalian steroidogenic P450 enzymes and result in the construction of new
more efficient metabolic pathways.
Wehave observed marked difference- 20 fold in the expression Ievel between the two
cytochromes - P45011Al and P4502B4, using the same expression system. This may be
attributed to the fact that only a minor form of the mP45011Al expressed in yeast is
presented by holo-form, while the majority ofthe produced protein is in the apo-form. We
can not at present quantify the exact proportion ofboth forms produced in our system.
The constructed expression system for cytochrome P4502B4 with the yields up to 1.5
mg of functionally active enzyme per Iiter of yeast cell culture can be used successfully for
the routine isolation of this protein for further bioelectronic applications. The presence of N-
terminal membrane anchoring signal besides possible benefitial effects on gene expression
and protein isolation procedures, can be also used as efficient means for controlled
orientation on biomembrane-like supports for the bioelectronic applications, as shown
recently for cytochrome b5 (Ramsden el al., 1995).
Studies are in progress towards more thorough characterization of the created
expression systems, purification and analysis of recombinant proteins.

ACKNOWLEDGMENTS

This research was supported in part by the INCO-COPERNICUS grant PL965070 and
by the Russian Foundation for BasicResearc hgrant 97-04-49133.

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Black, S.D., 1992, Membrane topology of the mammalian P450 cytochromes, FASEB J. 6: 680.
Black, S.M., Harikrishna, J.A., Szklarz, G.D., Miller, W.L., 1994, The mitochondrial environment is
required for activity ofthe cholesterol side-chain cleavage cnzyme, cytochrome P450scc, Proc Nat/
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102
THE MOLECULAR ROLE OF THE PUFX PROTEIN IN BACTERIAL
PHOTOSYNTHETIC ELECTRON TRANSFER

Francesco Francia 1, Paola Turina2, B. Andrea Melandri 2, and Giovanni

Venturoli 2

1 Department ofMembrane Biochemistry,

Max-Planck-Institute for Biochemistry,
82152 Martinsried, FRG
2 Department of Biology, Laboratory of Biochemistry and Biophysics,

University ofBologna,
40126 Bologna, Italy

INTRODUCTION

The occurrence of photosynthetic activity in bacteria offers an excellent opportunity

for studying the primary events of photosynthesis at the molecular Ievel, due to the greater
simplicity of these organisms with respect to eukaryots. In both classes of organisms the
light reactions of photosynthesis are carried out by a series of multiproteic complexes
embedded in specialized membranes. The generat strategy for transducing light energy into
a chemical potential consists in a sequence of electron transfer reactions: shuttling of
electrons involving different membrane complexes results in an electrochemical potential
difference for protons across the photosynthetic membrane. This electrochemical gradient is
used primarily by the ATP-synthase to produce ATP from ADP and Pi, thus satisfying the
metabolic energy request ofthe bacteria.
The primary event in this transduction chain is a photoinduced charge separation
across the membrane dielectric, which, in bacteria, is accomplished by a single specialized
pigment-protein complex, the photosynthetic reaction center (RC). The RC is sorrounded
by highly pigmented proteins (antenna systems) which amplify its light capturing ability by
efficient transfer of excitation energy. The RC and the antenna form an ordered
supramolecular arrangement of pigment-protein complexes, referred to as the
photosynthetic unit.

Structure of the Bacterial Photosynthetic Unit

Typically, the photosynthetic bacteria contain two types of antenna complexes. One,
named light harvesting complex 2 (LH2), is arranged peripherally with respect to the RC

Biophysics of Electron Transfer and Molecu/ar Bioelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 103
and the other, light harvesting complex 1 (LH 1), is intimately associated with the RC to
form the so-called "core complex".

Figure l. LH2 octameric complex of Rhodospirillum molischianum (Koepke et al, 1996). The left panel
displays a top view with C-terminal pointing upward; the apoproteins are represented as Ca-tracing strands
in light grey; the a.- and ß-apoproteins form the inner and outer ring respectively. The BCW-a molecules are
in black and the lycopenes in deep grey. The right panel shows a lateral ribbon view ofthe apoproteins; the
C-terminal ends, facing the periplasmic side of the membrane, point upwards; atoms of the tryptophan
residues are shown as spacefilling spheres in deep grey. Figures were produced with the programs WHAT
IF and RasMoL

The amount of LH2 relative to the RC depends strongly on growing conditions,

whereas the LH1 is present in a fixed stoichiometry. The antenna complexes are formed by
two small hydrophobic polypeptides, named a and ß in both LH1 and LH2; the prosthetic
groups are bacteriochlorophyll-a and carotenoid molecules. Recently, the structure of the
LH2 system has been determined at atomic resolution by X-ray diffraction crystallography
in two species (McDermott et al., 1995; Koepke et al., 1996). In both of them the aß
dimers are associated into a ring-like structure, with the pigments arranged to maximise the
energy-transfer processes (Freer et al., 1996). A top and a lateral view of LH2 from
Rhodospirillum (Rs.) molischianum are shown in Figure 1.
Structural data have been reported for the LH1 complex in Rhodobacter (Rb.)
sphaeroides (Nunn et al. , 1992), Rs. mo/ischianum (Boonstra el al., 1994),
Rhodopseudomonas marina (Meckenstock et al., 1994) and Rs. ruhrum (Karrasch et al.,
1995). A model ofthe LH1 complex of Rs. rubrum, based on electron microscopy image
analysis, is represented in Figure 2. The ring like structure appears to be formed by 16
repetitive units. Each unit consists of three electron density peaks: the inner and outer ones
are attributed to the a and ß polypeptides respectively, the third resulting most likely from
the bound bacteriochlorophylls (BChl).
The reaction centre 3D atomic structure has been determined in two species of
photosyntethic bacteria. In both species, two subunits, L and M, each spanning the
membrane with five hydrophobic helices, bind the cofactors responsible for the primary
photochemistry. A third subunit, H, is anchored by a single hydrophobic helix to the LM
complex and caps it with a large hydrophilic domain facing the cytosolic side of the
membrane (for a review see Lancaster et al, 1995).

104
Figure 2. The 16-fold rotationally fittered image extracted from a projection map oftwo-dimensional
crystals from LHI of Rhodospirillum rubrum. Peaks of the inner and outer ring have been suggested to
represent the <X- and ß-subunits. The scale bar corresponds to 20 A. The contour line inside the LH1 ring
represents an approximate van der Waals projection of the RC of Rhodopseudomonas viridis (Deisenhofer
et al., 1985). A top view ofthe porphyrin rings ofthe BChl-b specialpair is also shown. Taken from Fig.6
of Karrasch et al ( 1995).

As Figure 2 shows, the RC fits well the size of the internal ring. Moreover, image
processing of solubilized RC-LHI core complexes from Rs. molischianum (Boonstra el
al., 1994) and of 2D-crystals of the complex from Rs. ruhrum (Walz and Ghosh, 1997)
reveal a ring-like structure surrounding a central nucleus attributed to the RC.
On the basis of the structural information available, summarized above, an overall
picture of the entire photosynthetic unit is emerging, consisting of a ring-shaped RC-LHI
complex in contact with a variable number of LH2 antennae (Papiz et al., 1996; Hu et al,
1997; Hu and Schulten, 1997).

The Interaction between the Reaction Centre and the bc 1 Complex

The RC cofactors, bound to the hydrophobic helices of the L and M subunits, are
arranged in two arms, A and B, associated with the L and M subunit respectively. Both the
cofactors and the L and M subunit show a high degree of local twofold symmetry with the
symmetry axis perpendicular to the membrane plane. Starting from a BChl couple ( called
special pair or P), located on the periplasmic side, the cofactor tree branches, each arm
consisting of a monomeric BChl, a bacteriopheophytin (Bpheo) and a quinone (Q)
molecule. A non-heme iron atom is placed halfway between the two quinone molecules, QA
and Qs, on the cytoplasmic side. The symmetry is broken by a single carotenoid molecule
present on the M subunit. Only the A-branch is active in the electron transfer and the final
acceptor molecule is the quinone bound at the Q8 site, more closely associated with M
subunit.
Following the absorption of a first photon, the excited P dimer reduces the BPheoA
molecule forming the state P+BPheoA·; the electron is subsequently transferred from
BPheoA· to the quinone in the QA site. Finally, QA- reduces the secondary quinone acceptor

105
generating the state P+Q8 ". The photoxidized primary donor p+ is rapidly re-reduced by a c-
type cytochrome (soluble cytochrome c2 (cyt) in Rb. sphaeroides). The absorption of a
second photon in the state PQa· induces the transfer of another electron along the same
pathway and the state P+Q8 2" is produced. The doubly reduced quinone on the Qs site is
stabilized by protonation, leaves the Qs site and is replaced by an oxidised quinone molecule
coming from a Iipid soluble quinone pool (McPherson et al., 1990; for reviews see Feher et
al., 1989, Woodbury and Allen, 1995).
The RC drives a cyclic electron transfer chain: the reducing equivalents delivered to
the Qs site are retumed (via cyt c2) to p+ through a second membrane embedded
multienzyme system, the ubiquinol-cytochrome c2 oxidoreductase complex (bc 1 complex).
This complex is analogaus to that operating in mitochondria, although simpler in structure.
The atomic structure ofthe mitochondrial bc 1 has been recently reported (Xia et al., 1997).
In purple bacteria, the bc 1 consists of three proteins: a b-type cytochrome which binds two
heme groups (heme bs66 and heme bs 61 ), a Rieske type iron-sulphur protein (Fe2S2) and a c-
type cytochrome ( cyt c,). The complex accomodates three different catalytic sites: a
ubiquinone reductase site (Qi), at the membrane-cytoplasm interface, a ubiquinol oxidizing
site (Qo) and a cytochrome c2 reductase site, bothat the opposite, periplasmic side of the
membrane.
According to the Q-cycle model (for a review see Crofts and Wraight, 1983),
oxidation of the doubly reduced and protonated quinone, generated by the photochemical
activity of the RC, occurs at the Qo site in a two-step concerted reaction: the first electron
is delivered through the high potential chain of the complex, which includes Fe2S2 and cyt
c~, to the cyt c2 reductase site and the second electron enters the low potential cyt b chain
(cyt bs66 and cyt b561 ) and finally reduces quinone to semiquinone at the Qi site.
When a second quinol molecule interacts with the Qo site, the same events take place
and a fully reduced quinone is formed at the Qi site. Summarizing, the absorption of four
photons by the RC generates two quinol molecules, subsequently oxidised at the Qo site of
the bc, complex, which retums one fully reduced quinone molecule to the quinone pool.
The uptake of protons upon reduction of quinone molecules takes place on the citoplasmic
side (at the Q8 and Qi sites), while the release of protons upon oxidation of quinol occurs
on the periplasmic side (at the Qo site); part ofthe light energy is thereby transduced in an
electrochemical potential difference of protons across the membrane.
The recent models of the photosynthetic unit raise a problern that must be taken into
consideration. Ifthe antenna ring-like structure ofLH1 continuously surrounds the RC, the
hydrophobic part of the aß dimer will create a barrier contrasting the free diffusion of the
quinoVquinone molecule out/in the Q8 site. In some bacterial species, as Rb. sphaeroides
and Rb. capsulatus, it has recently been shown that one additional protein (Putx) is
associated with the RC-LH1 complex (Farchaus et al., 1992, Lilbum et al., 1992). It has
been proposed that the pujX protein plays a crucial role in facilitating the lateral movements
ofthe quinone molecules (Barz et al., 1995a; Barz et al., 1995b).
It is the aim of this work to review what is known about this protein with special
emphasis on the experimental evidence supporting its role in the electron transfer chain.
Predictions of the secondary structure and of the membrane topology of PufX will be
presented and discussed in relation to its function.

THE PUFX PROTEIN

The puf Operon

In most of the photosynthetic purple bacteria two operons contain the genes for the
LH2 and LH1-RC complexes, the puc operon and the puf operon respectively. Some

106
differences are present in the gene composition of this latter. In all species studied so far,
the puf operon contains the genes for the a.ß dimer of the LH1 and for the L and M
subunits of the RC; in Rb. sphaeroides and Rb. capsu/atus, however, two additional genes,
pufQ at the 5' end and pujX at the 3' end, are present (see Figure 3). PufQ has been
suggested to code for a protein involved in regulating the biosynthesis oftetrapyrroles in the
BChl pathway (Bauer and Marrs, 1988), whereas the pufX gene is essential for
photosynthetic growth (Farchaus et al., 1992, Lilbum et al., 1992).

Q BA L M X

1000 bp

Figure 3. Schernatic representation of the pufoperon frorn Rb. sphaeroides. Modified frorn Barz and
Oesterhelt (1994).

Consistent with the multimeric structure of LH1, the transcriptional Ievel of its a.ß
dimer largely exceeds the one of the RC subunits; in Rb. sphaeroides this is due to the
presence of an intercistronic terminator located between pujA and pujL that blocks the
transcription before the pujL gene with high efficiency (DeHoff et al., 1988). In Rb.
capsulatus the mechanism is different: the region between pufA and pujL could generate
two alternative stem-loop structures that apparently block the 3' exoribonucleases; in this
case the short mRNA containing only the pujB and pufA genes is derived from the partial
digestion of a Ionger mRNA (Belasco et al., 1985). These data indicate that the fixed
amount of RC and LHI in the core complex is basically under transcriptional or post-
transcriptional control, although recent work shows that also translational steps can
influence the amount ofLHI and RC (Gong and Kaplan,l996).

Phenotypic Effects of PufX Deletion

The deletion of the pufX gene in Rb. capsulatus (Lilburn et al., 1992) and Rb.
sphaeroides (Farchaus et al., 1992) gives a non photosynthetic phenotype, indicating that
the pujX protein is required for photosynthetic growth. In the absence of pujX, bacteria can
grow only after a very long lag phase. In this case it has been shown that second-site
suppressor mutations take place (Barz and Oesterhelt, 1994).The involvement of pufX in
the conversion of light into chemical energy was directly demonstrated in Rb. sphaeroides,
since light driven ATP synthesis in vivo could not be detected in pufX deleted cells of Rb.
sphaeroides (PufAX) (Barz et al., 1995a), suggesting that the energy Ievel of the bacteria
lacking PufX was too low for replication under light growing conditions.
Curiously, deletion of pujX dramatically affects the morphology of the internal
cytoplasmic membrane (ICM) system which carries the photosynthetic apparatus. Rb.
sphaeroides and Rb. capsulatus are good model systems for such mutational studies, since
the synthesis of the photosynthetic ICM can be induced during aerobic dark growth simply
by lowering the oxygen tension. In this way the photosynthetic apparatus can be studied in
spite of its inability to support growth. In Rb. capsulatus pujX deleted strains grown under
low oxygen tension in the dark, ICM exhibit invaginations that are 50% larger in diameter
than those ofthe wild type bacteria (Lilbum et al.,l992). In Rb. sphaeroides, the isolation
of ICM fragments from pufX deleted strains yields a large amount of open vesicles that are
not obtained from wild type cells (Barz et al., 1995a). In Rb. capsulatus suppressor mutants
a large amount of inverted or open vesicles was found (Lilburn et al. 1995).

107
 This amazing change in the ICM morphology, due to the absence of this small protein,
points to the possible role of an integer photosynthetic unit in organizing the overall
architecture of the photosynthetic membrane.

ROLE OF PUFX IN THE ELECTRON TRANSFER CHAIN

Reduction Kinetics of Cytochrome b561 in the PufX Deleted Chain

As mentioned above, pujX deleted cells are unable of performing light-driven ATP
synthesis even at high irradiance (Barz et al., 1995a). Experiments on intact cells had shown
that the Iack of photosynthetic growth was not due to a Iimitation in energy transfer from
the light harvesting complexes to the RC (Farchaus et al., 1992). These observations had
suggested that a lesion in light-induced cyclic electron transfer was responsible for the non
photosynthetic phenotype. Since, following a single-turnover photoexcitation, ICM from
the Pufi\X strain display a normal primary photochemistry, leading to formation of stable
semiquinone and quinol at Q8 , we focused on subsequent secondary electron transfer events
involving the bct complex.
The redox interaction between the reaction centre and the bc 1 complex can be
investigated in ICM fragments by studying the kinetics of QH2 oxidation at the Qo site,
under appropriate redox poise of the quinone pool. This reaction can be analyzed by
monitoring spectrophotometrically the reduction of cyt bs 61 induced by a single turnover
flash in the presence of antimycin, which inhibits its re-oxidation at the Qi site of the
complex. Cyt bs6t reduction exhibits a lag period before reaching maximal rate (Figure 4). In
wild type ICM vesicles, the lag increases from 200 J.I.S to about I ms, upon varying the
redox poise from 100 mV to 200 mV, at pH 7.0. Over this range of ambient redox
potentials, the initial rate of the reaction decreases in parallel by approximately one order of
magnitude. In the framework of the Q-cycle model, this kinetic behaviour has been
attributed to the progressive pre-oxidation of the quinone pool (Em=90 mV at pH 7.0
(Takamiya and Dutton, 1979)) and, consequently, to the decreasing availability of reduced
quinone as substrate at the Qo site (Crofts et al., 1983). This interpretation has been verified
quantitatively by increasing or decreasing the size of the quinone pool and, therefore, the
concentration ofQH2 in the membrane at a given value ofthe redox poise (Venturoli et al,
1986).
At redox potentials ::::: 180 mV (when the quinone pool is fully oxidised before light
excitation), the only QH2 molecule reacting at the Qo site isthat generated by the reaction
centre after the flash and just released in the membrane phase from the Q8 site. The maximal
lag duration observed under these redox conditions, includes, in principle, contributions
from the dissociation of QH2 at the Q8 site, its diffusion in the membrane phase and
association at the Qo site of the bc,. Therefore, under oxidizing conditions, the kinetics
monitor the rate limiting steps in the RC-bc, redox interaction.
The kinetics of reduction of cyt b561 has been investigated in ICM isolated from the
Pufi\X strain (Barz et al., 1995a). When the quinone pool is significantly reduced before the
flash, the reduction kinetics of cyt b561 is comparable to that observed in wild type ICM,
demonstrating that the affinity of the Qo site for quinol is unaffected. On the contrary, as
shown in the lower panel ofFigure 4, under oxidizing conditions (Eh= 200 mV) the initial
rate is decreased by more than one order of magnitude and the lag is increased from one to
several ms. These results indicate that the loss of the pujX protein severely impairs the
redox interaction ofthe bc1 complex with the quinone-reductase site ofthe RC and point to
a critical roJe of the pujX polypeptide in assisting the release of QH2 by the RC-LHI
complex (Barz et al.,l995b).

108
 2,5
2,0 lag= 1.1 ms
1.5

1,0
0
X
0>
~
<(iii.o5
, WT
<1
- -1,0
Q) 0 2 4 6 8
Ol
c time(ms)
<0
..c
(.)
Q)
(.)
2,5
c lag = 4.8 ms
<0
.D
..... 2.0
0
(f)
.D 1.5
<0
1,0

0 ,5 P ufl'lX

-0,5 L_____L-~---J..,..-~--'--~--'--~---'---'
0 10 20 30 40
time(ms)
Figure 4. Kinetic traces of cyt b 561 reduction induced by a single tumover flash in pseudo-wilde type (upper
panel) and PufßX (lower panel) ICM inhibited with antimycin. The kinetics were measured in a N2
atmosphere under controlled redox conditions following the procedure outlined in Venturoli et al (1986).
ICM fragments were suspended to a final BChl concentration of 30 JlM in 100 mM KCI, 50 mM MOPS
(pH 7.0) in the presence of 10 J.!M valinomycin, 7 JlM nigericin and 10 JlM antimycin. The ambient redox
potential (Eh) was poised at 180 mV and at 170 mV during measurements performed in WT and PufßX
ICM respectively. Traces are the average of 25 measurements with a time constant equal to 100 J.lS. The
continuous curves are best fits to an exponential function. Details of the numerical procedure used to
determine the duration ofthe lag are given in Barz et al (1995b).Continuous and dashed verticallines
indicate the time ofthe flash and the lag period respectively.

In ICM from the Pufi1X strain the reduction kinetics of cyt bs6 1 showed the same
dramatic impairment, when continuous light was used for the excitation of the RC (Figure
5, upper panel). Under these experimental conditions, although the bc 1 complex is inhibited
by antimycin, a few turnovers of the RC photochemistry are possible, since a !arge
complement ofsoluble cyt c2 is available. At Eh=235 mV, the lag duration, which is close to
3 ms in ICM from the wild type strain, increases to 12 ms in Pufl'lX and the initial rate of
reduction decreases by about one order of magnitude. It is noteworthy that in ICM from
SuplOI a partial recovery of the wild type kinetics was observed (Figure 5, lower panel).

Multiple Turnover Experiments on Wt, PufcSx and Suppressor Mutants from

Rhodobacter Sphaeroides

The results described above have been complemented by studying the reaction centre
activity in multiple turnover experiments performed in ICM and intact cells of WT, Pufl'lX

109
and suppressor mutants (Barz et al., 1995a, b). In wild type ICM, over a !arge range of
redox poises (20-240 mV), at the onset of continuous illumination, the primary donor P
reached a maximum Ievel of photoxidation within a few tens of milliseconds. This Ievel of
p+ was steadily mantained during all the photoxidation period (1 s). Different kinetics were
observed in Puf.::\X ICM, where, below approximately 100 mV, a transient maximum of P
oxidation is attained in tens of milliseconds after the onset of illumination, followed by
relaxation to a lower steady state value. This behaviour suggests an impairment in the
QH:z/Q exchange at Qs, leading to inhibition of RC photoactivity when the fraction of
oxidized quinone molecules in the membrane pool is decreased.
Analogaus experiments, using continuous illumination or multiple flashes, have been
carried out in intact cells, where extreme reducing and oxidizing redox conditions can be
tested. Under oxidizing conditions (cells suspended in oxygen saturated medium) no
difference could be detected between WT and Puf.::\X in the Ievel of light-induced P+. On
the contrary, when cells where incubated in an argon atmosphere, again the photochemical
activity in the pujX deleted strain was impaired following a few turnovers of the system
(Barz et al, 1995b). It appears that, in anaerobiosis, i.e. when the concentration of oxidized
quinone in the pool is very low, the replacement of QH2 at Q8 with oxidized Q from the
pool is critically impaired in the absence of the pufX protein.
In an attempt to determine quantitatively the effect of PufX on the kinetics of QH2/Q
exchange at Qs, the recovery of photochemistry following a couple of closely spaced single
turnover flashes was studied in triple flash experiments. In intact Puf.::\X cells, under
reducing conditions, the recovery required a darktime between the 2nd and the 3rd excitation
in the hundreds ofms (half time :::500 ms) as compared to a half time ofrecovery close to a
few ms in wild type cells (Barz et al, 1995b).The behaviour ofthe Sup101 suppressor was
intermediate between that ofWT and Puf.::\X (Barz et al., 1995b).
The dramatically slow exchange of QH2 with Q observed in anaeobic Puf.::\X cells is
consistent with the impaired release of QH2 by the Q8 site revealed by the reduction kinetics
of cyt bs6J in ICM (cf Figures 4 and 5); the rapid QH:z/Q exchange promoted by the pufX
protein critically controls the rate of cyclic electron transfer under anaerobic conditions.
Consistent with this molecular description of the action of PufX, it has turned out that
the addition of terminal electron acceptors of dark anaerobic growth, ~ike dimethylsulfoxide
(DMSO), to the pufX deleted bacteria restored the generation of light-driven membrane
potential to a Ievel comparable to that of the wild type; pufX deleted bacteria were able to
grow photosynthetically at normal rates in a medium supplemented with DMSO, showing
that the oxidation of the Q pool by these electron acceptors overcomes the absence of the
pufX protein (Barz et al., 1995b).

Phenotypic EtTects of Other Antenna Mutants

The proposed roJe of PufX is confirmed by further sturlies based on the use of mutants
in which whole components of the antenna system were absent or not properly assembled in
the photosynthetic unit.
Jones et al (1992) have built mutants deleted of LH1, LH2 or both (RC-only
mutants). Interestingly, in strains lacking only the LH1 complex, a faster growth rate was
found with respect to the RC-only mutant indicating a direct energy transfer from the LH2
to the RC. The possible direct roJe of pufX in facilitating the ubiquinone/ubiquinol exchange
between the Q8 site of the RC and the Qo site of the bc1 complex was tested using RC-only
mutants with and without pufX (McGlynn et al., 1994). In the absence of any light
harvesting system, RC-only mutants could grow at high light intensity and no difference in
the rate of photoheterotrophic growth could be observed in the presence or absence of the

110
 20 40 60 80 100

1,0
0

X
., 0,5
~
~
<( 0,0 -IIM'klf-~LVY.Jj.-.:-~--....;_--------l
<1
light on off --;

0,6

~
~ 0,2

<(
<1

20 40 60 80 100
time (ms)
Figure 5. Kinetics of cyt b561 reduction induced by a short period of continuous illumination (50 ms) in
thepresence of antimycin in ICM from WT, Puf.t\X and a suppressor mutant (Sup10 1). Traces are
normalized to the same concentration of total photoreducible cyt b561 . Assay composition as in Figure 4. The
redoxpotentialwas poised at 235±15 mV. Signalsare the average of8 measurements with an instrument
time constant of 100 J.!S. Sup lO 1 strain, described in detail in Barz and Oesterhelt ( 1994), is characterized
by a non-sense mutation oftryptophan 43 in the a subunit ofLHI. The following initial rates of reduction
have been estimated from the traces: 62.0 (WT), 5.3 (Puf.t\X), 8.5 (Supl01) cyt b561 '"d complex· 1 s' 1 ; the
corresponding lag periods are 3 ms (WT), 12 ms (Puf.t\X) and 8 ms (Sup101).

pujX protein. Lack of pujX deletion phenotype was also observed when the LHl structure
was disarranged by C-terminal truncation of the u chain (McGiynn et al., 1996). The
important message is that the pufX protein is necessary only when LH 1 is present and
integer.
The analysis of suppressor mutants has yielded results consistent with this conclusion.
In Rb. sphaeroides, phenotypic suppression of the pufX deletion takes place at high
frequency by point mutations in the pujB or pufA genes (Barz and Oesterhelt, 1994). Three
different groups of suppressor mutants were found: (1) the u43W~* and ß44W~*
nonsense mutations (leading to the truncation ofthe polypeptide) did not assemble the LH1
complex; (2) the ß47W~R mutant did not assemble the LHl complex in a correct
stoichiometry of the pujBA genes and no absorption band attributable to the inner antenna
was detectable; (3) the spontaneous suppressor mutants ß20H~R, u47S~F and the
engineered ß47W~G assembled LH1 in the membrane with altered spectral properties. It
should be noted that these last suppressors exhibit a low growth rate under photosynthetic
conditions. In Rb. capsulatus, other suppressor mutants were characterised weil (Lilbum et
al., 1995). These mutants (u2S~P and u2S~F) fall in the third group, being able of
assembling LH1.

111
 From these works, it is clear that the most frequent mutational events in the
suppression phenomena are alterations in the genes encoding the LH1 system. However, it
should be noted that also some alterations in the LH2 system were observed. In Rb.
sphaeroides two mutants mapped outside the puf operon (Barz and Oesterhelt., 1994). In
one case, the LH2 peak was absent from the absorption spectrum of the cells. Also in Rb.
capsu/atus a mutant lacking the LH2 absorption peak was isolated (Lilburn and
Beatty, 1992). Most workers in the field agree now that the pujX protein is located in the
LH1-RC complex and facilitates the quinollquinone exchange through the barrier to lateral
diffusion represented by the antenna ring. The alterations in the antenna structure which
characterize the suppressor strains appear to be a compromise between two requirements of
the photochemistry: the LH1 energy transfer to the RC and the fast escape of the reducing
power generated at the Qs site.
A putative model for the location ofPufX in the LH1 has been proposed in which the
PufX is present as a dimer which interrupts the continuous LH1 ring, facilitating the escape
of quinone molecules from Q8 (Codgell et al., 1996). The concept that the PufX chain breaks
the symmetry of the LH1 ring by substituting for a or ß subunit(s) is consistent with the
higher complement of LH1 BChl in the pujX deleted strains, when normalized to the RC
content (McGiynn et al., 1994). These data suggest that, in the absence of pujX, a higher
number ofaß dimers is necessary to complete the LH1 ring.

Predicted Secondary Structure of the Pufx Protein

As outlined above, several complementary lines of evidence indicate that the pujX
polypeptide arranges the RC surroundings and prevents the LH1 ring from blocking lateral
movement of quinones to and from the Q8 site. We have looked for structural features of
PufX which could be compatible with this molecular action.
Each a or ß subunit of LH2 forms one a-helix across the membrane, the whole
structure being arranged in two multiprotein concentric rings coordinating the pigment
molecules (see Figure 1). An analogaus arrangement is likely for LH1 (see Figure 2) on the
basis of the primary structure of pufA and pujB proteins (the a and ß subunits of LH 1) and
oflow resolution electron diffraction data.
As shown in Figure 6, the analysis of the primary structure of PufX from Rb.
sphaeroides predicts a single transmembrane helix as weil; the secondary structure
prediction contains however a strong indication for a second a-helix on the C-terminal end
(residues 63-82) with a very marked amphipathic character. The membrane topology and
secondary structure predictions of PufX from Rb. capsulatus yield similar results, i.e. a
transmembrane helix extending from residue 28 to residue 47 and a second, amphipathic,
helix on the C-terminal extramembrane domain. An attracting model for the inclusion of
PufX in the LH1-RC complex would therefore be the insertion of the protein into the
antenna ring as a substitute of one or more a or ß monomers, being the amphipathic helix
layered close to one ofthe surfaces ofthe membrane.
A ring of lipid-exposed tryptophan residues is present in the LH2 structure placed close
to the periplasmic side ofthe membrane (cf. Figure 1, right panel). Similar tryptophanrings
appear tobe present in the LH1 and RC complexes. In modeling the LH1-RC structure of
Rb. sphaeroides, Hu et al (1997) have found that the minimum energy conformation ofthe
complex corresponds to a coplanar arrangement of the lipid-exposed tryptophan side
groups. In order to build a structural model of the entire photosynthetic unit, Hu et al.
(1997) have positioned LH2 by aligning its tryptophan ring with those of the LH1-RC
complexes. Noteworthy, the resulting structure is optimal for energy transfers, due to the
coplanar arrangement ofBChls ofLH2, LH1 and ofthe specialpair P in the RC.

112
 100
CDCD®©
© / /' @
)(

Ci)
80
® -/ 9
I
.c:
...0 CD
-- 60 T

-...
~
:::J
a.
:::J
0
40
E
E ®
H

-
0
3: 20
Q)
r::::

0 20 40 60 80

residue number
Figure 6. Predicted membrane topology and secondary structure of Rb sphaeroides PufX. The aminoacid
sequence oftheRb sphaeroides PufX was subjected to neural network analysis for the like1ihood of
Iransmembranehelices (continuous line. a) and ofhelices in general (dashed line, b) (Rost and Sander,
1993; Rost and Sander, 1994; Rostet al, 1994; Rostet al, 1995). In this lauer case on1y the region of
significant likelihood is shown for clarity. The results of the prediction indicate a high probability for
Iransmembranehelix in the middle region ofPufX (residues 35-50) and for an additional helix in the C-
terminal extramembrane region ofthe sequence (residues 63-82). As evidenced by the helical wheel
representation (shown on the left), in which apolar residues are circled, this predicted helix is markedly
amphipathic.

Two tryptophan residues (22 and 34) are present close to the N-terminal end of the
transmembrane a-helix of PufX in Rb. sphaeroides (one of them is conserved in Rb.
capsulatus at position 21 ). These residues could offer a criterion for inserting the PufX in a
RC-LH l model. As a consequence, the amphipathic helix of PufX would be located on the
cytoplasmic side, i.e on the same side ofthe Q8 site ofthe RC. It is possible that this domain
ofPufX is related to its functional roJe in the quinone/quinol exchange at the Q8 site.
Among all second-site mutations that suppress the Pufi:\X phenotype, the most
frequent ones are non-sense mutations of the lipid-exposed tryptophans in the a and ß
subunits of LH l. The function of regular arrays of tryptophans in organizing the quaternary
structure ofthe antenna-RC complexes (Hu et al. , 1997) is emphasized by the fact that the
assembly of the truncated subunits of LHl is prevented in these suppressors (Barz and
Oesterhelt, 1994).

CONCLUSIONS

Recent crystal structures of the LH2 antenna from photosynthetic bacteria reveal a
highly ordered protein scaffold which positions the pigments responsible for the exciton
transfer at the appropriate mutual distance and orientation. On this basis, Iow resolution
data have been used to model the LH l antenna as a ring sorrounding the RC. Mutational

113
and functional studies have shown that in the absence of a small antenna polypeptide (PufX)
the LHI antenna ring represents a physical barrier for the incoming and outcoming quinone
molecules at the Q8 site of the RC. The pujX protein overcomes the problern most likely by
replacing a or ß subunit(s), thus interrupting the continuity ofthe LHI ring. The analysis of
the aminoacidic sequence ofPufX is compatible with such a structural roJe in organizing the
supramolecular arrangement ofthe RC-LHI system. It turnsout that a small polypeptide is
pivotal in promoting the fast electron transfer between complexes necessary to sustain the
metabolic energy request of the bacteria.

ACKNOWLEDGEMENTS

We are grateful to Drs. Wolfgang Barz and Dieter Oesterhelt for stimulating and
extensive discussions. This work was supported by the Ministero della Universita' e della
Ricerca Scientifica e Tecnologica of Italy and by the University of Bologna. F.F. is the
recipient ofa M.P.I. post-doctoral fellowship.

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Karrasch, S., Bullough, P.A., and Ghosh, R, 1995, The 8.5 A projection map ofthe light-harvesting
complex I from Rhodospirillum ruhrum reveals a ring composed of 16 subunits, EMBO J. 14:631-
638.
Koepke, J., Hu, X., Muenke, C., Schulten, K., and Michel, H., 1996, The crystal structure of the light-
harvesting complex II (B800-850) from Rhodospirillum molischianum, Structure 4:581-597.
Lancaster, C.RD., Emler, U., and Michel, H., 1995, The structures ofphotosynthetic reaction centers from
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Lilburn, T.G., and Beatty, J.T., 1992, Suppressor mutants ofthe photosynthetically incompetentpuj.X
deletion mutant Rhodobacter capsu/atus DRC6(pTL2), FEMS Micr. Lett. 100:155-160.
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Lilburn, T.G., Prince, RC., and Beatty, J.T., 1995, Mutation ofthe Ser2 codon ofthe light-harvesting
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McDermott, G., Prince, S.M., Freer, A.A., Hawthornthwaite-Lawless, A.M., Papiz, M.Z., Cogdell, R.J., and
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McGlynn, P., Westerhuis, W.H.J., Jones, M.R, and Hunter, C.N., 1996, Consequences for the organization
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McPherson, P.H., Okamura, M.Y., and Feher, G., 1990, Electron transfer from the reaction center of Rb.
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Nunn, RS., Artymiuk, P.J., Baker, P.J., Rice, D.W., and Hunter, C.N., 1992, Purification and
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116
SINGLE ELECTRON AND QUANTUM PHENOMENA IN ULTRASMALL
PARTICLES

Victor Erokhin 1, Sandro Carrara1, and Claudio Nicolini1. 2

1 Fondazione El.B.A.,
Salita Superiore della Noce 35, 16132 Genova, Italy
2 Institute ofBiophysics, University ofGenova,

Corso Europa 30, 16132 Genova, ltaly

ABSTRACT

Principles, responsible for the single-electron phenomena, are considered. Different

ways of the junction formation are discussed, emphasising that, based on the utilization of
nanometer size granules. Model, describing the single electron properties as weil as the
appearance of the differential negative resistance regions, is proposed. It is also discussed
the technical approaches for the formation of ultrathin semiconductor layers and
superlattices based on the aggregation of ultrasmall semiconductor particles.

SINGLE ELECTRON PHENOMENA

Single electron phenomena attract the attention of many researchers both from
fundamental and applied points of view during last years.
Coming from early attempts of explanation on the basis of very simple model (Giaever
and Zeller, 1968; Zeller and Giaever, 1969), formally a theory of single charge phenomena
was outlined in 1986 (Averin and Likharev, 1986a).
D.V. Averim and K.K. Likharev developed a theory for describing the behaviour of a small
tunneling junctions basing on the electron interactions. They had started from previous
works on Josephson junctions (Likharev and Zorin, 1985; Ben-Jacob, 1985; Averin and
Likharev, 1986b) and established the foundamental features of the single charging
phenomena. Their work is based on a second quantisation theory and handles the tunneling
phenomenon as a perturbation, described by annihilation and creation operators of a
hamiltonian.
The theory foresees the possibility of Coulomb Blockade phenomenon in such
junctions. Averim and Likharev had investigated the conditions of vanishing for the
Josephson tunneling and demostrated the possibility to have normal electrodes in the

Biophysics of Electron Transfer and Molecular Bwelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 117
junction. That is, no superconducting electrodes are necessary and, therefore, Coulomb
Blockade is possible to observe, in principle, even at room temperature.
The work of Averim and Likharev had suggested to consider two junction systems to
trap the electron inside it, basing on the ideas ernerging from the theory frame.
Following this idea, the two junction systems were investigated and a step-like
behaviour was observed. 1t was related to the exclusion of the next incoming electron into
the intermediate granule due to the electric field created by the previous entering ones
(Mullen et al., 1988}.
The researcheres coninued to consider the idea, that the modern STM (Scanning
Tunneling Microscopy} could be the proper utility for the formation oftwo junction systems
when working with very small particles. This consideration had related the sturlies of the
single electron phenomena to the concepts of quantum dots (Giazman and Shechter, 1989).
In particular, considering a ballistic model for the charge trasport through a dot, it was
possible to demonstrate that the current trhough it should be represented as a series of
equidistant peaks whose position corresponds to the steps in the Coulomb Staircase.
Moreover, the possibility to consider the single electron phenomena in a frame of a dot-
based system theory allows to consider even semiconductor nanoparticles as quantum dots,
useful for the single electro junctions (Averin et al., 1991 ).
The modelling of junctions based on these semiconductor quantum dots reveals as their
behaviour in terms of single electron phenomena can even result in current-voltage
characteristics with differential negative resistance regions (Gritsenko and Lazarev, 1989).
The fact was connected to the possibility of resonat tunneling through quantised energy
Ievels inside the dot (Guinea and Garcia, 1990; Beenakker et al., 1991; Sumetskii, 1993;
Groshev et al., 1991}.
On the other hand, some works on the topic consider the presence of the negative
differential resistance in the current-voltage characteristics and the possibility of Coulomb
staircase as different output of the very same phenomenon and, therefore, have tried to
consider both of them in the very same conceptual frame (Beenakker, 1991; Stone et al.,
1992; Prigodin et al., 1993}. This approach seems tobe sucessful; in fact, it was possible to
see models describing current-voltage curves presenting both stairs and negative resistance
along them (He and Das Sarma, 1993; Carrara et al., 1996).
The whole theoretical work proposed during these last ten years forces the
experimental researchers to develop real systems to observe the described phenomena. In
reality, only a year after the very first work of Averim and Likharev, the first measurements
ofthe Coulomb Gap and the Coulomb Staircase phenomena were pubblished.
The Likharev group performed the first measurements of an oscillation of the right
periodicity in respect to the signle electron blockade in an indium granule based junction
(Kuz'min and Likharev, 1987}.
Few monthes after, the observation ofthe Coulomb Gap appeared (Fulton and Dolan,
1987). In a case of Coulomb Staircase only one additional mouth was required (Barner and
Ruggiero, 1987}.
Working with electrodes configured as two or three junction systems, it was possible to
observe Coulomb Staircase (Kouwenhoven et al., 1991 }, coulomb Gap and the eharge
oscillations in normal and superconductive systems (Lafarge et al., 1991) and was, also,
possible to obseve the real existence of the cooper pairs in junctions with superconductive
electrodes (Lafarge et al., 1993}.
On the other hand, working with particle systems, it was possible to observe differential
negative resistance (Reed et al., 1988) and the characterisation of such kind of single charge
system begun with the STM (Weiner et al., 1991}. In particular, even in particle systems it
was observed the Coulombgap (van Benturnet al., 1988a} and the Coulomb staircase (van
Benturn et al., 1988b}, some non linear effects were observed in the current-voltage

118
characteristics (Wilkins et al., 1989) and behaviour related to the quantised energy Ievels
inside the particles was described (Crommie et al., 1993; Dubois et al., 1996).
However, the main research results of these years were the descovery of the room
temperature single electron phenomenon. In the ninenties, STM experiments on liquid
christal molecules had shown very weak staircase (Nejoh, 1991); only one year later, the
clear Observations of Coulomb Gap and Coulomb Staircase were demonstred on Au
nanoparticles (Shönenberger et al., 1992a) and the roJe of the system symmetry on the
appearance ofthese two phenomena was outlined (Shönenberger et al., 1992b).
The effect of the structure parameters on the feature of the junctions was studied. For
example, different materials were tested as insulator for spacing the two junction systems
(Shönenberger et al., 1992b); Dorogi et al., 1995), or the roJe ofthe barrier width in such
systems was tested as possible parameter to create regions with differential negative
resistance in the curren-voltage characteristics (Erokhin et al., 1995), or the roJe of different
semiconducting materials in contstructing the particles useful for the experimental system
(Erokhin et al., 1996).
Finally, it was possible to build-up the first standing along room temperature signle electron
junction depositing a semiconducting particle directly onto the tip of a very sharp electrode,
avoiding in this case the usage of an STM microscope, and it was possible to observe on
such sytstem the Coulomb staircase (Facci et al., 1996).
In order to understand the basic principles, Iet us consider the following scheme (Fig.
1). Small granule is placed between two electrodes and is separated from them by tunnelling
gaps. The structure is assymetric. Tunnelling barriers are characterized by their hights and
widths. These parameters determine the tunnelling probabilities for each barrier. Therefore,
it is possible to attribute to each of them the time t1 and t2, characterizing the time of
electron passing through them. The time, which electron can stay in the granule is tg.
The following inequation must be valid:

From the other hand, these junctions can be also characterized by the capacities.

CdS particlc
nanopartich: STM tip
{
under STM
invcsligalion ~
I
I
I
disc of radius R
electrostatic
{ mllthcmltliCIII I
modcl

·rri ~;:
poinl ~
I I .,.;"";_,-.,
barrier encrgy
model
{ ,,..gyi
.,x axis

r--:-
d --+-t- H-d
clcctrical
model
{
----l H Co C

Figure 1. Scheme ofthe single-electronjunction.

119
 Let us considere what will happen when the valtage is applied to this system.
When the valtage value is rather small the electron will be tunnelled from the electrod 1
to the granule after the time t 1· lncome of the electrone will result in the appearance of the
electric field equal to e/C, where C is the capacity of the junction. This electric field will be
directed oppositely to the bias valtage and will prevent the income of new electron before
the biasvaltage will be more then e/C. Therefore in a time 'tl the electrone will be tunnelled
back to the electrode 1. Thus, it will be no current in the circuit (Coulomb Blockade). When
the bias valtage will overcome the value of e/C one electrone will stay always at the granule
and, therefore, after the time 't2 will be able to tunnell to the second electrode, providing a
constant current, whose amplitude will be reversly proportional to the tz. When the bias
valtage will overcome the value 2e/C, two electrones will stay in the granule and after 't2
will be tunnelled independantly to the electrode 2, providing a constant current of the
doubled amplitude. Increasing futher the bias valtage it will be possible to come to the
situation when 3, 4 and more electrones will stay in the granule, providing stearcase
behaviour ofthe valtage current characteristics (Coulomb Stearcase).
Described behaviour can be observed in systems, when thermal excitation is less then
electrostatic energy, namely:

e2
->-kT
2C

Therefore, it always excist the tempetature, when the single electron phenomena are
not anymore observable for each geometry of the junction. Increase of this temperature
demands the decrease ofthe capacity value and, therefore, the granule size. In order to make
possible the observation of such phenomena at room temperature, the granule size must be
less then 3 nm. Therefore, the single electron conductivity on the circuits, fabricated with the
traditional microelectronic technology, was observed at very low temperatures, as the
granule sizes are much more then 3 nm. However, the approach of continious reduction of
the element sizes with the development of the traditional technology approaches is still the
main stream in the fabrication of single electron devices.
Apart from the main stream of the element formation there were carried out several not
traditional technological approaches for the formation of elements with nanometer sizes and
their utilization for construction of single electron elements (Wilkins et al., I 989;
Shönenberger et al., I 992a; Dorogi et al., 1995, Erokhin et al., I 995).
In 1988 it was suggested a method of the formation of CdS particles in Langmuir-
Biodgett (LB) matrix (Smotkin et al. 1988). LB film of cadmium arachidate was exposed to
the atmosphere of H2S. During the reaction the head groups of arachidie acid were
protonated and CdS was produced according to the following reaction:

Several experimental techniques were applied to characterize these objects. It was foud
that CdS was formed as small particles inside LB film with the sizes in the nanometer range.
Similar works were carried out resulting in the formation of PbS, CuS, HgS etc. Sizes of the
particles produced by such approaches turned out to be rather similar to that of CdS. The
observed sizes allowed to suggest, that the objects can be useful for formation of
nanogranules for room temperature single electron junctions.

120
 2nm

Onm

Figure 2. STM images of the CdS particles formed in LB films.

However, the formation of the junction is not a trivial task even if the granule is
formed. It is necessary to provide contacts to it.
Scanning tunnelling microscope (STM) was choosen as a tool for realization of this
task (Wilkins et al., 1989). CdS nanoparticles were formed in a bilayer of cadmium
arachidate deposited onto the surface offreashly cleaved graphite (Erokhin et al., 1995). The
graphitewas used as the first electrode. Initially, STM was used for localizing the position of
the particles. Fig. 2 shows the images of different areas of the sample. The particles are
visible as wells in corrugated matrix. Their sizes correspond weil to the sizes estimated for
these objects with other techniques. lt allows to suggest that the wells in the images could be
related to CdS particles formed in the LB film of the arachidie acid. The reaction process
disturbs strongly the structure of the film, resulting in an increased corrugation of the
arachidie acid matrix, at least in some regions surrounding the particles. Taking into account
the hydrophilic properlies of CdS, we can suppose that the matter in the bilayer is re-
distributed after the reaction in such a way that CdS particles are not covered by the
arachidie acid molecules. These consideration can account for negative features that are
visible in the STM images and therefore could be related to CdS particles.
As the second step, the STM tip was locked uper the desired particle, feedback was
temporally switched off and voltage-current (V-1) characteristics were measured. Tipical
trend ofthe V-1 characteristics is shown in Fig. 3. Current steps are clearly observable in the
presented curve, indicating that the single electron junction was formed. lt is worth to
mention, that the characteristics, observed in areas without particles demoostrate a normal
tunnelling behaviour (see Fig. 4).

121
 1.2

-
;(
c
...... 0.8

c
~ 0.4
::I
0

-
'S 0
...Q.
::I
0 -0.4

-0.8
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75
Bias Voltage [V]

Figure 3. V-I characteristics with single-electron steps, obtained over the forrned particle.

Apart from the characteristics, similar tothat shown in Fig. 3, other characteristics with
periodic current oscillations were observed. V-1 characteristics measured by placing the
STM tip over particles 1 and 2 in Fig.2a are shown in Fig. 5 a and b respectively. Regions
with negative resistance, visible in these characteristics, are not usual for Coulomb staircase.
However, several features of these curves are similar for single electron junctions, namely,
equal steps in voltage corresponding to the current steps. Moreover, there is a dependance
of the voltage step width upon the particle size over which the tip was locked. It is clearly
visible that the voltage step in the curve, obtained over the object with smaller sizes (smaller
capacity), is bigger, corresponding qualitively weil to the value of a voltage steps e/C.
Nevertheless, the appearance of regions with negative resistance is generally not typical
for Coloumb staircase phenomenon. However, several works reported similar features both
theoretically (Beenakker, 1991 ; Stone et al., 1992; Prigodin et al. , 1993)and experimentally
(Reed et al., 1988).

0.9

;(
.=.
-c 0.5
......CD
::I
u

-
'S 0.1
Q.

5 -0.1

-0.5
-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
Bias voltage [V]

Figure 4. V-1 characteristics with single-electron steps. obtained over the region without particles.

122
 The observed phenomena can be explained starting from the consideration that different
behaviours in the V-1 characteristics ofthe same granule (staircase and negative resistance)
are measured when different values of current are locked by the STM feedback. This fact
implies, of course, that different tip - granule distances are attained in the two cases. By
considering the structure as a two-barrier system, we can suggest that one barrier, namely
that between the graphite and the granule, is fixed (cannot be varied), while the second can
be adjusted by displacing the vertical position of the tip. From these considerations, we can
conclude that, by varying the locked current, we can change the assymetry ofthe structure.
Asymetry of the structure is one of the basic parameters responcible for the Coulomb
staircase phenomenon (Shönenberger et al., 1992b). As we have considered previously, it is
absolutely necessary to have asymetric system in order to observe step-like V-1
characteristics.
In a case of rather symetric system tunnelling probabilities become to be of the same
order of magnitude for both barriers. Therefore, the total current through the system is
controlled by both barriers. Before reaching the threshold value e/C for a bias voltage, a
normal tunnelling curent through both junctions is present. This is a reason why a marked
suppression of conductivity araund zero bias valtage was not observed. When the bias
valtage matches e/C, the charge excisting in the granule unbalances the valtage distribution
through the junctions, preventing the new ingress of electrons to the granule and facilitating
their drain. This behaviour can be the possible reason of the appearance of regions with

a 80
60
I
c....a.
..
..
c
CD

..
:I -1.7 -1.2

..
0
:I -40
a.
:I -60
0
-80
-100

Bias Voltage [V}

2
b

c....c 1.5

....
.c
CD

..
0
:I

..
:I

0
a.
:I
-0.5 0.3 0.5

-1

-1.5

Bias Voltage [V]

Figure 5. V-1 characteristics with negative resistance regions, obtained over the formed particles (a- particle
I ofFig.2a; b- particle 2 ofFig. 2a).

123
negative resistance in the measured V-I characteristics. More detailed consideration of the
model, describing both single electron phenomena and differential negative resistance is
presented in the Appendix I and here we will use only the results, obtained from the
application of the model.
Starting from these consideration, the fitting was performed taking into account two-
barrier system where the voltage is re-distributed when successive electron enters the
granule. The results of the fitting correspond to the experimental data shown in figure (Fig.
5) and describe weil the observed behaviour.
The results listed above clearly show the possibility of the organization of single-
electron junctions. Nevertheless, it cannot be still considered as stayin along junction, as it is
necessary to find the particle with STM. Moreover, if to go out of tunnelling and then to
land the tip once more, the probability of coming to the same place will be practically zero
due to several factors, such as the thermal drift, mechanical vibrations, etc.
The good solution of this problern was performed by synthetizing the particle directly
on the tip ofmetal stylus (Facci et al. 1996}.
Junction preparation involves several steps. The first step, Fig. 6a, consists of stylus
etching. Styli were prepared from a 0.5 mm tungsten wire by electrochemical etching in 1 M
KOH (pH 12.4) with a special etcher, designed for the STM tip preparation, by applying a
20 V peak-to-peak a.c. voltage between the wire and a toroidal graphite electrode and
setting a shut-off current of 0.5 A. After etching, tips were washed in pure water and in
isopropanol. The tip surface turned out to be hydrophobic, as indicated by the downward
bending of the water meniscus when the stylus was immersed into the trough and, hence,
provided a suitable surface for depositing an even nurober of monolayers.

...

Figure 6. Scheme ofthe formation ofstand-along single electronjunction: a) Stylus etching, b) Covering of
the sylus with; c) Formation of the CdS particles at the tip of the sylus.

124
 The second step, Fig. 6b, consists of the covering of the styli with cadmium arachidate
LB films. Monolayers of arachidie acid (in principle, it is possible to use also stearic or
behenic acids with practically the same results) were spread over the surface of 104 M
CdCI2 water subphase in Langmuir trough. The monolayer was compressed till a surface
pressure of 27 mN/m and transferred onto styli by a vertical dipping technique. Up to 6
monolayers were deposited.
The third step, Fig. 6c, consists of the formation of CdS nanoparticles inside the LB
film by exposing precoated tips to the atmosphere of H 2S for a time, sufficient for
compleating the reaction according to the film thickness (Facci et al., 1994).

------- ,-------- I
1 I
I I
I I
I I
I I
I I
I I
I
I
_I

Figure 7. Experimental set-up for single-electron cunductivity measurements.

The treated tips were connected to the experimental set-up (Fig. 7) which is able to
bring them into the close proximity to the atomically flat electrode (plates of freshly cleaved
highly oriented pyrolytic graphite in our case). Practically, the tips were connected to the
one-dimensional piezo mover controlled by a feedback circuit.
After landing the tip in a random place and typically locking a current of 0.1 nA at the
bias voltage of 1 V to avoid tip-substrate impact, voltage-current characteristics were
measured at room temperature by switching off the feedback system, sweeping the bias
voltage, and recording the current flowing through the system.
Two typical characteristics, obtained in such prepared junctions, are shown in Fig. 8
and Fig. 9 (Facci et al., 1996). These curves are representatives of the overall behaviour of
the set of studied junctions. It is clearly observable, that their behaviour is highly nonlinear,
displaying several interesting features. In both cases it is possible to see a marked depression
of the current around zero-baised voltage, as it is typical for single electron junctions,
pointing out so called Coulomb blockade. Moreover, in both types of characteristics, the
current revealed the step like behaviour upon the bias voltage. Figure 8 shows rather ideal
symmetric characteristics. The steps in voltage are equidistant as in Coulomb staircase
phenomenon allowing the estimation ofthe junction capacitance, which turned outtobe 1.5
I0-19 F (estimated from the voltage step of 0.54 V). This value is consistent with that
allowing to observe such phenomena at room temperature. In fact, electrostatic energy of
the junction, calculated from this values is about 540 meV, what is much higher then thermal
excitation energy at room temperature (26 meV).
Another typical behaviour ofvoltage-current characteristics is reported in Fig. 9. These
data are different from those in the previous case mainly because of the fact that the curve is
asymmetric in the value of offset voltage. This behaviour, together with the possibility of

125
 200

<
.....
100

...c
CL

..
2! 0

...
~
0
-100
...
~
CL
~
0
-200

-2 -1 0 2

Bias Voltage [V]

Figure 8. VII characteristics with rather ideal single-electron cunductivity.

observing wider depression of the conductivity around zero bias voltage has been reported
earlier (Kuz' min and Likharev, 1987; Wilkins et al., 1989; Shönenberger et al., 1992b). It is
likely due to the charge trapping in the particle by means of impurities. In fact, being initially
nonneutral, the particle will shift the voltage offset around zero due to the variation in the
electrostatic energy of the granule itself.
Reported trends (step-like characteristics of both types) were registered in about 60%
of all prepared samples, a percentage that is consistant with the yild of the good tip
production (good tip means that it allows to obtain atomic resolution when used in STM).
The other 40% of samples displaied usual tunnelling characteristics without any steps.
The described approach represents one more step toward the realization of a
compleatly stand-along single-electron junction based on nanoparticles, produced in organic
matrix. Quantum dot synthesis directly on the tip of meta! stylus allows to avoid using of
STM for localizing the particle position and requires only the use of atomically flat
electrodes and a feedback system for maintaining a desirable double barrier structure.

5
<c
-......
.....
cCl)
4

3
~

...
0
2
...
~
CL
~
0
0
0 0.2 0.4 0.6 0 .8
Bias Voltage [V]
Figure 9. VII characteristics with asymmetric single-electron cunductivity.

126
 Futhermore, the reproducibility of the results in this case is much better than that
obtained by measuring voltage-current characteristics on CdS nanocrystals formed onto a
flat electrode and localized by the STM tip. Indeed, the present approach allows a good
repeatability of voltage-current characteristics since the mutual position of the tip and the
granule is fixed and the only adjustable gap is that between the granule and the flat electrode,
which is feedback controlled. lnstead, in a usual STM approach, even disregarding the
problern of localizing the particle position, thermal drift and external noise can affect the
stability and reproducibility of the data, since the positioning stage (x-y mover) is not
equiped with feedback system, e.g., it is practically impossible to achive tip repositioning in
different tip-substrate approaches.
Thus, previously described experiments had demonstrated the possibility of realization
of single electron junctions based on CdS nanoparticles. Nevertheless, as only one type of
the particles was tested, the question about the roJe of the material properties for successfull
single electron junction formation was still open.
The aim of the next stage of the work was to study the possibility of the realization of
structures that would exhibit single electron phenomena, forming the nanoparticles with the
same technique but from different materials. Camparisan of the properties of such structures
with those built up with CdS particles will make more clear the roJe of material
characteristics on the final properties of the structure.
PbS was chosen as the object ofthis study (Erokhin et al. 1997). In fact, CdS is a wide
band semiconductor (2.42 eV), while PbS is a narrow band semiconductor (0.37 eV) and,
thus, the differences in properties of the structures based on them, if any, should be easily
distinguishable.
Structures were prepared in a way, similar to that used for the CdS nanoparticle
preparation. The only difference was that Iead arachidate LB films were deposited, after
spreading and transferring of arachidie acid monolayers at the surface of 1o-4 M of
Pb(N0 3h
As in the case ofCdS granule two types ofV/1 characteristics were registered, namely,
one with negative resistance regions (Figure 10) and the other with step-like behaviour of
current upon the bias valtage (Figure 11) (Erokhin et al., 1997a). The value of the valtage
step depends upon the capacitance of the junction which is related to the size of the granule.

100
......
c(
.....
...c. 0
c
......Cll ·100
:I

...
0
:I
,S-·200
:I
0
·300

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8

Bias voltage
Figure 10. VII characteristics, obtained over PbS particles, with negative resistance regions.

127
This dependence was observed experimentally providing one more evidence of the
monoelectron nature ofthe phenomena. The typical voltage step is about 0.1 - 0.2 V, which
corresponds to the capacity of8- 16 lQ-19 F. Such value demands to deal with the particles
with characteristic sizes of 2 - 3 nm, that corresponds to the sizes previously estimated by
different methods on CdS particles.

3.5
C('
.....
c
...c 3

2.5
...2!
::::s 2
...6. 1.5
0

...
::::s 1
0
0.5

0
0 0.2 0.4 0.6 0.8
Bias Voltaga [V]

Figure 11. VII characteristics, obtained over PbS particles, with single-electron cunductivity.

The appearance of one or the other type of characteristics was shown to be connected
with the asymmetry of the system which is controlled by the value of the locked tunnelling
current, exactly as it was in a case of CdS particles.
When the locked current was small (0.5 nA), giving rise to a large barrier between the
granule and the tip, staircase-like V-1 characteristics were registered (Figure 11). Instead,
when the value of locked current was higher (1 nA), characteristics with negative resistance
took place at constant voltage steps (Figure 10).
Similar results were obtained also when MgS and CuS particles were used for the
junction formation.
The similarity of the results obtained on systems based on nanogranules made of
materials with different bulk properties allows to conclude that phenomena at issue are
connected only with the decreased size of the granules and not with the bulk properties of
the material. In fact, when dealing with such small sizes, it is, probably, impossible to
attribute to them bulk properties, as surface states begin to play a dominant role in the
electrical properties of such objects.
Several possible applications of the this phenomenon are under the discussion. The
easiest on is to consider it as an analog-digital transducer. In fact, contineous sweepping of
the voltage applied to the junction results in the digital output of the current, providing,
therefore, a fixed value of the current to the different voltage intervales. Moreover, it is
possible to vary the unit step of the digitization, taking the granule of different sizes. Next
steps to the practical realization of such transducer will be in a synthesis of the granule
between preformed sharp metal electrodes. The electrodes can be prepared using the
selective etching technique of the thin and nerrow metal strips, deposited onto insulating
substrates.
Several possible applications are proposed for systems, using 3 electrodes. In this case,
2 of them with a granule between them are served as analogues of source and drain in field
effect transistor. lnsteade, the third one, analouge of the gate electrode, is served for the

128
application of the electric field to the granule, which varys the character of the current flow
between the source and the drain. Such structures can be realized with the traditional
microelectronic approaches, even if still now the technological possibilities do not allow to
form structures, capable to work at room temperature. It is more difficult to realize such
structures using the nanogranules. Nevertheless, the modelprototypewill can be realized if
it will be possible to synthetise the granule between two sharp metal electrodes. In this case,
the electrodes will be formed at the surface of conductor (or semiconductor), covered by
thin insulating layer (for example, an organic monolayer, deposited by LB technique). In this
case the conductive substrate will be used as third electrode.
Apart from the transistor like devices, single electron junctions can be usefull also for
sensor applications. The simplest one can be the monitaring ofH2S. As the formation of CdS
nanogranule takes place when initial cadmium arachidate layer is exposed to this gas, we can
expect the appearance of single electron conductivity only when it is present in the
atmosphere.
The other sensor applications can be considered if some sensitive biological molecules
(such as antiborlies or receptors) are attached to the nanogranule. If, for example, an
antibody molecule is attached to it. The granule is placed between two electrodes. Single
electron current flows between them. Step value ofthe Coulomb staircase depends upon the
capacity ofthe junctions. When the antibody molecule will bind specific antigen, the capacity
value will be changed, and , therefore, the step value of V/I characteristics will be also
change.

ULTRATHIN SEMICONDUCTOR LAYERS AND SUPERLATTICES

Apart from the described single-electron junctions, there is another excttmg

technological possibility to use these nanoparticles. lt tumed out that the particles can be
aggregated into very thin polycrystalline layers (Facci et al., 1994). This part will descibe
some aspects ofthis phenomenon.
The prosedure starts, as in a previous case, from the deposition of the LB film of fatty
acid salts with bivalent metals. Than, the film was exposed to the atmosphere ofH2S for the
formation ofthe particles. It is worth to mention, that the compleatment ofthe reaction was
controlled by quarz balance measurements (Facci et al., 1993).
Quartz balance is a tool, allowing to detect the increase (or decrease) of the film mass,
deposited onto the surface of quartz resonator, connected to the driving circuit, reqistering
the shift in a frequency. The dependence is expressed by Sauerbrey equation (Sauerbrey,
1964):

M
-=---
~m

fo AP.

where ~f is a frequency shift, f0 is an initial frequency of the oscillations, ~m is difference in

mass, and A, p and I are the area of the resonator, covered by the film, quartz density and
resonator thickness respectively.
Using this equation, the flow ofthe reaction was followed by registering the frequency
shift as a function ofthe reaction time. Typical dependances are shown in Figure 12 (Facci et
al., 1994). As one can see, the curves are tending to come to the saturation (saturation time
depends upon the film thickness). On the other hand, the plateau Ievel in alt cases
coresponds weil to the value resulting from a simple calculations of the number of cadmium
atoms available for the reaction in each sample. Numerical data of such estimations are
presented in Table I. The available amount of cadmium atoms was estimated in this case
taking into account that one atom coordinates two fatty acid head groups, and in the pH

129
 140

......
120 ·-
C'l
.s 100
Q)
(/)
«< 80
...
Q)
(.)
c:
(/) 60
(/)
«<
:E 40

20

0
10 100 1000 10000
Times [s]
Figure 12. Dependence ofthe mass increase on time during the CdS particle formation.

(6.5-6.8) range used in this work, the amount of salt in the film must be about 70% (the
reast 30% are ofthe pure fatty acid) (Hasmonay et al., 1980).
The next step consists in the selective removal of the fatty acid from the film. It turned
out that the washing of the samples with cWorophorm and their drying with nitrogen flow
results in the removal of mass amount corresponding to the amount of fatty acids present on
the resonator surface (Table I) (Facci et al., 1994). This procedure, however, did not
remove compleatly the film from the quartz surface, leaving the residual mass. Taking into
acount both the amount of reacted H2 S and the mass of the residual film (nonsoluble in
chlorophorm, contrary to hydrocarbon chains), it could be argued that the residual film is
made ofCdS structures which were formed during the reaction.

Table 1. Nanogravimetrie Assay Results.

number of reacted HzS reacted HzS fatty acid fatty acid residual film residual film
bilayers (theory) (exptl) density density (theory) (exptl)
{theo!l} {ex[!tQ
20 3.86 3.4±0.2 101.16 97±5 14.3 12.4±0.6
15 2.89 2.5±0.1 75.87 72±4 10.7 9.3±0.5
10 1.93 1.6±0.1 50.58 47.8±2 7.1 6.5±0.3

X-ray measurements in a small-angle region had also demonstrated the formation ofthe
semiconductor layers. X-ray pattern of initial fatty acid salt LB film contain both Bragg
reflections and Kiessig fringes. After the reaction their angular position was changed,
indicating the increase of the spacing and the total thickness. After washing with chloroform,
both Bragg reflections and Kiessig fringes disappear (Facci et al. , 1994).
The formation of the layers was also checked by optical absorption measurements and
ellipsometry. After the reaction the absorption spectrum was blue shifted with respect to the
bulk spectrum of CdS, indicating the formation of very small particles. Their sizes was also
possible to estimate using Rama Krishna and Friesner theory (Rama Krishna and Friesner,
1991 ). After washing with chloroform the blue shift becomes to be smaller (but still
remaining), indicating the aggregation ofthe particles in the layer (Facci et al. , 1994).

130
 Results of ellipsometric study are presented in Table 2. As it is clear from the table, the
resultant average thickness of the semiconductor layer, obtained from one bilayer
precoursor, is about 0.8 nm. This value can be considered as the thickness resolution ofthis
technique. It is worth to mention, that amang the available techniques, only molecular beam
epitaxy allows to reach such resolution. However, the proposed technique is much simpler
and does not require complicated and expansive equipment.

Table 2. Ellipsometric Assay Results

before H2S afterH2S after chloroform

film thickness per 2.7±0. 1 3.3±0.1 0.4±0.1
monolayer

STM imaging was performed in order to achive a more direct understanding of the
structure of resultant semiconductor layers. For these reasons, the films were deposited onto
atomically flat surface (freshly cleaved graphite). The image ofthe rather large area is shown
in Figure 13. Rough surface of the sample corresponds to the aggregates of nanoparticle,
forming the layer. Sizes of individual particles are of the order of 3-5 nm, what corresponds
weil to the results of other studies. There are also rather flat areas in such films, where it is
possible to see atomic resolution. Figure 14 sows such image of CdS layer (Facci et al.,
1994). It is clearly visible the lattice of the CdS with hexagonal symmetry. Disturbance of
the lattice at boundaries is connected to the finit (small) sizes of the granules, compositing
the layer. Figure 15 shows the image ofthe layer, formed by the aggregation ofPbS particles
(Erokhin et al., 1997a). Symetry of the image and lattice parameters correspond to that of
bulk PbS.

Figure 13. STM image of the large area of aggregated CdS particle layer.

Figure 14. STM image ofthe aggregated CdS particle layer with atomic resolution.

131
 Having such a technological possibility it was logic to try to apply it for the forrnation of
complicated heterostructures - semiconductor superlattices.
Superlattices were prepared in three different ways. The essential steps of each are
listed below:
a) deposition of the fatty acid salt LB film (with meta! I); reaction for forrnation of
nanoparticles; aggregation; deposition offatty acid salt LB film (with meta! li); reaction for
forrnation of nanoparticles; aggregation (scheme of the process is shown in the Fig. I).
Then, all the steps were repeated several times (2 or 3).
b) deposition of the fatty acid salt LB film (with meta! I); reaction for forrnation of
nanoparticles; deposition offatty acidsatt LB film (with meta! li); reaction for forrnation of
nanoparticles. Then, all the steps were repeated several times (2 or 3). Aggregation was
perforrned when all the sample layers were deposited and exposed to the reaction.
c) deposition ofthe fatty acidsalt LB film (with meta) I); deposition offatty acidsalt LB film

l.
(with meta! II). Then, all the steps were repeated several times (2 or 3). Partide forming

. ..... ..
reaction and aggregation were perforrned when all the sample layers were deposited.

..
~
.. "' ,. "
. ..;r ... e ,,'-

~· · ··.-...........
~
•. • ' • •••• , ••• •i
~ .. .. -~~ ........'N

t...........
'~•··~,.,
·~-
~
.....
~
.
.......
.. . ._,
. ,.

Figure 15. STM image ofthe aggregated PbS particle layer.

a b

Figure 16. SEMimage ofPbS-CdS-PbS superlattice: a) uniform region, b) disturbed region.

132
 First of all, only the samples, prepared according to the proccdure "a" were suitable für
scanning electron microscope (SEM) measurements. All the other samples were very
unstable under the electron beam. These results allow to suppose, that in a case of the
sample preparation according to the procedure "b" and "c" the removal of fatty acid matrix
was not completed. The residual fatty acid molecules resulted in the decrease of the sample
stability.
SEM images of the superlattice of the type PbS - CdS - PbS are presented in the Fig.
16 (Erokhin et aL, 1997b). Fig. 16a demonstrates rather uniform part of the superlattice,
while Figure 16b demonstrates the disturbed region with some defects formed during the
particle formation or aggregation processes. The thickness of the individual layers, estimated
from the Figure 16a is about 50 nm, giving the average thickness of the aggregated film
corresponding to the bilayer of initial LB film of about 0.5 nm, what is consistent with the
data already obtained by ellipsometry measurements. In a case of Figure 16b the average
thickness is slightly bigger, what can be due to the fact, that this region is disturbed.
SEM image of the CdS - MgS - CdS - MgS - CdS superlattice is presented in the
Figure 17. It is possible clearly to distinguish these layers in the image. The average
thickness öf each layer in the superlattice is about 60 nm, what is again consistent with the
ellipsometry measurements of average thickness corresponding to the bilayer of the initial
film.

Figure 17. SEM image of CdS-MgS-CdS-MgS-CdS superlattice.

The proposed technique seems to be rather prommising for the formation of electronic
devices of extreamly small sizes. In fact, its resolution is about 0.5-0.8 nm, what is
comparable with that of molecular beam epitaxy. However, molecular beam epitaxy is a
complicated and expansive technique. All the processes are carried out at I 0"10 vacuum and
demand to use extra pure materials. In the proposed technique the layers are synthetized at
normal conditions and, therefore, is much less expansive. The presented results had
demonstrated the possibility of formation of superlattices with this technique. The next step
will be the fabrication of devices based on these superlattices. To begin with, two types of
devices will be focused on. The first will be a resonant tunnelling diod. In this case the
quantum weil will be surrunded by two quantum barriers. In a case of symmetric structure,
the resonant Ievel will appear in a quantum weil, what will result in the resonant current
maximum in V/I characteristics, when the bias voltage will correspond to the Ievel energy.
The other possible device can be a semiconductor Iaser. The Iaser will be similar to that

133
fabricated with molecular beam epitaxy (Capasso et al., 1995). lt does not require the
recombination of the carriers, but is based on the transitions of electron through resonant
Ievels within quantum wells in a semiconductor superlattice. Realization of such device will
demand the formation of complicated superlattice with different composition and thickness
of the layers in it.
Summarizing the material, it is possible to concloude, that the technique of the
ultrasmall semiconductor particle formation turned out to be a powerfull tool for building up
single-electron junctions, working even at room temperature, as weil as thin semiconductor
layers and superlattices with structural features, reachable in a past only with molecular
beam epitaxy.

APPENDIX 1

TheModel

A two junctions system can trap <n> electrons in granule between them. Moreover, a
two junction system can be also modelled by a two capacitors system. In this case, if <n>
are in the granule, then the two capacitance valtage became (Carrara et al., 1996).

V CCs e V e
Vc =----<n>-=--<n>-
s Cs C + Cs 2Cs 2 2Cs

(1)
V CCs e V e
Vc =---+<n>-=-+<n>-
C C+Cs 2Cs 2 2Cs

Obviously, the approximation is only valid if it is possible to neglect the difference in

capacitance of the two capacitors.
In a situation like that, the voltages in equation (I) are connected to the valtage applied
to the system, which is redistributed taking into account the presence of electrons in the
granule.
In fact, the energy of the n incoming electrons the granule is equal to (Lafarge et al.,
1991)

csv2
(2)
2
Now, considering a Bolzmann distribution of the electrons in the granule, it is possible to
write:

I;ne
< n > _,_V_.,n_ _ _---=_ (3)
(V- Vn)2
CT2
I:e
Vn
where

(4)

134
 Introducing equation (3) into equation ( 1) enalbe us to describe the voltage potential of
the two barriers in relationship with the total bias voltage ofthe whole two junction sytem.
This fact is important, as it gives the possibility to try to model the current flowing
through the whole system as due to two tunneling processes in the two junctions. In fact,
considering the probability of tunnel in a barrier system as

P oc e
-P~if>d
h (5)

It is possible to write for the single barrier of a two junction system

(6)

where Vj is indicating the single capacitance voltage.

The current flowing through a two barrier system is proportional to the electtron
tunnelling probability of the first and the second barrier and, therefore (Carrara et al., 1996)

2ml
--
h
[V e J1 2ml
2 (> 5 -e--<n>- ds- -if>-e(-+<n>-)d
2 2C 5 h2 2 2C
V ej
I oc Ve e s (7)

It was already shown that the equation (7) can describe V/I characteristics with the
shape tipical for the staircase (Carrara et al, 1996). Of course, this happens if and only if the
average number of electrons <n> in the granule is not vanished.
This could happens if the electrostatic energy of the trapped electrons is not lower then
their thermal exitation; that is

e2
cs <2kT
- (8)

In that condition, the apperance of staircase could be found in the current-voltage

characteristics of a two junction system. In particular, reffering to the normal condition of a
system like ones possible to create with nanoparticles, the order of magnitude of the capacity
coming from the equation (8) is about I0-18 F.
At the same time, it was also observed the phenomenon of differential resistance
regions along the current-voltage characteristics in nanoparticles systems. Nevertheless, it
was shown (Carrara et al., 1996) that the equation (7) can describe weil even such
behaviour.

(9)

According to the experimental results, Coulomb Gap is observable in junctions with the
Capacity value ofabout w-18 F (van Benturnet al., 1988a), Coulomb Staircase in I0-18_10-
19 F junctions (Wilkins et al., 1989; Shönenberger et al., 1992b; Facci et al., 1996) and
regions with differential negative resistance in I0-19 F ones (Erokhin et al., 1995).
The condition (9) can be satisfied on1y starting from the particular value of the number
<n>. lt means that different features can appear along the current voltage characteristics.

135
The staircase phenomena can be observed for the firsts steps and regions with differential
negative resistance will appear for higher values of the bias voltave V (He and Das Sarma,
1993; Carrara et al., 1996). Such behaviour oftwo junction systemswas really observed in
some experimental studies (Reed et al., 1988; Erokhin et al., 1995).
Now, ifthe two junction system is realised by placing nanoparticles between electrodes,
it is also possible to model its capacitance in terms of the nanoparticle sizes. This model
depends on the nanoparticle shape. However, the structures of nanoparticles prepared with
different techniques were found tobe different: shaped like tubules (Röder et al., 1993; Tian
et al., 1995), spheres (Röder et al., 1993; Yang et al., 1995), or disks (Erokhin et al., 1991;
Erokhin et al., 1995). In fact, different authors suggest different models for the nanoparticles
shape (Shönenberger et al., 1992b; Dorogi et al., 1995; Wilkins et al., 1989; Erokhin et al.,
1995). In reality, models referring to spherical of disk-shaped nanoparticles are both useful
for modelling single-electron two junction sytems.
Therefore, if a spherical particle is considered for two junction systems then its
capacitance will be (Ruggiero and Barner, 1991)

(10)

otherwise (Carrara et al., 1996)

(11)

In reality, considering nanosize particles, both equation (10) and (11) provide quasi the
same values and, therefore, it is still remained not clear wich kind of Cs-R relationship we
have in such systems (Ruggiero and Barner, 1991 ).
However, summarizing only the experimental data, it is possible to say that no single
electron phenomena were observed in systems based on nanoparticles of more than 10 nm in
sizes (Kuz'min and Likharev, 1987; Wilkins et al., 1989; Ruggiero and Barner, 1991),
Coulomb Staircase was recorded for particles ranging from 2 up to 5 nm (Shönenberger et
al., 1992b; Erokhin et al., 1995, Erokhin et al., 1996; Facci et al., 1996) and the differential
negative resistance was obtained only for 5 nm sizes particles (Erokhin et al., 1995; Erokhin
et al., 1996).

ACKNOWLEDGEMENTS

Authors would like to acknowledge Mr. Andrea Rossi and Mr. Fabrizio Nozza for their
help in the preparation ofthe manuscript.

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138
ELECTRON CORRELATION IN QUANTUM MOLECULAR BIOPHYSICS: THE
CASE STUDY OF HEMOCYANIN

Piero Fariselli 1 and Rita Casadio2

1 Lab. ofBiocomputing
Centro Intertipartimentale di Ricerche Biotecnologiche
via S. Giacomo 9/2, 40126 Bologna
tel. +39-51-354005 e-mail: farisel@alma.unibo.it
2 Lab. ofBiophysics

Dept. ofBiology University ofBologna

via Irnerio 42, 42 40126 Bologna
tel. +39-51-351284; e-mail: Casadio@alma.unibo.it

The underlying physical laws necessary for the mathematical theory of a !arge part ofphysics and
the whole of chemistry are thus completely known, and the difficulty is only that the exact
application ofthese laws Ieads to equafions much too complicated tobe soluble.
--P. A. M DIRAC, 1929

INTRODUCTION

One of the most interesting prob1ems of modern molecular biophysics is the study of the
relationship between structure and function in proteins. The data base of proteins known at
atomic resolutions (the Brookhaven Protein Data Base (PDB), release 19/9/1997) presently
comprises 5900 structures, obtained from X ray diffraction patterns of ordered crystals or in
some cases from twodimensional NMR spectroscopy. Weil resolved structures include
enzymes of several different type.
Proteins form the class of biological macromolecules that have the most well-defined
physicochemical properties and have therefore been easily isolated and characterized since
the earliest days ofbiochemistry. Furthermore proteins, particularly in the form of enzymes,
play a central roJe in biological processes. lt is weil known that the most relevant
biochemical functions are catalysed by enzymes which include in their three-dimensional
structures meta) ions and/or prosthetic groups whose atomic resolution is also detailed in the
data base. This is so for several metalloproteins, including different types of copper proteins
(132 structures in the present PDB release), whose function has been described at length in
the Iiterature (Voet and Voet, 1995).

Biophysics of Electron Transfer and Molecular Bwelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 139
 In structural studies, the disclosure of the protein structure at atomic resolution is
routinely paralleled by the proposal of a putative catalytic mechanism for the protein
enzyme. Several enzyme catalysed reactions have been recently described (or re-described)
based on the refined folded structure of the corresponding protein, enlightening which
residue/s or groups (including meta! ions) are involved in the mechanist steps at the active
site and in their regulation (Holm et al., 1996). By this procedure, enzymatic mechanisms of
catalysis, which have ample precedent in organic catalytic reactions, are related to the three
dimensional structure of the proteins and relevant residues and groups essential for the
function can be determined, suggesting possible experiments of site directed mutagenesis.
The procedure is however approximate since it is based on the recognition in the structure of
the proximity of groups with chemico-physical properties sufficient and specific for a given
organic catalytic reaction and is not theoretically grounded on a quantomechanical
description ofthe reaction pathway.
The so called modern "ab initio" approaches of quantum chemistry are indeed to be
used in order to give an accurate description of the electron structures and properties of
molecules and their bonds (Szabo and Ostlund, 1989). This accurate Ievel of description is
however hampered in the case of proteins by the atomic dimension of the system under
investigation, that often comprises some hundred atoms only in the active site. Most of the
schemes described in the literature, phenomenologically referring to the protein resolved
structure, are therefore based on the only possible approach that can be presently used to
bridge the gap between the quantomechanical description of reactivity and the dimension of
the active site. In addition to this, it must also be noticed that even in the most weil resolved
structures hydrogen atoms, being transparent to X-ray radiation, are not present in the
electron density maps and are assigned by means of computational methods. As a
consequence, for example, reaction intermediates of simple hydrolytic reactions, based on
acid-base catalysis, are necessarily putative.
Given these considerations, a few general questions arise.
To which extent the so far proposed enzyme-catalysed reaction mechanisms are
accurate? Is it possible to use ab initio calculations for describing pathways of biochemical
reactions? Can eventually this approach provide a tool to refine both the structure and the
reaction mechanism, once the folding of the protein enzyme has been described with enough
accuracy?
In the following we will review some of the work published so far in this perspective
focusing on the density functional theory, which presently allows the only feasible approach
to treat ab initio systems whose dimensions are similar in magnitude to those of enzyme
active sites.

STATE OF THE ART

Early attempts to study the electronic structure of biochemical compounds date back to
the fifties and sixties (Pullman and Pullman, 1963), when relevant biomolecules were
recognized as conjugated molecules and the role of the electronic delocalisation was first
described in biological processes. Two methods were available in quantum organic chemistry
for defining the electronic structure of molecules: the valence-bond method ( or resonance
theory) and the molecular-orbital method. Both represent approximate procedures for
obtaining approximate solutions ofthe Schroedinger equation relative to molecules and were
successfully applied in organic chemistry. However it was evident that, given its basic
principles, the resonance theory was far too complex and too difficult to handle to be used
for describing the electronic structure of biochemical molecules. Early sturlies deal with
applications of the molecular orbital theory to biomolecules (Pullman and Pullman, 1963).

140
After almost fifty years, the power of both mathematical techniques, based on the molecular
orbital theory, and of computing machines has tremendously increased. The study of the
electronic structure of a system is generally performed with refined mathematical methods
based on the molecular orbital theory (referred to as ab initio methods). An empirical
valence-bond theory has been developed and successfully applied to computer modelling of
chemical reactions in enzymes and solutions (Warshell 1991 ). This approach, which
massively relies on experimental phenomenological data for each systemtobe modelled (and
is therefore termed empirical), has been used to describe electrostatic interactions at the
active sites of some protein enzymes, including papain, serine protease and carbonic
anhydrase. More recently ab initio methods at various Ievels of accuracy (see below) have
been applied to model the copper(II)-oxygen interaction in the Superoxide dismutase (Rosi
et al., 1986), the electron structure of the active site of aspartic proteinases (Beverdge and
Heywood, 1993), and the coordination chemistry of the structural zinc ion in alcohol
dehydrogenases (Ryde, 1996).
A different approach to the solution of the Schroedinger equation is based on the
density functional theory (DFT) originally formulated in the mid sixties and applied to solid
state problems (Ziegler, 1991). During the past ten years it has become increasingly evident
that DFT has great potential in the study of molecular systems and chemical problems. DFT
performances in describing the electronic structures of the active site of metalloproteins and
its comparison with traditional ab initio methods at the most accurate Ievel of computation
have recently been tested by our group using hemocyanin as a benchmark (Bernardi et al.,
1996a, Bernardi et al., 1996b). A similar approach has been also applied to blue copper
proteins (Ryde at al., 1996) and indicates that the cupric geometry of the catalytic site of
plastocyanin is not strained, as it would be expected by considering the entatic state
(Williams, 1995) and the induced-rack hypotheses (Malmstrom, 1994), according to which
the protein backhone of metalloproteins may provide pre-formed sites that strain the the
metal ion into a catalytically poised state.

THEORETICAL BACKGROUND

Modern quantum chemistry generally deals with computational methods necessary to

find approximate solutions to the non relativistic time-independent Schroedinger equation in
the case of a many body problem, namely a system with a number of electrons and nuclei
greater than that of hydrogenoids (Szabo and Ostlund, 1989). According to the farnaus
Dirac's aphorism the whole body of chemistry (and necessarily biochemistry) can be obtained
by means of an appropriate mathematical development of quantum mechanics, provided that

Maximum Maximum

Minimum

Figure 1. The electronic potential surface ofthe reaction A+B <=> C+D with the E transition state.

141
solutions are found to the complicated equations necessary to describe the systems (Dirac,
1929).
Solutions are found to the classical Schroedinger equation:

Hlfi=Eifl (1)

where H is the Hamiltonian of the system, 1f1 is the wavefunction and E is the correspondent
eigenvalue. Fora molecular systemHisset equal to:

(2)

with:

(3)

where V is the Coulomb interaction potential, T. and T. are the kinetic energies of electrons
and nuclei, respectively and n is the Plank's constant. V in turn is the total sum of three
components, namely an electrostatic repulsion term, an electron-nucleus attraction term, and
a nuclear repulsion term; q is the electron charge and ry, r;n and Qnm are respectively the
distances between the ;th and the /h electrons, between the ;th electron and the n'h nucleus and
between the n'h and the m'h nuclei.
Exact solutions of equation Eq.l are known only for hydrogen and hydrogenoids. When
this formalism is applied to molecules, the resulting equations become so complicated that
cannot be analytically solved. This is so due to the fact that the full description of the
molecular system include the momenta of both electrons and nuclei. Possible solutions are
found with approximations such as the adiabatic approximation of Born and Oppenheimer
(Born and Oppenheimer, 1927), which is central to quantum chemistry (Szabo and Ostlund,
1989). Since nuclei are much heavier than electrons and move more slowly, their motion can
be neglected with respect to that of electrons, and solutions can be first found for the
electronic Hamiltonian describing the motions of electrons in the field of nuclear fixed
charges.
Accordingly:

H"1 t/J(r,QJ= {T. + V(r,QJ} t/J(r,QJ = E(QJ t/J(r,QJ (4)

{ TN + E(QJ} A.(QJ = E A.(QJ (5)

The electronic state of the system can be evaluated from a local topological study of the
multidimensional E(QJ surface by computing the maxima, the rninima and the saddle points
(Figure 1). According to this procedure, a minimum corresponds to a stable states for the
molecule (reagents, products, and intermediates of the reaction). A saddle point of the first
order corresponds to a transition state, namely a point in the energy surface from which only
one trajectory Ieads to the reaction completion.

142
AB INITIO METHODS

Ab initio methods are based on the orbital molecular theory (Szabo and Ostlund, 1989}.
The appropriate wavefunction describing a single electron is a spin orbital and the simplest
wavefunction describing a collection of N electrons, including the antisymmetry or Pauli
exclusion principle, can be written as a determinant, called a Slater determinant

(6}

where P is a permutation operator that permutes the column indices and the sum runs over
all n! permutations ofthe indices.
The spatial molecular orbital in the Slater determinant is expanded in a set of known
spatial basis functions:

(7)

To obtain exact molecular orbitalsaninfinite number ofterms is needed. The basis set is
however truncated for computations. The most frequently implemented basis set is that of
the Gaussian type.
Exact solutions to the Schroedinger equation are impossible even for as simple
molecules as H 2+. Finding and describing approximate solutions for many-electron problems
has been a major preoccupation of quantum chemistry since the birth of quantum mechanics.
The first step towards more accurate approximations is achieved with the Hartree-Fock
(HF) self consistent field method (SCF) based on the variation principle. The HF method
optimises the coefficients ofEq. 7, in order to obtain the best wavefunction (in the form ofa
Slater determinant (Eq 6)} for the description of the system. The method is self-consistent
since the procedure for solution-searching is re-iterated until the fields and the spin-orbitals
no Ionger change.
HF is restricted when, for closed-shell systems (with an even number of electrons) the
spatial orbitals are restricted to be the same for a. and ß spins, and each spatial orbital is
occupied by two electrons with different spin. For a molecule which has one or more open-
shell (unpaired) electrons, HF can be restricted or unrestricted. In the restricted open-shell
formalism, all electrons, except those that are explicitly required to occupy open shell
orbitals, occupy closed shell orbitals. In the unrestricted HF, electrons of a. spin are
described by a set of spatial orbitals and electrons of ß spin are described by a different set of
spatial orbital. A restricted HF description is inappropriate at long bond Iengths for
molecules (like H 2) which dissociate to open-shell species.

ELECTRON CORRELATION

The Hartree-Fock method treats electrons as being independent of each other and
introduces the idea of self consistent field, namely the interaction field that an electron
experiences when a spatial average over the positions of all the other electrons is considered.
This approximation, which is remarkably successful in many cases, has however its
limitations. For example HF predicts incorrect results for the ordering of ionisation
potentials of N 2 . The restricted HF cannot describe the dissociation of molecules into open-
shell fragments. The unrestricted version gives a qualitatively correct prediction of such
dissociation but the resulting potential energy curves (or surfaces) arenot accurate.

143
 A problern centrat to ab initio methods is the accurate description of electron-electron
interaction referred to as electron correlation. The HF energy (EHF) obtained in the Iimit that
the basis set approaches completeness is an upper bound to the exact nonrelativistic energy
of the system (E0 ). The difference between Eo and EHF defines the correlation energy (Ecorr)
and is negative. The so called post HF methods deal with an appropriate evaluation of the
correlation energy.
The conceptually simplest methods for obtaining the correlation energy is the method of
configuration interaction (CI), based on the idea of diagonalizing the N-electron Hamiltonian
in a basis of N-electron functions (Siater determinants) (Szabo and Ostlund, 1989). The
exact wavefunction is represented as a linear combination of N-electron trial functions
(multiconfigurational wave functions) and a linear variation method is used for determining
the coefficients. If the basis were complete, the exact energies of the ground and excited
states of the systems would be obtained and CI would provide an exact solution of the
many-electron problem. Practically, only a finite set of N-electron trial functions can be
handled and consequently CI provides only upper bounds to the exact energies.
Unfortunately, even for small molecules and moderately sized one-electron basis set, the
number of N-electron determinants is enormous, the trial function must be truncated and
only a fraction of all possible N-electron function is used (CID and CIDS). Truncated CI is
suited to treat systems comprising a !arge number of electrons; its application to chemical
reactivity is however hampered by its Iack of size-consistency (a generat definition of size-
consistency is that the energy of a many particle system even in the presence of interactions
becomes proportional to the number ofparticles N in the Iimit ofN tending to infinity).
An alternative approach to CI is the so called multi-configuration self consistent field
method (MCSCF), known also as complete active space self consistent field method
(CASSCF) (Szabo and Ostlund, 1989). MCSCF is based on a multiconfigurational
wavefunction description of the electron system and not being truncated, is size-consistent.
Molecular orbitals are classified into three types: core orbitals which are doubly occupied,
valence orbitals which can have occupancy of two, one or zero electrons (these orbitals
define the active space and their choice is somewhat arbitrary) and unoccupied orbitals
which have zero occupancy. In treating orbitals of the active space, MCSCF is similar to a
full CI. Electron-electron interaction is however limited to the active space.
A different approach for determining electron correlation energy is based on a form of
perturbation theory (popular as many-body perturbation theory). In this approach the total
Hamiltonian of the system is divided in two parts: a zeroth-order part which has known
eigenfunctions and eigenvalues and a perturbation term. The exact energy is then expressed
as an infinite sum of contributions of increasing complexity. Depending on the Ievel of
approximation the method is termed MP2 (the most frequently used), MP3, etc. This
method is size-consistent, but not being variational, does not provide an upper bound to the
exact energy.
More recently, a version of CASSCF, which includes MP potentiality, has been
developed (CASPT2) (Andersson et al., 1990, Andersson et al., 1992). This method is
presently the most accurate for evaluating the electronic structure of a system including
electron correlation. Due to its high computational cost, it can be applied only to small
systems.
The development of powerful techniques based on energy gradient methods has made it
possible to routinely carry out a full structural determination of stable molecules. An energy
gradient technique relies on an analytical evaluation ofthe firstorderderivatives with respect
to the nuclear coordinates (Ziegler, 1991 ). A recent extension to the second order of energy
derivatives allows also calculating vibrational frequencies. Developments of energy gradient
based methods can provide perspective on reaction mechanisms. Such methods can be used
to determine structures related to the transition state and estimations of the energy profile

144
and activation barriers. The HF method gives rather satisfactory reasonable geometric
parameters for molecules involving main-group elements, whereas the frequencies calculated
by HF for the same type of molecules are in general too large. Results become satisfactory
only when ab initio methods including electron correlation are used. In transition-metal
complexes, the electron correlation must be taken into account in determining structure and
frequencies (Ziegler, 1991). The application of ab initio methods to kinetic problems has
indicated that the HF method overestimates activation barriers. When electron correlation is
taken into account, barriers in good agreement with experimental data are obtained.
However the high computational cost of correlated ab initio methods restricts their
usefulness to rather small systems (Szabo and Ostlund, 1989, Ziegler, 1991).

THE DENSITY FUNCTIONAL THEORY

The approximate density functional theory (DFT) gives an alternative approach to solve
the Schroedinger equation (Ziegler, 1991). The basic notion in DFT isthat the energy in the
electronic system can be expressed in terms of its density according to the following
equation

(8)

where the different contributions are respectively the kinetic energy (T.), the nuclei-electron
Coulomb interaction (Vne), the Coulomb repulsive electron-electron interaction (Vc) and the
exchange-correlation energy (Exc). Solution to Eq.8 are found by means of the variation
method (c5E.1 =0), and similarly to the HF method, the eigenvalue equations are:

(9)

h~a includes an exchange-correlation potential given by

V [ ]= OExc[P] (10)
XC p Op

However the analytical form of Exc is unknown and therefore approximations of Vxc are
introduced.
The first DFT-based scheme used in studies of systems with more than one atom was
the Xa method. In this approximation the exchange-correlation potential is represented by a
function which is proportional to 1/3 power of the electron density and includes an
adjustable parameter a which is fitted to observable quantities, making this method suitable
to modelexperimental data bothin solid state physics and molecular spectroscopy.
More recent approximations for Vxc are ofthe local density (LDA) and non local density
(NLDA) types. In the LDA it is assumed that at a given coordinate the exchange-correlation
effects depend only on the value of the electron density. For example using the
parametrization ofVolsko et al. (Unichem DGauss, 1994), the exchange-correlation energy
can be described in analytic form as a function of electron density. In the general spin-
polarized case, the local spin density (LSD) exchange-correlation energy can be written as:

Exc =Jp(r)exc[ Pa (r),p p (r)] · dr (11)

145
where Bxc is the exchange-correlation energy per particle of a uniform electron gas with spin
density Pa and pp.. The LSD approximation of the exchange-correlation energy has been
used in numerous studies of molecular, surface and solid state systems with surprisingly
good results. Structures, vibrations and electrostatic properties are typically predicted weil
by LSD calculations. The energetics of chemical reactions, including molecular dissociation
energies are however poorly predicted. To overcome these difficulties non local corrections
in the form of density gradient expansions were developed. In this approach the exchange-
correlation energy ofEq 10 is redefined by including the local values of density derivatives in
addition to that of the density itself The exchange-correlation energy with non local
corrections (NLSD) can be written in the form.

(12)

where Ex0 and Ec0 are the non local or gradient correction to the exchange and correlation
energy, respectively. In the following an application of the Becke-Perdew (Ziegler, 1991,
Uniehern Dgauss, 1994) approximation is shown. DFT is therefore interesting since it
includes dynamic correlation and holds for ionic, covalent as weil as metallic bonds.
Moreover it can be applied to relatively )arge systems because of its N3 scaling (N being the
number of basis function) as opposite to the N 5-N 7 scaling of the post HF methods (Table
1).

Table 1. Accuracy and computational expedience ofthe different ab initio methods.

Method Electron correlation Accuracy Size consistency Scaling

Factor
HF NO YES NJ
HF+CI YES(Partial) NO >Ns-N6
CASSCF YES(Partial) YES >Ns-~
CASPT2 YES YES >N7
OFT YES YES NJ
N= number ofbasis functions.

THE CASE STUDY OF HEMOCYANIN

Hemocyanins are )arge multi-subunit proteins containing copper ions in the active site.
Hemocyanins transport oxygen in a variety of invertebrates (more than 80% of all living
species). Their molecular weight can be as high as 3500 kD and a single subunit comprises
647/628 amino-acidic residues with local motifs ofsecondary structures.
The oxygen binding centre is a dinuclear copper site, embedded in a protein matrix, with
three histidine residues coordinating each copper ion (Figure 2). Recently the three-
dimensional structures of deoxygenated exameric hemocyanins from Panulirus interruptus
(Volbeda et al., 1989) and those of the deoxygenated and oxygenated form of the
hemocyanin subunit II of Limulus poliphemus (Magnus at al., 1994) were refined at atomic
resolution (3.2 and 2.4 A, respectively). This crystal indicates that the oxygen is bound in the
side-on (J.L-1'{11 2) configuration, with respect to the Cu-Cu axes (Figure 5). The copper-
copper distance and the dioxygen length are 3.6 A and 1.41 A, respectively, with the four
atoms that lie almost in the same plane. The copper ligands are three histidines for each
metals, differentiated in proxymal and distal, as shown in Figures 3 and 4.
Raman, UV, EPR and EXAFS data are also available for these proteins (for review see
Solomon et al., 1992). EXAFS data show the existence of a vacancy in the copper ion d
shell of oxyhemocyanin. They also indicate that the two copper atoms in oxyhemocyanin are

146
 Figure. 2. Three dimensional structure of oxyhemocyanin (Magnus et al., 1984; PDB entry: 1oxy).

Figure. 3 Active site of deoxyhemocyanin from Panu/irus interruptus (Volbeda et al., 1989; PDB entry:
lhc6).

147
Figure. 4. Active site of oxyhemocyanin from Limulus poliphemus (Magnus et al., 1994; PDB entry: loxy).

both in the cupric C(II) oxidation state, demonstrating that , upon oxygen binding, an
electron transfer takes place from the two Cu(I) cations to the dioxygen (Solomon et al.,
1992). Accordingly the Raman spectra reveal a very low energy 0-0 Stretching vibration
(750 cm" 1) as compared to an isolated oxygen molecule (1550 cm"\ This agrees with the
electron transfer hypothesis from the cations to the 0 2 Iigand. Another relevant experimental
result is the absence of any EPR signal in oxyhemocyanins. Magnetic susceptibility studies
show that this behaviour is due to a strong antiferromagnetic coupling between the two
C(II) ions to form a singlet and triplet states, with the triplet higher in energy than the singlet
by a value greater than 600 cm· 1. This large antiferromagnetic coupling requires orbital
overlap between the two Cu(II) cations. Since the distance between them is about 3.6 ~ this
is indicative of a superexchange mechanism which implies the presence of a bridging Iigand
(the dioxygen) responsible for the Superexchange pathway.
All these data univocally characterise the electronic properties of the protein active site
and the coal of any computational method should be their simulation.
The active site of hemocyanins is therefore amenable to test the reliability of ab initio
methods in describing the electron structure of a system very weil characterised both
structurally and functionally. Moreover modelling of the copper ions and their ligands are
also a benchmark for describing transition metals in a protein environment.
Before the crystallographic data were available, only a limited number of theoretical
investigations were performed. Semiempirical calculations were carried out on different
(Cu •h-0 2 complexes (Figure 5) with four in plane NH3 ligands to emulate the histidine
residues ofthe protein (Solomon and Lowery, 1993). The dicopper-dioxygen complexes in
the end-on and side-on configurations (without histidine ligands to the copper ions) were
also described with ab initio methods at the CASSCF Ievel with only two orbitals in the

148
active space (Maddaluno and Giessner-Prette, 1991,). Both studies indicated that the side-on
configuration has the most likely geometry, with the singlet state more stable than the triplet.
Moreover it was concluded (Maddaluno and Giessner-Prette, 1991) with the ab initio
calculations that the simple side-on dicopper-dioxygen model used to simulate the active site
of the protein was sufficient to describe all the electronic features, even though the
computed singlet-triplet difference was below of 600 cm·' and not in agreement with the
strong antiferromagnetic coupling described between the two copper ions (Solomon and
Lowery, 1993).

\1
HN O-O NH
3\/ 3
Cu Cu
1--o--\
H3N H NH3

end-on (cis J..L 1,2)

Figure. 5. End-on and side-on models for the active site of hemocyanin 3.

The question then poses as to whether it is sufficient to use the two copper ions and
their interaction with the dioxygen molecule to simulate the active site of the protein without
including any specific copper-ligand interaction.

SIMULATION OF THE ACTIVE SITE OF THE HEMOCYANIN AT DIFFERENT

LEVEL OF ACCURACY

In the following computational studies of the active site of hemocyanins performed at

different Ievel of accuracy will be presented focusing on two aspects:
* the Ievel of complexity ofthe model used to emulate the enzyme active site
* the relevance ofthe electron correlation in describing the reaction mechanism
Some aspects of this study can also be found in Bemardi et al., 1996a and Bernardi et al.
1996b.

CASSCF STUDY OF THE HEMOCYANIN ACTIVE SITE

Due to the demanding scaling factor of CASSCF (Table 1), only two models of
increasing complexity were used to evaluate the wave function of the system at this
computationallevel (Model 1 and Model2 shown in Figure 6 and Figure 7, respectively).
Starting with Model 1, after an exhaustive search of the all possible active spaces, we
found that the necessary active space consists ofthe orbital depicted in Figure 8, namely:
1) the two dxy orbitals of the copper atoms in the in phase and the out of phase
combinations;
2) the two singly occupied orbitals Ttx• e Ttz• ofthe oxygen molecule;
3) the two cr e cr· orbitals ofthe oxygen molecule.
Using this active space a total number of 105 configurations is obtained.

149
 Figure. 6. Model 1.

Figure. 7. Model2

Three different basis sets of increasing computational cost were tested: LANLIDZ,
MIDI-4 and DZVP (EMSL web page). All the basis sets gave similar results; the best trade
off between the number of basis functions and accuracy in this kind of computation was
however MIDI-4.
Three degree of freedom for the different electronic configurations were minimised.
During the geometry optimisations, the Cu-Cu distance were kept equal to 3.6 A, the
original Cu-Cu distance in the active site of the oxygenated form of hemocyanin. Bond
length (r), distance from the dicopper plane (R) and rotation angle (ro) around a normal to
the dicopper bond axis of the dioxygen molecule were optimised. The results are listed in
Table 2. The results clearly indicate that the energetic difference AE(S-T) for all basis sets is
in agreement with the magnetic susceptibility data (Solomon et al., 1992).
The dioxygen distance obtained at this computational Ievel is however in disagreement
with that in the crystal (1.76 A versus 1.41 A, Table 2). Taking into account what described
above, the discrepancy may indicate that the electron correlation is not properly evaluated by
the CASSF method.
At least three different states are necessary to describe the electronic configuration of
Model 1, namely two triplets (T 1 and T2) and one singlet (So). A detailed description of the
different states can be found in Bemardi et al., 1996a. Briefly, the wave function of So is
characterised by two main resonant components Cl> 1 and Cl>2 . In Cl> 1 the lj/2 orbital is doubly
occupied and lj/3 is empty, while in Cl>2 the situation is reversed. The other two configurations

150
 a• - ---

Figure. 8. Correlation diagram illustrating the orbitals used to describe the wave functions.

of So (cl>3 and cl>4) are similar to the former two, with the cr orbital empty and the cr· doubly
occupied. The cr ~cr • transfer is the main reason for the negative charge of the oxygen. The
T 1 triplet spin density is more localised on the dxy orbitals of the copper atoms, while T2 is
closer to the free oxygen triplet with the 1t;
(mixed with dxy+dxy) and 7tz• singly occupied.
Computations at the CASSCF Ievel, carried out on the )arger Model 2 including four
NH3 ligands to the copper ions, are in agreement with those obtained with Model I (Table
3).
In order to describe the binding mechanism, we computed the potential energy curve for
the three states (So, T. and T2) using Model 1 with the MIDI-4 basis set (Figure 9).
Considering T2 as the starting state (R==lO A) and looking at Figure 9 from the right to the
left, it can be concluded that when the oxygen molecule approaches the plane of the copper

Table. 2. CASSCF computations ofModel I using different basis sets

E AE(S-T) R r(0-0) (1) q(Cu) q(O)

MIDI-4
So -3423.31300 0.00 0.00 1.76 90.0 1.64 -0.64
T1 -3423.3074 I 3.51 0.00 1.79 90.0 1.65 -0.65
T2 -3423.23450 45.75 0.00 1.48 90.0 1.13 -0.13

DZVP
So -3426.05372 0.00 0.00 1.62 90.0 1.66 -0.66
T1 -3426.04353 6.39 0.00 1.69 90.0 1.70 -0.70

1LANLDZ
So -249.89102 0.00 0.00 1.80 90.0 1.61 -0.61
T1 -249.88644 2.87 0.00 1.82 90.0 1.63 -0.63

E = energy in Hartree; DE(S-T)= energy difference between singlet (S) and triplet (T) in Kca1/mol.
q=Mulliken charge.

151
 Table 3. CASSCF computations on Model 2

E AE(S-T) R r(0-0) ro q(Cu) q(O)

ILANLDZ
So -475.02143 0.00 0.00 1.81 89.6 1.31 -0.68
T1 -475.01764 2.38 0.00 1.83 89.6 1.32 -0.69

E=energy in Hartree; AE(S-T)=energy difference between singlet (S) and triplet (T) in Kcal/mol;
q=Mulliken charge.

50

~
40
0
E 30
::::,.
10
~ 20
UJ 10

0------~----~------+------r----~
0 2 4 6 8 10
R
Figure. 9. Potential energy curves computed with Modell for the, S0 , T1 and T2 states. R= oxygen
approaching to the Cu-Cu plane.

atoms, first there is an electron transfer that switches the ground state form T2 to T 1 and then
a spin inversion takes place (through spin-orbit coupling) towards the So state.
Although this description seems good enough to capture the essence of the binding
mechanism, two results do not correspond to the experimental data. The first is the value of
the energy barrier in relation to the experimental kinetic constant of3x10 7 M 1s" 1 (Solomon
et al., 1992). The second result is the overestimation ofthe dioxygen distance (1.76 A versus
1.41 A in the crystal).

RELEVANCE OF ELECTRON CORRELATION: THE CASPT2 COMPUTATION

LEVEL

As discussed above, CASPT2 is a powerful ab initio method (Table 1). However its
main drawbacks are the high computational demand and the Iack of an automatic
minimisation procedure, which makes necessary a manual parabolic interpolation.
The results of CASPT2 calculations are shown in Tables 4 and 5 for Model 1. The
inclusion of a more refined description of the electron correlation completely changes the
energetic of the system. This is due to the fact that the correlation among the electrons in the
d orbitals l<?wers the \j/2 and \j/3 energetic Ievels as compared to the CASSCF description
(compare the correlation diagram of Figure 8 with that of Figure 10). Noticeably the rtz"
energetic Ievel becomes higher as compared to the CASSCF description, and closer to the
\113 Ievel favouring the T2 global configuration of the system. This is in agreement with the

152
relation between the siglettriplet stability and the HOMO (highest occupied molecular
orbital) and LUMO (lowest unoccupied molecular orbital) separation: the more HOMO and
LUMO are apart, the more the singlet is stable; on the contrary the more HOMO and
LUMO are closer the more the triplet is stable.
It is therefore evident that the dicopper-dioxygen model (Model 1) is definitely not
appropriate to describe the hemocyanin active site. The T2 triplet remains the ground state
all along the reaction coordinate and as a consequence no inter-system crossing takes place
between singlet and triplet, as shown in Figure 11.
Due to the high computational cost, Model2 is untractable at this Ievel ofaccuracy.

MODELLING THE ACTIVE SITE OF HEMOCYANIN WITH TUE DFT

APPROACH

Among the ab initio methods, DFT is the most expedient to treat molecular systems of
!arge size (Table 1). This approach allows then the comparison of different models of

Table 4. CASPT2 computations on Model 1 for the T 1 state at different approaching

distances.

R r(o-o) AE Energy Energy (Co) Charges

CASSCF CASPT2
Cu 0
0 1.79 -3423.30851 -3434.12252 (.85) 1.65 -.65
0 1.60 -3423.30036 -3434.12082 (.86) 1.67 -.67
0 1.90 -342330626 -3434.11724 (.85) 1.64 -.64
OPT 1.72 61.6 -3423.30765 -3434.12382 (.85) 1.65 -.65
0.5 1.65 -3423.28574 -3434.11471 (.85) 1.63 -.63
0.5 1.75 -3423.28808 -3434.11282 (.85) 1.62 -.62
0.5 1.60 -3423.28248 -3434.11390 (.85) 1.64 -.64
OPT 1.66 67.3 -3423.28620 -3434.11472 ~.85} 1.63 -.63

R= 0 2 distance from the Cu-Cu plane, r(0-0)= 0 2 inter atomic distance

AE= in Kcal/mol, referred to T 2 energy in the plane (R=O). Energy = in Hartree.
(Co)= coefficient ofthe unperturbed wave function .. OPT= value optimised by means ofa parabolic search.

es• - - - - - - - -

\113 1C.
z

dxy + dxy +t
dxy - dxy +t ----- \llz---- tt
Yf
C~u->x
0
\111 tt
z/~ csft---ft
Figure 10. Correlation diagram for Model 1 as evaluated with CASPT2.

153
 Table 5. CASPT2 computations on Model 1 for the T2 state at different approaching
distances.

R r(o-o) DE Energy Energy (Co) Charges

CASSCF CASPT2
Cu 0
0 1.50 -3423.21656 -3434.22200 (.81) 1.26 -.26
0 1.40 -3423.20664 -3434.21585 (.80) 1.25 -.25
0 1.55 -3423.21468 -3434.21970 (.80) 1.28 -.28
OPT 1.50 0.0 -3423.21656 -3434.22200 (.81) 1.26 -.26
0.5 1.50 -3423.16000 -3434.18616 (.79) 1.31 -.31
0.5 1.45 -3423.13418 -3434.18507 (.77) 1.29 -.29
0.5 1.52 -3423.15639 -3424.17323 (.79) 1.34 -.34
OPT 1.48 19.7 -3423.16214 -3434.19066 (.79) 1.31 -.31
1.0 1.30 -3423.27751 -3424.19644 (.85) 1.0 .00
1.0 1.50 -3423.27060 -3424.19546 (.84) 1.0 .00
1.0 1.20 -3423.26209 -3424.17638 (.85) 1.0 .00
OPT 1.39 13.4 -3423.27816 -3424.20068 (.77) 1.0 .00
2.0 1.40 -3423.29702 -3424.19316 (.85) 0.96 .04
2.0 1.20 -3423.29130 -3424.18465 (.86) 0.97 .03
2.0 1.30 -3423.30175 -3424.19691 (.86) 0.97 .03
OPT 1.33 15.7 -3423.30155 -3424.19704 (.86) 0.97 .03
3.0 1.40 -3423.28958 -3424.17845 (.86) 0.98 .02
3.0 1.20 -3423.28662 -3424.17365 (.86) 0.98 .02
3.0 1.30 -3423.29569 -3424.18406 (.86) 0.98 .02
OPT 1.30 23.8 -3423.29569 -3424.18406 (.86) 0.98 .02
10 1.20 -3423.28460 -3424.16886 (.86) 1.00 .00
10 1.30 -3423.29244 -3424.17753 (.86) 1.00 .00
10 1.40 -3423.28509 -3424.17015 (.86) 1.00 .00
OPT 1.30 27.9 -3423.29244 -3424.17753 (.86) 1.00 .00

The legend is as in Table 4.

70
60
~50
0
E 40
::::,. ---so
.;z30
lll
--.--- T1
w 20 ___._ T2
10

0.-----~----~-----+----_,----~
0 2 4 6 8 10
R

Figure 11 Potential energy curve computed for Model 1 for the, S0, T 1 and T 2 states. R= oxygen
approaching distance to the Cu-Cu plane.

154
 Table. 6. CASPT2 computations on Model 1 for the So state at different oxygen
approaching distances.

R r(o-o) AB Energy Energy (Co) Charges

CASSCF CASPT2
Cu 0
0 1.60 -3423.30795 -3424.15109 (.85) 1.64 -.64
0 1.70 -3423.31332 -3434.14850 (.85) 1.64 -.64
0 1.50 -3423.29574 -3434.14982 (.85) 1.64 -.64
OPT 1.58 44.4 -3423.30614 -3424.15118 (.85) 1.64 -.64
0.5 1.50 -3423.27574 -3434.13412 (.84) 1.62 -.62
0.5 1.45 -3423.26685 -3434.13373 (.84) 1.61 -.61
0.5 1.60 -3423.28675 -3434.13339 (.84) 1.62 -.62
OPT 1.51 55.1 -3423.27722 -3434.13417 (.84) 1.62 -.62
1.0 1.40 -3423.25364 -3434.15289 (.86) 0.98 .02
1.0 1.50 -3423.25097 -3434.15222 (.86) 0.98 .02
1.0 1.30 -3423.24983 -3434.14576 (.86) 0.98 .02
OPT 1.44 43.1 -3423.25307 -3434.15331 (.86) 0.98 .02
2.0 1.20 -3423.25529 -3434.14201 (.86) 0.97 .03
2.0 1.30 -3423.26891 -3434.15694 (.86) 0.96 .04
2.0 1.40 -3423.26799 -3434.15665 (.86) 0.96 .04
OPT 1.35 40.12 -3423.26965 -3434.15806 (.86) 0.96 .04
3.0 1.20 -3423.25150 -3434.12814 (.87) 0.98 .02
3.0 1.38 -3423.26307 -3434.13938 (.87) 0.98 .02
3.0 1.40 -3423.26198 -3434.13830 (.87) 0.98 .02
OPT 1.34 51.05 -3423.26434 -3434.14064 (.87) 0.98 .02
10 1.20 -3423.24902 -3424.12733 (.87) 1.00 .00
10 1.30 -3423.26025 -3424.13913 (.87) 1.00 .00
10 1.40 -3423.25695 -3424.13570 (.87) 1.00 .00
OPT 1.33 51.9 -3423.26031 -3424.13923 {.87) 1.00 .oo
The legend is as in Table 4

increasing complexity in emulating the hemocyanin active site. In addition to Models 1 and
2, two other models including three NH3 or three imidazoles per copper ion (Model 3 and
Moldei 4, respectively) can be considered (Figures 12 and 13). The basis set selected for
these computations is the DZVP (EMSL web page).
The results (shown in Table 7) indicate that for Model 1, the DFT and CASPT2
approaches gives the same energetics (the triplet more stable than the singlet and a correct
evaluation ofthe dioxygen bond length). This prompted us to use the DFT approachalso for
Models 2, 3 and 4, which are computationally untractable at the CASPT2 Ievel. Our results
can be summarised as follows:
* at increasing the system size, the singlet stability also increses with respect to that
of the triplet.
* Model 2 is the minimum model required in order to obtain this trend, which is in
agreement with the experimental results (Solomon et al., 1992).
Using Model2 it is possible to study the binding mechanism ofthe oxygen molecule. In
Figure 14 and in Table 8 a cross-section of the potential energy surfaces of the triplet (T)
and singlet (S) states are computed for the 02 approaching distance. The two curves do not
show an energy barrier and both states have an electron transfer charge from the copper to
the oxygen atoms. The triplet state has a minimum at 0.75 A above the Cu-Cu plane, with an
energy value equal to that of the singlet at the same distance. The system reaches its energy

155
minimum in the plane ofthe copper atoms after an inter-system crossing and the most stable
singlet state is reached after trespassing the unique barrier due to spin change. The values of
the energetic barrier as weil as that of the final 0-0 distance ( 1. 47 A vs. 1.41 A in the
crystal) are in good agreement with the experimental data (Solomon et al., 1992).
In Figure 15 the new \j/ 1 and \j/3 orbitals obtained for Model 2 with DFT are
characterised by the presence of the nitrogen p-orbital components. It appears that \j/1 is
more stabilised by the ligands, whereas the nonbonding \j/3 is furthermore destabilised by the
nitrogen atoms. This Ieads to a big HOMOILUMO splitting and consequently to a more
energetically favoured singlet state.

SUMMARY AND PERSPECTIVES

Our results indicate that the DFT approach is expedient in treating at a high Ievel of
accuracy !arge molecular systems such as the dicopper active site of hemocyanin. At least
two in plane ligands per copper ion are necessary to simulate the electronic properties of the

Table 7. NLDA Calculations on different models ofhemocyanin.

Mod Mult E AE R r(o-o) Charges

Cu 0
s -3430.54058 0.00 0.00 1.41 1.28 -0.28
T -3430.54684 -3.90 0.50 1.39 1.21 -0.21
2 s -3657.23919 0.00 0.00 1.42 0.68 -0.38
T -3657.22053 11.70 0.60 1.38 0.60 -0.27
3 s -3770.41870 0.00 0.00 1.42 0.65 -0.37
T -3770.39744 13.34 0.60 1.38 0.58 -0.24
4 s -4788.63950 0.00 0.00* 1.41* 0.63 -0.39
T -4788.59673 26.84 0.00* 1.41* 0.58 -0.34

Mod=Model on which the computation is perfonned. Mult= spin multiplicity. E=

energy in Hartree. AE = Energy difference between singlet and triplet in Kcal/mol.

Figure 12. Model 3.

156
 Figure 13. Model 4.

0 2 4 6 8 10
R

Figure 14. NLDA: Potential energy cmve computed with Model2 for the, Sand T states. R= oxygen
approaching distance to the Cu-Cu plane.

157
 Table 8. NLDA computations on Model 2 at different approaching distances.

R Spin state E AE r(o-o) Charges

Cu1 Cu2 01 02
0 s -3657.25673 0.0 1.47 0.76 0.78 -0.46 -0.41
T -3657.21159 28.3 1.46 0.76 0.78 -0.43 -0.38
0.5 s -3657.24750 5.8 1.44 0.69 0.71 -0.39 -0.34
T -3657.23420 14.1 1.42 0.69 0.71 -0.39 -0.28
0.75 s -3657.23611 12.9 1.42 0.64 0.66 -0.32 -0.29
T -3657.23800 11.8 1.40 0.64 0.66 -0.31 -0.25
1.0 s -3657.21959 23.3 1.40 0.59 0.61 -0.26 -0.25
T -3657.23396 14.3 1.38 0.59 0.60 -0.23 -0.20
1.5 s -3657.17720 49.9 1.34 0.54 0.56 -0.18 -0.17
T -3657.20835 30.3 1.32 0.51 0.52 -0.14 -0.12
2.0 s -3657.14682 69.0 1.30 0.49 0.50 -0.11 -0.10
T -3657.18711 43.7 1.28 0.45 0.46 -0.05 -0.03
3.0 s -3657.11944 86.1 1.25 0.43 0.45 -0.03 -0.02
T -3657.17676 50.2 1.25 0.42 0.43 0.01 0.02
10 s -3657.11582 88.4 1.24 0.43 0.44 0.00 0.00
T -3657.17504 51.3 1.24 0.43 0.44 0.00 0.00

R= 02 height from the Cu-Cu plane, r(0-0)= 0 2 inter almnie distance. AE= in KcaVmol,
referred toS energy in the plane (R=O). OPT= optimised value with parabolic search.

Figure 15. The DFT \j/ 1 e \j/3 orbitals for Model 2.

molecular oxygen-copper complex at the active site. In our approximation, a NH3 molecule
is a good substitute ofthe histidine Iigand (Model 2). However with the DFT approach it is
possible to evaluate the effect ofthe increasing complexity ofthe molecule on the stability of
the singlet relative to the triplet upon oxygen binding (Model 3 and 4). It appears that the
inclusion of three ligands per copper atom, and particularly the inclusion of the histidine
rings promote an increasing stabilization of the oxygen binding at the active site. With our
study, we show that the inclusion of the electron correlation is essential for the correct
evaluation of the energetics of the copper ions in a protein active site and for the first time
describe the roJe of ligands at a quantum mechanical Ievel of computation. It is therefore
tempting to speculate that with this approach it will be possible in the near future to describe
reaction pathways common to relevant enzymes containing metal ions in the active site in
order to contribute to a better understanding of the structure to function relationship in
proteins.

158
REFERENCES

Andersson, K., Malmqvist, P.A., Roos, B.O., Sadley, A.J., and Wolinski, K., 1990, Second order
perturabation theory with CASSCF reference function, J Phys Chem 94:5484-5488.
Andersson, K., Malmqvist, P.A., Roos, B.O., 1992, Second-order perturbation theory with a complete
active space self-consistent field reference function, J Chem Phys. 96:1218.
Bernardi, F., Bottoni, A., Casadio, R., Fariselli, P., and Rigo, A., 1996a, An ab-initio study of dioxygen
binding site ofhemocyanin: a comparison between a CASSCF and DFT approach, Int J Quant
Chem 56:109.
Bernardi, F., Bottoni, A., Casadio, R., Fariselli, P., and Rigo, A., 1996b, An ab-initio study ofthe
mechanism ofthe binding porocess oftriplet 02 to hemocyanin, Inorg. Chem .. 35:5207.
Beverdge, A.J., and Heywood, G.C., 1993, A quantum mechanical stduy ofthe mechanism ofthe active
site of aspartic proteinases, Biochemistry. 32:3325.
Born, M., and Oppenheimer, R., 1927, Ann Phys 84:457.
Dirac, P.A.M., 1929, Proc Roy Soc (London) Series A. 123:714.
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Holm, R.H., Kennepohl, P. and Solomon, E.I., 1996, Chem Rev 96:2239.
Maddaluno, J., and Giessner Prettre, C., 1991, Structural and functional spects ofmetal sites in biology,
Nonempirical calculations on (cu+)2-02: a possible model for oxyhemocyanin and oxytyrosinase
acitve sitesjnorg Chem 30:3439.
Magnus, K., Hazes, B., Ton-That, H., Bonaventura, C., Bonaventura, J., Hol, W.G.J., 1994,
Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows
unusual differences, Proteins Struc Fun Gen 19: 302.
Malmstrom, B.G., 1994, Back-induced bonding in blu-copper proteins, EurJ Biochem. 223:711.
Pullman, B., and Pullman, A., 1963, Quantum Biochemistry. Wiley & Sons.
Rosi, M., Sgamellotti, A., Tarantelli, F., Bertini, I., Luchinat, C., 1986, Ab-initio calculations ofthe Cu2+_
0 2 interaction as a model for the mechanism of copper/zinc Superoxide dismutase, Jnorg Chem.
25:1005.
Ryde, U., 1996, The coordination chemistry ofthe structural zinc ion in alcohol dehydrogenase studied by
ab initio quantum chemical calculations, Eur BiophysJ. 24:213.
Ryde, U., Olsson, M.H.M., Pierloot, K., Roos, B.O., 1996, The cupric geometry ofblue copper proteins is
not strained, J Mol Bio/ 261: 586.
Solomon, E.D., Baldwin, M.J., and Lowery, M.D., 1992, E1ectron structures ofactive sites in copper
proteins: contribution to reactivity, Chem Rev 92:521.
Solomon, E.I., and Lowery, M.D., 1993, Electron structure contributions to function in bioorganic
chemistry, Science 259:1575.
Szabo, A., and Ostlund, N.S.,. 1989, Modernquantum chemistry, McGraw-Hill.
Unichem, 1994, Chemistry codes: DGauss, Version 2.1 and version 2.3.1. Cray Research Inc.
Voet, D., and Voet, J.G., 1995, Biochemistry, second edition, John Wiley and Sons, Inc. New York.
Volbeda, A., and Hol, W.G.J., 1989, Crystal structure ofhexameric haemocyanin from panulirus
interruptus refined at 2.3 A resolution, J Mol Bio/. 209:249.
Warshell, A., 1991, Computer modelling ofchemical reactions in enzymes and solutions. Wiley & Sons.
Williams, P.J.R., 1995, Energised (entatic) states of groups and of secondary structures in proteins and
metalloproteins, EurJ Biochem. 234:363.
Ziegler, T., 1991, Approximate density functional theory as a practical tool in molecular energetics ands
dynamics, Chem Rev 91 :651.

159
ELECTRON TRANSFER REACTIONS IN MULTICOPPER OXIDASES

Lilia Calabrese

Department ofßiology
University ofRoma TRE
Rome, Italy

INTRODUCTION

The copper ion is essential for all biological systems and, with the only exception of
storage proteins, all copper proteins are involved in electron transfer (ET) processes.
Electron transfer in proteins shares common features with that occurring in small molecules,
however some features distinguish the two systems. Small molecules exchange electrons in
solution at dose proximity or direct contact to afford inner sphere mechanisms. In proteins
redox partners are held in fixed positions and in case of long donor-acceptor distances for
long range electron transfer, the intervening medium is the protein matrix. ET in proteins is
characterized by a weak interaction between electron donors and acceptors, prevented from
coming in contact by the polypeptide chain. Different evolutionary mechanisms have evolved
to overcome this hindrance in the different dasses of proteins. The blue multicopper
oxidases use a structural motif of the polypeptide chain to provide efficient electronic
coupling between two redox partners, a mononudear copper site, referred to as the blue
site, and a trinudear copper site. Blue sites have evolved in blue copper proteins as fast one
electron transfer device, while the trinudear duster is specially suited to oxygen binding and
reduction. The efficient catalytic function of multicopper oxidases results from the
synergistic use of these two sites in an intramolecular electron transfer process which
couples one electron oxidation of substrates to four electron reduction of dioxygen.
Therefore they are among the few enzymes that utilize the full oxidizing capacity of oxygen,
which is reduced to two water molecules.

THE STRUCTURE OF MULTICOPPER OXIDASES

The multicopper oxidases are a dass of enzymes that can be defined by the unique
spectroscopic properties and reactivity of their copper sites (Malkin and Malmstrom, 1970;
Malmstrom 1997). All contain at least one blue site, the primary acceptor of electrons from
substrate, and one oxygen reducing site, the trinudear duster. The better defined

Biophysics (!(EI<•ctron Tran,fer and Molecu/ar Biol'iectronics

Edited hy C. Nicolini, Plenum Press, New York, 1998 161
multicopper oxidases are ceruloplasmin(CP}, ascorbate oxidase (AAO) and laccase (LC).
They are characterized by extensive sequence homology and distinctive subdomain structure.
AAO and CP are structured in multiple domains, three and six respectively.The blue copper
is located in intradomain site(s), one in domain 3 of AAO, three in domains 2, 4, and 6 of
CP. The trinuclear duster is an inter-domain site, which is formed by domain 1 and domain 3
in AAO, and by domain 1 and domain 6 in CP. Each domain is structured with the
cupredoxin fold, an eight-stranded Greek key ß-barrel typical of cupredoxins, small single
copper blue proteins, such as plastocyanin or azurin (Figure 1}.

Figure 1. The structure ofthe plastocyanin molecule and ofthe Tl copper binding site. The copper is
depicted as a black circle together with its two His, one Cys and one Met, ligands. The geometry ofthe
copper site is shown to the right.

Multicopper oxidases have evolved from an ancestral two-domain protein, the product
of gene duplication of a cupredoxin (Ryden, 1988}. They followed different evolutionary
pathways, which led, through the selective loss of some intradomain copper sites, to the
diverse functionalities of nowadays proteins. The widely distributed Iaccases are 60-80 kDa
molecules and catalyze either oxidative polymerization of cathecol derivatives of Iatex, in
some plants, or Iignin degradation in fungi (Reinhammar, 1997). Ascorbate oxidase, is a
homodimer of 70 kDa, three-domains, subunits. It performs a still unknown function in
higher plants by oxidizing ascorbate to semidehydroascorbate radical, which then
disproportionates to dehydroascorbate (Messerschmidt, 1997). Ceruloplasmin, 130 kDa, is
the most complex among blue oxidases (Ryden 1984). Beside the minimal functional unit, the
four copper atoms of the blue and trinuclear sites, it contains two additional blue copper
centers. Although it is capable of oxidizing a variety of organic compounds, the
physiologically relevant substrate is Fe(II}, which is oxidized to Fe(III) for a better uptake by
iron transporter(s) (Huber and Frieden, 1970}. Ceruloplasmin is· ubiquitous among
vertebrates and is found either in plasma, as soluble protein, or in some areas of the brain,
anchored to the cell membrane (Pate! and Oavid, 1997}. Its complete absence in humans,
resulting from mutations of the gene, Ieads to diabetes, retinal degeneration and neurological
disease with concomitant tissue iron accumulation (Harris et al. , 1995)

Spectroscopic Properties of Copper Sites

Spectroscopy has played a major roJe in the understanding of copper centers of

multicopper oxidases and their interactions with substrates. Multicopper oxidases contain a
combination of three different classes of Cu centers. Differences in spectral features defines

162
 these classes, or Types, of copper sites which exhibit different reactivity (Solomon and
Lowery, 1993). Type l(Tl) has very intense absorption at 600 nm (e = 5000 M-1 cm-1), and
unusually narrow hyperfine coupling in EPR spectroscopy. The copper is coordinated by
three strong ligands and one or two weaker ligands. Type 2(T2), "normal copper", has much
weaker absorption, broader hyperfine interactions and is generally coordinated by four or
five ligands, histidines and water molecules. Type 3(T3) is a pair of copper atoms
antiferromagnetically coupled, usually coordinated by three histidines and a bridging Iigand.
High resolution X-ray crystal structure of AAO (Messerschmidt et al., 1992) and CP
(Zaitseva et al., 1996) has recently confirmed that blue oxidases combine the T2 copper to
the T3 couple in a common site, to form the trinuclear duster, T2/T3, necessary for binding
and reducing dioxygen. This result is in agreement with the concept of the close proximity of
these sites as anticipated by the results of several spectroscopic studies performed on these
proteins.

A B
0.9

w
0
z
:i 0.6
a::
0
tJ)
ID
ct 0.3

400 500 600 700

NANOMETERS TESLA
Figure 2. Spectroscopic properlies of ceruloplasmin. (A), optical spectra of chicken, (a), and sheep, (b), CP.
(B) EPR spectra of CP at the end ofthe purification procedure, (a), and after storage and manipulations, (b).
(Reprinted from Ref Calabrese et al. , 1988. With permission)

The absorption spectrum of multicopper oxidases (Figure 2,A) has two dominant
features, an intense band at ca. 600 nm and a less pronounced absorption at 330 nm. The
band at 600 nm is the spectroscopic signature of the T 1 site and obscures those of other
sites, which also contribute in this region but have much lower intensity. The 330 nm band is
associated with the oxidized T3 center. The EPR spectra of laccase and AAO exhibit the
Superposition of contributions from the Tl and the T2 site. Since the pair of copper atoms is
strongly antiferromagnetically coupled, T3 does not contribute to EPR spectrum. The
consequence is that the fraction of copper detectable by EPR is only 50%, with equal
contributions from Tl and T2. The EPR spectrum of CP is different. Based on a
stoichiometry of six copper atoms per molecule, in human CP, and the presence of three T 1
sites and of one T2/T3 site, the fraction of the EPR detectable copper should approach a
value of ca. 70%. However such a value can hardly be reached in native protein, it rather
appears typical of the denatured protein. The state of copper atoms in ceruloplasmin has
been the matter of a big debate, this holds in particular for the T2 copper. The operational
definition ofthe Type 2 copper in trinuclear enzymesisthat it is the EPR-active center ofthe
T2/T3 duster. In a search Iasted 10 years we measured hundreds of CP specimens from

163
different sources, isolated by a very rapid single-step procedure, and invariably found only
the resonances of blue copper in the EPR spectrum (Figure 2, B). A signal with magnetic
parameters typical of T2 copper, the spectroscopically "normal" copper, was discernible
only when samples were subjected to manipulations (Calabrese et al., 1989) or when plasma
had been withdrawn from individuals over 60 years (Musci et al., 1993). Same results were
obtained with on ceruloplasmins with a content of only five copper atoms per protein
molecule, isolated from different organisms, other than humans (Calabrese et al., 1988). The
observation that chloride elicits an increase of the extinction coefficient at 600 nm (Musci et
al., 1995) and the results of anaerobic reduction experiments and computer simulations of
EPR spectra ( Musci et al., 1993) led us to the condusion that the state, and not only the
stoichiometry, of copper atoms of ceruloplasmin is different with respect to other blue
oxidases. First of all T2 copper of ceruloplasrnin is EPR inactive, in the resting enzyme.
Unless one ofthe three T2/T3 copper atoms is reduced, and assuming that antiferromagnetic
coupling is possible between more than two coppers in the T2/T3 duster, unusual magnetic
interactions operate in the dioxygen reducing site of CP. Moreover the three Tl sites are
unequivalent, and a fraction ofthem stays permanently reduced.

Geometry of Copper Sites

Crystallographic data on AAO ( Messrschrnidt et al., 1989) and on human CP (Zaitseva

et al., 1996) point to a structural sirnilarity of the blue sites and of the T2/T3 duster. Blue
centers in domain 3 of AAO, (Figure 3), and domains 4 and 6 of CP, (Figure 4) all have the
four canonical Tl ligands (His-N, Cys-S, His-N, Met-S), in the coordination best described
as trigonal bipyrarnidal also found in plastocyanin (Figure I, 8) , with the methionine sulfur
bond quite long, 3 A, and the cysteine sulfur bond quite short, 2 A (Solomon and Lowery,
1993). Studies on small blue proteins have shown that much of the electronic character of
the Type I copper is due to the fact that this copper is bound to the electron-rich cysteine
thiol through a very strong, highly covalent bond, and has a tetrahedral rather than the
tetragonal geometry preferred by normal Cu complexes (Holm et al., 1996). These are the
basis for the unusually narrow parallel Cu hyperfine splitting of the EPR spectrum and of the
intense low-energy charge transfer transition, the band at 600 nm.

Figure 3. The structure ofthe AAO molecule. (A), schematic representation ofthe structure of AAO
subunit. The copper atoms are depicted as black circles. (B), structure of the copper binding sites. T 1 site is
shown on the top, together with its two His, one Cys and one Met ligands. The T2ff3 site is shown to the
right.

164
 The relatively high reduction potential of blue proteins have been attributed to the
distorted tetrahedral geometry and to weaker donor interactions at the axial Iigand which
destabilizes Cu(II) relative to Cu(I). The mononudear copper of domain 2 of CP Iacks the
axial methionine which is replaced in the sequence by leucine. This non coordinating residue
is also present in blue sites offungallaccases characterized by higher redox-potential among
blue sites (Reinhamrnar, 1972).
Cristallographic results on AAO give a dear picture of the trinudear duster (Figure 3,
B) and ofhow it changes upon reduction or with a peroxide ligated, that can help to disdose
how the oxidase binds and activates the oxygen. The dose proximity of T2 and T3 within
the duster appears not mediated by comrnon (bridging) ligand(s). T2 Cu has a peculiar
geometry since it appears only three coordinate, to two histidines and an OH group, this
latter is oriented away from the duster. The two T3 Cu have three His and are bridged by an
OH group, responsible for the strong antiferromagnetic coupling. The two T3 coppers loose
their bridge upon reduction, move away from each other and become three coordinate.The
eight histidines occur in a highly conserved pattern of four HXH motifs. In one, X is the
cysteine bound to Tl copper and each of the histidines binds to one of the three T3 coppers.
The disposition of the trinudear duster and the nearest Tl copper, that of domain 6, of
ceruloplasmin (Figure 4, B) is dosely similar tothat found in the subunit of AAO. However,
the structure of the duster of CP is less defined, due to Iimitation in resolution (3 A). The
overall organization appears as that of AAO, however distances between copper atoms are
slightly different, and the presence of further ligand(s) in between copper atoms cannot be
discounted.

Figure 4. The structure of the CP molecule. (A), schematic representation of cp structure showing its
partition into six domains. (B), structure of copper binding sites. The trinuclear cluster is shown on the
bottom. The three Tl sites areshownon the top and are Iabeted according to the intradomain localization.

ELECTRON TRANSFER REACTIONS

The functional relationships between copper sites of blue oxidases have been analyzed
by a variety of spectroscopic studies. Transient kinetics has been applied to monitor the
different steps during the catalytic cyde. In the presence of both substrates, complex
patterns have been observed, therefore transient kinetics was carried out in the presence of

165
only one substrate in each case. Thus observations have been performed on the anaerobic
reduction of the enzyme and, thereafter on the reoxidation of reduced enzyme by oxygen.
When static anaerobic reduction of CP by ascorbate is followed by optical spectroscopy,
both chromophores, 330 and 600 nm, are reduced (Figure 5, A), the T3 site is apparently
fast er than Tl. When less than one electron per Cu atom is fed into the protein and the
reaction is performed by stopped flow technique (Figure 5, B), the 610 nm band decreases in
few seconds, then, when the absorption at 340 nm begins to decrease, it shows an increase
that parallels the disappearance of the 340 nm. Upon reoxidation of reduced samples by
oxygen the process takes place in the opposite direction (Carrico et al., I97I).

% A B
100 0.025

GI
75 g 0.002
ca
.c
50 0
Ul '
~ 0.015 ',,,
25 ...............

0.01
,_
---
o+---~~~~==~~ 0~---5~0----~
10
~0~--
1-50~--2~0-0~
0 25 50 75 100
Time (sec)
Time (min)
Figure 3. A: time courses of the optical absorbances at 330 nm, triangles, and at 600 nm, circles, and of
EPR detectable copper, squares, during the anaerobic reduction of ceruloplasmin. B: Partial anaerobic
reduction of ceruloplasmin by 0.6 electron equivalent of ascorbate. The solid and dashed lines refer to 610
and 340 nm absorption, respectively. The arrows mark the final absorption values for the closer curve. (B,
reprinted from Ref. Carrico et al., 1971. With permission)

Such early observations have suggested that the blue Type I is the entrance site for the
electrons into the enzyme and that the Type 3 is the oxygen reducing site. They also support
the hypothesis of a linear array of the redox center which undergo sequential reduction.
According to this hypothesis three kinds of electron transfer processes are involved in the
catalytic cycle of blue oxidases. The intermolecular electron transfer from reducing substrate
to TI site is followed by long-range intramolecular electron transfer (LRET) from TI site to
T3 site. Electrons are then redistributed within the duster to reduce bound dioxygen. There
are however many differences in the behavior of the oxidases and this may relate to protein
differences and, probably due to these discrepancies, many questions have remained
unresolved. One prominent question is related to oxygen-binding and to the oxygen
intermediates that are bound to the cluster du ring reduction of oxygen. Another is the roJe of
Type2, and this is related to the question ofwhether sites other than the blue site coupled to
the cluster can accept electrons directly from substrate.

Intermolecular Electron Transfer

The function of the blue sites in shuttling electrons from substrates to the clusters is
weil documented, and it is generally accepted that these sites have been recruited during
evolution as "fast centers" for electron transfer. An extensive research on the unique
properties of these sites, that are critical to their function, has attempted to clarify, at
experimental and theoretical Ievel, how in blue proteins the protein structure provides a site
with exceptionally enhanced reactivity in ET. Recent progress in crystallography and site-
directed mutagenesis on small blue proteins have documented that there are minimal

166
structural changes in Tl sites, upon reduction and reoxidation; in plastocyanin the copper-
ligands distances changes only slightly and the angles hardly change at all (Guss et al.,1986).
This is due to the fact that geometry of Tl sites is intermediate between the optimal
geometry of Cu(I), tetrahedral, and that of Cu(II), tetragonal. Therefore it has been
proposed that the protein moiety force the Cu(II) ion into the geometry of the Cu(I) to gain
a lessened extent of structural rearrangement. A small change in geometry gives, in the
frame ofMarcus theory (Marcus and Sutin, 1985), a small reorganization energy and thus a
high rate of electron transfer. The entatic state theory (Williams, 1995) and the induced -
rack theory (Malmstrom, 1994) have explained this phenomenon as a generat mechanism for
metalloproteins, which impose unusual geometry at the metal site to exert a strain that force
the metal into catalytically poised state. Other evidence disfavors the hypothesis of a strained
geometry. Results obtained on the optimal vacuum structure ofmodels ofblue sites have led
to the conclusion that blue proteins minimize the electron transfer reorganization energy
simply by an appropriate choice of metal ligands, in particular the cysteine thiolate(Ryde et
al., 1996).
The electron transfer from reductants on the TI sites of multicopper oxidases takes
place in bimolecular second order reactions with rates that depend primarily on the nature of
reductant, and on many other factors that can influence sites conformation, such as pH. The
redox-potential ofT1 sites of AAO (Kawahara, 1985) and tree laccases (Reinhammar, 1972)
are in the range of 320-350 mV, comparable to values determined for small blue proteins.
Taking into account the redox potential of the couple ascorbate/dehydroascorbate radical
the difference allows an estimate of a driving force of ca. 50 mV for the oxidation of
ascorbate. The bleaching of Tl site of AAO by ascorbate is very fast, the reaction is
assumed to proceed with a rate higher than the tumover number of the enzyme. In a
modeled encounter complex the ascorbate goes in proximity of a Iigand His, at a distance ca.
7 A from Cu, and a parallel arrangement of rings and good overlap 1t-electron density system
facilitates, the outer-sphere mechanism of a rapid electron transfer (Messerschmidt, 1992).
The reduction ofblue sites by Fe(II) is the fastest step (1.2x I0-6 M-1 s·l) in the redox
cycle of ceruloplasmin (Osaki and Walaas, 1967). Kinetic evidence for the formation of an
enzyme-substrate complex have been obtained in these early sturlies which are consistent
with recent findings of labile meta! binding sites in the crystal structure of the protein
(Lindley et al., 1997). These sites appears as islands of negative electrostatic potential values
in close proximity of Tl copper sites of domains 4 and 6 (Musci et al.l998). Such an
electrostatic potential minimum is not observed on the copper site of domain 2, which also
Iacks the methionine Iigand. Our idea is that this site stays permanently reduced in the native
protein and only enters in the redox cycle when chloride binds to the cluster. Cu of domain 4
and 6, although having the same geometry and Iigand set, display different spectroscopic
properties and redox potential values, 480 and 540 mV (Deinum and Vanngard, 1973), both
higher than those ofTI sites oflaccase and AAO. Calculations ofthe electrostatic effect of
the protein moiety on their relative redox-potentials explain that Cu of domain 6 should
have ca. 100 mV higher potential than that of domain 4. Results of Brownian dynamics
simulations shows that electrostatic potential drives Fe(II) diffusion preferentially towards
domain 6 such as this site was the preferential route of entry of electrons and/or a sink
collecting electrons from a reduced Cu of domain 4 (Musci et al., 1998).

Intramolecular Electron Transport

Long Range Electron Transfer in proteins is characterized by a weak interaction

between electron donor and acceptor, in this case the rate constant is proportional to the
square of the electronic coupling between the electronic states of donor and acceptor,
reflecting orbital overlap between sites, in the form of a matrix element HoA (Holm et al.,

167
1996). Both theoretical and experimental studies now demonstrate that the structure and
composition of protein medium intervening between donor and acceptor must be induded in
the analysis of HoA· The rate of electron tunneling through the protein is expected to decay
exponentially with increasing distance altematively specific superexchange pathways for the
electron through the protein can be considered, where the total electronic coupling is a
product of contributions from individual steps (Beratan et al., 1990). In this model the
protein medium is divided in small subunits Jinked by covalent bonds or hydrogen bonds or
through space contacts and each type of links is assigned a coupling decay factor. Thus one
electron propagating through a covalent bond is attenuated by ca. 0.6 while a hydrogen bond
is nearly effective as two covalent bonds. In order for a meta! center to couple into such
electron transfer pathways there must be covalency in the Iigand-metal bond and anisotropy
in this covalency (Lowery et al. , 1993). Given that covalency correlates with charge-transfer
transition at that Iigand, this should allow efficient coupling into Superexchange pathways.
As already described AAO, CP and laccase are all characterized by the structural motif
His-Cys-His, where the cysteine Iigand of the Tl site is flanked by histidines ligands of the
duster. The crystal structures of AAO and CP shows that this motif connects the two redox
center at a distance of ca. 13 A and fumishes a bifurcate pathway of electron transfer (Figure
6). A likely through-bond ET pathway proceeds from the TlCu ligating Cys 509 to either
His-508 or His-51 0 ligands of T3 coppers in AAO, the same situation is found for the Tl Cu
of domain 6 in CP. A theoretical pathway analysis (Kyritsis et al., 1992) indicate that the
most favoured routeisthat through Cys 507-His 506, wich indudes a hydrogen bond, since
it gives approximately three times more efficient electronic coupling. Detailed spectroscopic
and theoretical analyses of blue centers of plastocyanin and AAO by Solomon and
coworkers (1996) demonstrate that the unique electronic structure of the blue site reflects a
ground-state wave function that provides in both cases the same, highly anisotropic covalent
pathway involving cysteine sulfur. The covalency activates this residue for directional ET.
The low energy Cys-S to Cu CT transition provides a hole Superexchange mechanism for
electron transfer between T 1Cu and T2/T3 in multicopper oxidases, and a surface patch in
plastocyanin where a Tyr is covalently linked to the Iigand cysteine.

Figure 6. Proposed intramolecular electron transfer pathway in blue oxidases. The two covalent pathway,
from Tl Cu to the T3Cu couple, are shown for AAO, (a), and for CP, (b)

The question of whether this pathway and the structure of the trinudear duster can
exert a control of the ET process and, whether 0 2 binding to the duster modulates ET rate,
has been addressed by several different approaches. The anaerobic reduction of the
trinudear copper species, as monitored by the bleaching of the 330 nm band, the

168
reappearance of the 600 nm band and the behavior of the T2 EPR signal, appears to be a
multiphasic process in most cases. The unimolecular rate constant values, :::; 102 s·l,which
have been obtained in these studies, are one or two orders of magnitude lower than those
expected on the basis of the measured k.., values, representative of tumover numbers
(Messerschmidt, I997, and references therein). It should be pointed out that, in order to be
consistent with the tum-over kinetics these rates should be at least as )arge as the turn over
numbers.

Electron Transfer Within the Cluster

During the reoxidation process several, different, oxygen intermediates have been
identified and characterized. These findings lend to the condusion that oxygen binds to the
trinudear site and to the hypothesis that, during the catalytic cyde, this site may store three
electrons and transfers them to the bound dioxygen, followed by a final one- electron
transfer. More recently it has been shown that the 0 2 coordination to duster is a key factor
controlling the LRET rate. An enhancement of this rate has been observed in aerobic vs.
anaerobic reduction, which has been ascribed to a I 00 mV increase of the reduction
potential of the T3/02 complex with respect to that of T3. (Farver et al., 1994). The
reorganization energy for this LRET has been also calculated, on the basis of the activation
parameters determined for AAO. Using a through-bond distance of 13 A between Tl Cu
cysteine-thiolate bond and one imidazole nitrogen coordinated to a T3-Cu, a value of I 53 kJ
moJ·l was obtained, considerably higher than that of 99 kJ moJ·l calculated for
intramolecular ET of a small single copper containing blue protein, azurin (Mikkelsen et al.,
I993). The changes ofthe structure ofthe trinudear duster upon reduction are the obvious
reason of this difference. As already described there is a considerable local conformational
change in the three-dimensional structure of reduced AAO (Messerschmidt, I993). The
distances between copper atoms change and, more important, the antiferromagnetic coupling
is disrupted. More marked changes occur when dioxygen binds to duster and intermediates
are formed. However there is a complete disagreement between crystallography and
spectroscopy since the two techniques propose a very different picture of how the duster
can cope for oxygen complete reduction. The peroxide, a two-electrons reduction
intermediate of dioxygen, might either be bound as a bridge between T2 and one of the T3
coppers, according to spectroscopic, in solution, studies (Cole et al., I991), or altematively,
as proposed by crystallographic data on a peroxide adduct (Messerschmidt, I993) it might be
bound to only one T3 copper.

The Catalytic Mechanism of Blue Oxidases

The fully reduced and the peroxide derivatives are considered two important
intermediate states during the catalytic cyde of blue oxidases. Early proposals for the
catalytic mechanism were limited by the poor knowledge of the structure and spatial
arrangement of copper centers in the various states of the catalysis. More detailed catalytic
reaction schemes have been now formulated, for AAO and laccase, incorporating this new
information. As proposed by Messerschmidt and co-workers (I993) for AAO, the first step
is the reduction of TI Cu by one-electron transfer step, the electrons are transferred trough
the protein to the duster, by a through-bond or a through-space (or both) mechanism. When
four electrons have been taken up by the protein, the enzyme is fully reduced. Binding of
dioxygen and concomitant transfer of two electrons from T3 coppers induces a
hydroperoxide adduct which is held by a T3 copper atom for subsequent reduction. A first
water molecule is released when the 0-0 bond is broken by a third electron transferred by
the T2 reduced copper. Now an oxygen radical is formed that is released after further

169
reduction of Tl Cu and transfer of this electron to the oxidized T3 copper atom. This latter
transfers the fourth electron to the radical and a second water molecule is formed. The water
may remain bound, as a bridging Iigand, between the two T3 coppers in the case of single
turnover, then the enzyme goes to the resting form. Ifturnover is continued the water will be
released, and the duster will maintain a structure very dosetothat ofthe reduced form.
Solomon and co-workers (I996) have proposed a catalytic cyde for laccase based on
the different structure of the peroxide intermediate identified by spectroscopic techniques. In
this scheme the peroxide intermediate Ieads, upon rupture of the 0-0 bond and water
release, to a "native intermediate", where an OH group is bridging T2 and one T3 copper.
Two possible mechanisms for the reduction of the trinudear duster are outlined in this
reaction scheme. The Tl and the T2 sites together reduce the T3 pair or, alternatively, each
copper of the trinudear duster is sequentially reduced by electron transfer by TI.
In a proposal for the catalytic mechanism of ceruloplasmin, the presence of three
different Tl sites has to be taken into account. As shown by crystallography (Lindley et al.,
I997) the Cu of domain 6 is coupled to the duster through the Cys-His pathway. The
reduction of this copper is likely to be followed by transfer to the duster through Cys I 02I
and His I020 and /or I022. Both Cu of domain 4 and of domain 2 Iack the two histidines
flanking the Iigand cysteine, thus it is not dear whether and how these sites participate in the
electron transfer to the duster. A pathway from Cu of domain 4 could involve Cu of domain
6 or only the protein moiety, for a direct transfer to the histidines ligands of T3. The
difference in the redox potential of the two sites, as previously discussed, suggests that the
Tl of domain 6 may shuttle electrons from domain 4 to the duster. The roJe of the third Ti
copper is undear. Unfavorable electrostatic factors add to the Iack of axial methionine of
this site, making it possible that it is the blue center permanently reduced in the resting
enzyme (Musci et al., I993). We have shown that upon binding of chloride human
ceruloplasmin undergoes structural changes that Iead to displacement of an electron from
this site to the T3 pair, and that chloride is able to enhance up to ten fold the catalytic
activity (Musci et al.I995). Since chloride is present in the plasma at relatively high
concentration we have conduded that ceruloplasmin activity, in vivo, is under the control of
this anion.

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Guss, J. M., Harrowell, P. R., Murato, M., Norris, V. A., and Freeman, H. C., 1986, J. Mol. Bio/., 192:361
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Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A.,
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Messerschmidt, A., 1997, inMulti-Copper Oxidases, A. Messerschmidt, ed., World Scientific, Singapore
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Muscf,G., Bonaccorsi di Patti, M. C., Fagiolo, U., 1993, J. Bio/. Chem., 268:13388
Musci, G., Bonaccorsi di Patti, M.C., and Calabrese, L., 1995, J. Prot. Chem., 14:611
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and Ce/lu/ar Aspects, A. Leone and J. F. B. Mercer, eds.,
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Ryden, L., 1988, in Oxidasesand Related RedoxSystems, T. S. King, H. M. Mason and M. Morrison, eds.,
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J. Bio/. Jnorg. Chem., 1:15

171
THE OPTICAL BIOSENSOR STUDY OF PROTEIN-PROTEIN INTERACTIONS
WITHIN CYTOCHROMES P450 CONTAINING MONOOXYGENASE SYSTEMS

Alexander I. Archakov, Yuri D. Ivanov

Institute ofBiomedical Chemistry RAMS, Moscow, Russia, 119832

INTRODUCTION

The cytochromes P450 - containing liver microsomal monooxyganase systems play an

important roJe in the oxidation of drugs, toxins, carcinogens, mutagens and other
xenobiotics (Archakov and Bachmanova, 1990). It is known that microsomal cytochromes
P450 (P450), in particular P450 2B4 (2B4), function by interacting with their redox partners
- NADPH-cytochrome P450 reductase (Fp) and cytochrome b5 (b5). Mitochondrial
cytochromes P450 (particularly P450scc) hydroxylate cholesterol by acquiring electrons
from adrenodoxin (Ad), which in turn accepts them from adrenodoxin reductase (AdR).
Similar mechanism operates in the case of bacterial P450s (for example, P450cam), whose
electron donor is putidoredoxin (Pd), which acquires electrons from putidoredoxin reductase
(PdR). In some studies, protein-protein interactions were explored by the the spin
equilibrium shift (Backes et al., 1985; Tamburini et al., 1985; Lapko et al., 1991; Gerberand
Sligar, 1994) and the fluorescence quenching techniques (Davydov et al., 1996), based on
which the affinities and kinetic constants of P450 complexes were determined. In other
studies, kinetic electron transfer rate constants for interprotein electron transfer were
reported (Cush et al., 1993; Tamburini et al., 1985; Eyer and Backes, 1992; Kanaeva et al.,
1992; Wu et al., 1994; Voznesensky and Shenkman, 1992; Sevrukova et al., 1994; Gerber
and Sligar, 1994; Lambeth and Kriengsiri, 1985; Lambeth and Kamin 1977; Nakamura et al.,
1994). In present-day molecular interaction researches the ever-widening application is
finding the optical biosensor method. lt enables to study molecular interactions without
protein Iabeling in real time (Johnsson et al., 1991; Cush et al., 1993; Davies et al., 1994;
Yeung et al., 1995). The method was widely used in studies on complex formation kinetics
ofwater-soluble molecules (Shymko et al., 1994). In our biosensor-based studies, the kinetic
constants for complex formation of membrane proteins 2B4, Fp, b5 were determined. The
native environment of these proteins is the phospholipid bilayer membrane to which the
proteins are anchored. The rate constants determination in its presence is rather difficult. To
obviate this difficulty, in our Iabaratory the soluble monooxygenase system containing hemo-
and flavoprotein monomers was reconstituted in the absence of phospholipids (Kanaeva et
al., 1992). To elucidate the mechanisms whereby this system is operated, it was necessary to

Biophysics of Electron Transfer and Molecu/ar Bioelectronics

Edited by C. Nicolini, Plenum Press, New York, 1998 173
determine the redox partners' assoctatton and dissociation rate constants during their
complex formation and to compare the values obtained with the appropriate rate constant
values for electron transfer and hydroxylation. Redox partners interactions are accompanied
by formation of productive or unnproductive complexes - with intermolecular electron
transport either occurring or not occurring, respectively. Employing the optical biosensor
method, it was possible to measure the total nurober of complexes formed - both productive
and unproductive. Obviously, for effective protein-protein electron transport to occur the
life-time of a bimolecular complex must be higher than the interprotein electron transfer
time. Comparison of kinetic parameters obtained by the optical biosensor method as
employed in this study with electron transfer rate constants for the complexes formed
enabled us to estimate the proportion of productive complexes in reference to the overall
nurober of complexes formed. Analogaus approach was taken to determine the proportion
of productive complexes in the P450scc system containing membrane-bound and water-
soluble proteins, as well as for the water-soluble P450 cam system.
Full-length enzymes- cytochrome bS (d-bS), NADPH-cytochrome P450 reductase (d-
Fp) and cytochrome P450 2B4 (d-2B4) - are amphipatic proteins containing hydrophobic
membrane fragments capable of being incorporated into the Iipid bilayer. These fragments
may be removed by tryptic digestion- with the aim to obtain truncated proteins t-bS or t-Fp
(Omura and Takesue, 1970) or by gene expression - to stimulate t-2B4 production
(Lehnerer et al., 1995). Based on comparison of the biosensor-obtained complex formation
parameters for the full-length and truncated forms of 2B4, bS and Fp, the dominant roJe of
hydrophobic membranaus tails in productive complex formation was demonstrated.
The cytochrome P450scc-containing system includes the membrane protein P450scc
itself and soluble proteins - AdR, Ad. The P450cam-containing system includes only water-
soluble proteins PdR, Pd and P450cam itself.

EXPERIMENTALPROCEDURES

Chemicals. Chemicals. Glucose oxidase, xanthine, xanthine oxidase were purchased

from Serva (Heidelberg, Germany). Catalase, superoxide dismutase, NADPH were obtained
from Boehringer Manheim (Vienna, Austria). Emulgen 913 was purchased from Kao Atlas
(Osaka, Japan). 1- Ethyl-3 - (3-dimethylaminopropyl) carbodiimide and N-hydroxy
succinimide were obtained from Fisons (Cambridge, UK). Other chemieals were purchased
from Reakhim (Moscow, Russia).
Preparation of proteins. Phenobarbital- induced rabbit liver microsomes, d-2B4, d-Fp
and d-bS were prepared as described earlier (Karuzina et al., 1979; Kanaeva et al., 1985;.
Spatz and Strittmatter, 1971). Specific content and specific activity of d-Fp were 13-13.5
nmol Fp/mg protein and 40-43 nmol of cytochrome c/(min*mg)" 1 of protein at 30°C,
respectively. Specific content ofd-2B4 was 17-18 nmoVmg ofprotein and A276nmAusnm=0.5.
Specific content of d-5 was 48-52 nmoVmg of protein, A276nml~I4nm=0.6. Purified tryptic
fragments of b5 and Fp were obtained from rabbit liver microsomes following their
treatment with trypsin (Omura and Takesue, 1970). The proteins showed a single band on
SOS-PAGE. Truncated cytochrome P450 (t-2B4) lacking N- terminal residues 2-27 was
kindly presentedby Dr. P. Hlavica. Specific content oft-2B4 was 15 nmoVmg ofprotein.
Monomerization ofaggregates ofthe isolated Fp and 2B4 was carried out as described
previously (Kanaeva et al., 1992) except that in some cases instead of 100 mM K-phosphate
buffer (KP buffer), the 500, 50 mM KP buffers and 100 mM Na-HEPES (HEPES) were
used. Monomerization of aggregates of isolated d-b5 was carried out as follows: to 5 nmol
ofprotein 13 jJ.l of2% (w/v) Emulgen 913 solutionwas added. After the incubation for 10
min at room temperature, the concentrations of cytochrome d-bS and Emulgen 913 were

174
brought to 5 J.lM and 0.25 g/1, respectively, with 500 mM phosphate buffer, pH 7.4, and the
mixturewas incubated at 4°C for 24 h.
P450scc, Ad and AdR were kindly presented by Dr. S.A. Usanov; they were prepared
from bovine adrenal cortex mitochondria by use of special affinity adsorbents (Lepesheva
and Usanov, 1997)
P450cam, Pd and PdR were a gift from Dr. Hui Bon Hoa; they were expressed in
Escherichia coli, strain TB, isolated and purified to homogeneity as described elsewhere
(Jung et al., I992; Gunsalus and Wagner, I978).
The determination of proteins' binding parameters by optical biosensor. The "resonant
mirror" biosensor method is direct, i.e. it permits immediate real-time measurement of the
total amount of complexes formed. The principle and instrumentation of the resonant mirror
sensor have been described elsewhere (Jonsson et al., I99I; Cush et al., I993; Davies et al.,
I994). Determination of redox partners' binding parameters within the monomeric
reconstituted system was carried out in the IAsys biosensor cell (FISONS, UK) by
covalently immobilizing one of the partners, with the use of standard coupling chemistry
methods, to the carboxymethylated dextran - coated cuvette. The chemistry involved
coupling of Iigand amino groups to the carboxyl groups on dextran via I- ethyl-3 - (3-
dimethylaminopropyl) carbodiimide (EDC)/N- hydroxy succinimide (NHS). The sample cell
(volume 200 J.ll) was brought to thermal equilibrium and incubated at 25 _ C in potassium
tween buffer (PBS/t- IO mM Na-phosphate buffer, pH 7.4, I38 mM NaCI, 2.7 mM KCI,
0.05% Tween 20) for 30 min. Then a mixture ofEDC and NHS was introduced into the cell.
After cuvette activation, protein immobilization was conducted for I5 min in I 0 mM acetate
bufferat pH=5.5, 4.5 and 4 for 2B4, Fp and b5, respectively; at pH=7,7 and 4 for P450scc,
AdR and Ad, respectively, and at pH=4.8, 5.2 and 4.5 for P450cam, PdR and Pd,
respectively. Then Iigand solution was added. The uncoupled Iigand was removed with
PBS/t buffer. Those carboxyl groups on the dextran which remained uncoupled to the
biomolecules were deactivated with IM ethanolamine, pH 8.5, and the sample cell was
washed with PBS/t. The molecular surface concentrations of immobilized (im) enzymes d-
and t- 2B4, d- and t- Fp, d- and t-b5 were found to be (26±3)*I0"14, (6.0±0.6)I0"14,
(IO±I)*I0- 14 , (4.0±0.4)*I0" 14, (43±5)*I0" 14 and (14±2)*I0"14 moVmm2, respectively; those
ofP450scc, AdR and Ad were (35±3)*I0" 14, (Il±I)*I0"14, 6.9±0.T14 mol/mm2 and those of
P450cam, PdR and Pd were (8.8±0.9) 10"14, (5.6±0.5) 10" 14 and (2.8±0.3) 10"14 mol/mm2 ,
respectively. The binding of immobilized proteins with appropriate redox partners was
followed by adding aliquots ofligands in !arge excess over the immobilized ligate after buffer
washing. The association kon and dissociation koff rate constants were determined under
pseudo-first order reaction conditions. Kinetic data fitting was performed using FASTfit
program (FISONS, (Yeung et al., I995)) based on the equation:

R = Ro + R1 *(1-exp (f't)) (I)

where R is response ofthe device, t- time period, Ro- response at t=O,

j= kon*C + koff (2)

and c is Iigand concentration, R 1 is the response at t= a (at the equilibrium state) - relative
to the response at t=O. The association and dissociation rate constants were obtained from
the equations [I, 2].
The equilibrium constant, Keq was calculated as

(3)

175
and the life time of a complex was determined as

r- 1/koff (4)

The binding curve did not depend on the stirrer speed if it exceeded 30 device units;
therefore all measurements were carried out at 60 device units.

Analytical measurements. The concentration of 2B4 was deterrnined by the method of

Omura and Sato (Omura and Sato, 1964) using an extinction coefficient of ~so-490 = 91
mM 1 cm· 1 for the CO-complex of reduced 2B4 minus its reduced form in the difference
spectrum. The concentration ofb5 was determined using an extinction coefficient of ~24-408
= 165 mM 1cm· 1 for the Na dithionite reduced form minus its oxidized form in the difference
spectrum; the concentration of purified oxidized Fp, was estimated with the extinction
coefficient of ~s6 = 21.4 mM 1cm· 1 (French and Coon, 1979). Kinetics of NADPH-
dependent reduction of b5 and 2B4 in the presence of benzphetamine was measured under
anaerobic conditions at ~24-408 and ~so-490, respectively. The incubation mixture contained
500 mM K-phosphate buffer, pH 7.4, 0.25 g Emulgenll, 0.5 ~ 2B4 monomers and 0.5 M
Fp monomers, 2 mM benzphetamine. The anaerobic system included 40 mM D-glucose, 90
U glucose oxidase/ml, and 2500 U catalase/ml. The samples were bubbled with CO for I
min. The reaction was started by adding 1 mM NADPH. The kinetic curves were fitted
using the computer program "SpectraLab" based on the nonlinear regression method
(Davydov et al., 1995). In the presence ofb5, the second-electron transferrate constant was
calculated from the difference of the rate constants for NADPH oxidation and superoxide
anion generation during 1-electron reduction of 02 on 2B4. NADPH oxidation rate was
registered at 340 nm and 25 °C. The incubation mixture contained 500 mM K-phosphate
buffer, pH 7.4, 0.25 g Emulgenll, 0.5 ~ 2B4 monomers, 0.5 ~ Fp monomers and 0.5 M
b5 monomers. The reaction was initiated by addition of 0.3 mM NADPH. Molar extinction
coefficient for NADPH was 6.22 mM 1cm· 1. Superoxide anion generation rate was measured
by registering the superoxide dismutase (SOD)-sensitive reduction rate of 30 M succinilated
cytochrome c as described by Zhukov and Archakov {Zhukov and Archakov, 1985). The
reaction was started by addition of 0.3 mM NADPH. Registration of optical density was
carried out at 550 nm and 25 °C for 1 min, then 140 U SOD /rnl was added. The molar
extinction coefficient ofreduced cytochrome c at 550 nm was 21 mM" 1cm- 1; T=25 °C.
P450scc concentration was determined from difference absorption spectra upon
formation of the carbonyl complex of reduced hemoprotein using the extinction coefficient
91 mM 1cm· 1 (Omura and Sato, 1964). Concentrations of AdR and Ad were measured
spectrophotometrically with extinction coefficients 11 mM 1cm" 1 at 450 nm and 10 mM 1cm· 1
at 414 nm, respectively (Chu and Kimura, 1973).
Concentration of P450cam (substrate-bound oxidized form) was determined with the
extinction coefficients 102 mM 1cm· 1 at 391 nm (Nakamura et al., 1994). Concentrations of
PdR and Pd were determined spectrophotometrically with the extinction coefficients.
10mM 1 cm· 1 at 454 nm and 10.4 mM" 1cm· 1 at 455 nm, respectively (Gunsalus and Wagner
1978).

RESULTS AND DISCUSSION

284 Containing System

Earlier we have shown that while the aggregates of cytochrome P450 2B4 and
NADPH-cytochrome P450 reductase are susceptible to monomerization in low ionic

176
strength KP buffers, the monomerization of b5 is only possible with 500 mM KP buffer
(8achmanova et al., 1995). Since the data on buffer ionic strength influence upon
interpretein electron transfer in d284/d-Fp are controversial (Voznesensky and Shenkman,
1992; Lapko et al., 1991 ), the preliminary experiments were set up to study the influence of
KP concentration on the electron transfer rate and catalytic activity of cytochrome P450 284
and the stimulatory effect of cytochrome b5 in the monomeric reconstituted system. The
data are presented in Tab1e 1.

Tablel. Influence of d- or t-b5 on benzphetamine N-demethylation rate, k in monomeric

reconstituted system (MRS)

MRS/ d-284 + d-Fp, t-284 + d-Fp,

KP buffer k .• k ·I
's 's
lOOmM 500mM IOOmM 500mM
-b5 0.11±0.02 0.09±0.02 0.027±0.002 0.024±0.00 I
+d-b5 0.16±0.01 0.14±0.01 0.034±0.003 0.032±0.00 I
+t-b5 0.11±0.01 0.10±0.02 0.025±0.002 0.023±0.001

Note. Incubation mixture contained 100 or 500 K-phosphate buffer, pH 7.4, with 0.25 g Emulgen 913/1, IM
of each protein, 2 mM benzphetamine, 2 mM NADPH, T=25°C.

1t will be seen that both the hydroxylation rate and the electron transfer rate from d-Fp
to d-284 are unaffected - up to 500 mM concentration - by KP buffers of varying ionic
strength, in full accord with the data of Voznesensky and Shenkman (Voznesensky and
Shenkman, 1992). Taking into account the Iack of buffer ionic strength dependence and the
possibility ofb5 monomerization only at high ionic strength (500 mM), the kinetic constants
for complex formation of284, Fp and b5 were measured in 500 mM KP buffer, pH 7.4. The
binding curves for these proteins (Figures 1-3) have many features in common: there is an
increase of the association signal after the addition of a Iigand (d-Fp or d-284) into the
cuvette followed by its decrease, indicating the dissociation to occur after buffer addition.
To clarity the roJe ofpositively charged groups ofproteins in complex formation, each ofthe
partners was alternatively immobilized, through its amino groups, on dextran layer. If the
calculated kinetic constants upon alternative immobilization of either protein partner in a
given pair did not differ, within the experimental error, their means were presented in Tables
2 and 3 as one line. If the differences were essential, the constants were presented in the
tables as two lines; corresponding to one or the other of immobi1ized partners.
The 284 - Fp complex formation The binding curves for the full-length and truncated
forms of 284/Fp pairs are shown in Figures 1,2 and the kon and koff constants, in Table 2.
Irrespective ofwhether the proteins were full-Jength or truncated, their kon values werein the
order of (0.5-4.0)* 106 M 1 s·'; indicating the complex formation rate to be diffusive- 1imited
(Janin 1995). The dissociation rate constant for the 284/Fp pair was found to be 0.2- 0.4
-I
s .
The binding curves obtained upon d-284 immobi1ization for the full-Iength d-284im /d-
Fp pair and upon d-Fp immobilization for the full-length d-Fpim /d-284 pair are presented in
Figs. 1 and 2a, respectively. Table 2 presents the kon and koffvalues calculated from the redox
partners' binding curves. The kon and koffva1ues for the d-284im /d-Fp and d-Fpi",/d-284 pairs
were similar. The fact that kinetic constants for d-284/d-Fp did not depend on whichever of
the partners was immobi1ized was interpreted as indicating the absence of charge interaction
upon comp1ex formation between d-284 and d-Fp in 500 mM KP buffer pH 7.4; and as
evidence for the dominant roJe of hydrophobic interactions in complex formation - in accord
with Iiterature data (Voznesensky and Shenkman, 1992). To elucidate the roJe of
membraneaus fragments of 284 in productive complex formation of the redox partners, a

177
comparative study oft- and d- 2B4 with their full - length and truncated redox partners was
undertaken (Table 2). In experiments with immobilized t-2B4, no t-2B4im complexing with
d-Fp (the binding curve is not shown) was observed- while their full-length protein partners
were able to form complexes. On the other hand immobilized d-Fp does interact with t-2B4.
This means that unlike in the case of the d-2B4/d-Fp pair, the charged groups of t-2B4 are
absolutely necessary for complex formation of d-Fp with t-2B4. The same situation was
observed with the complexes of d-2B4 with t-Fp and t-2B4 with t-Fp. Complex formation
occurred regardless ofwhether d- or t-Fp was immobilized while no complex formation with
immobilized t-2B4 was registered. From this it follows that positively charged groups oft-
2B4 are absolutely necessary for its complex formation with d- or t-Fp.

RESPONSE

40
buffer
30 I

20

10 d-28:::-··i ___,..., .......... -:,.........,.,...._~--~..,..,..-.""""~

0L_~~--~--~~--~--~-------....
20 60 100 140
TIME,(S)

Figure 1. The binding of d- Fp or t-Fp to the immobilized d-284. The incubation mixture contained 500
mM K-phosphate buffer, pH 7.4, with 0.25 g Emulgen 913/1; T= 25° C. The d- or t-Fp (0.5 ).LM) was added.
Solid line -full length protein, broken line - truncated protein.

lt is of interest that d- or t- 2B4 may form complexes between themselves, with the rate
constants of formation and decay comparable with those for the 2B4 /Fp pair. The kinetic
constants for complex formation between d-ort- 2B4 are represented in Table 2. For d- or
t-Fp/d- or t-Fp no complex formation was registered.
Presented for comparison with the above data are the electron transfer rate constants
for the protein pairs studied. The mean koff value for the d-2B4/d-Fp pair is 0.2±0.1 s· 1 and
the electron transferrate constant, k•• is 0.47±0.25 s· 1. Thus the characteristic life span ofthe
complex is 5.0±2.5 s and the electron transfertime is 2.1±1.1 s. Comparing these values, one
can see that the life span of the compex is sufficient for 2 electron transfer realization. The
probability of charge transfer (P = t!t.) from d-Fp to d-2B4 within the life span of the
complex is equal to I (P can not exceed I by definition); hence all the complexes formed are
100% productive. If one of the partners is truncated, the quantity of productive complexes is
not higher than 6%. Thus the hydrophobic tails of d-2B4 or d-Fp are essential for productive
complex formation. Other authors also stressed the decisive contribution of hydrophobic
interactions of these pairs to the electron transport reaction (Voznesensky and Shenkman,
1992).
The 2B4-b5 complex formation. The binding curves for 2B4/b5 in 500 mM potassum
buffer are presented in Figures 3,4. The kinetic constants measured for the full-size and
truncated forms of2B4/b5 pairs are presented in Table 3. The kon values werein the order of
(1.2-4.3)*106 M 1 s· 1 suggesting that the complex formationrate is diffusive limited (Janin
1995). The koffvalue was estimated tobe 0.4-0.5 s· 1.
With the the full- length d-b5/d-2B4 pair, the complex formation took place
irrespective of which of the partners was immobilized. Accordingly, the kon and k off values

178
 a
RESPONSE buffer

30
I
~rffer
20
d-284 .;"' ~ ~~ , ~- ~ N "' ~ V'- \J
10 " t-284 ~

20
I ~~--~~--
o~~~~L_~---------------------~
60 100
' 140
TIME,(S)

b
RESPONSE

40

60 80 100
TIME,(S)

Figure 2. The binding of d- or t-284 to the immobilized d- Fp (a) and to the immobilized t-Fp (b). The
incubation mixture contained 500 mM K-phosphate buffer, pH 7.4, with 0.25 g Emulgen 913/1; T=25° C.
The d-ort- 284 (0.5 I!M) was added. Solid line -fulllength protein, broken line- truncated protein.

Table 2. Relation between complex formation and electron transfer constants for 284/Fp
redox partners.

Pairs k.ft 106 k.ffi 't, K.,*I06 ke, %

M·' s' s·' s M·' s' produclive
coml!lexes
d-284+d-Fp 0.45±0.20 0.20±0.10 5.0±2.5 2.2±0.98 0.47±0.25 100
d-284m.+t-Fp 0
0.003±0.001
t-Fpm.+d-284 0.50±0.20 0.30±0. 10 3.3±1.0 1.67±0.80
t-284m.+d-Fp 0
0.020±0.003 6
d-Fp.."+t-284 4±2 0.30±0.10 3.3±1.0 12.5±7.8
t-284m.+t-Fp 0
0.003±0.00 1 0.8
t-Fpm.+t-284 1.5±0.4 0.40±0.10 2.5±0.6 3.7±1.3
d-284+d-284 0.06±0.04 0.13±0.07 7.7±4. 1 0.45±0.38
d-284+t-284 0.3±0.1 0.3±0.1 3.3±1.0 1.0±0.5
t-284+1-284 1.8±0.2 0.20±0.05 5.0±1.3 9.1±2.5
d{Q-Fe+d{Q-FE 0

179
for this pair did not depend on the partner being immobilized either The Iack of kinetic
constants' dependence on the partner immobilized demonstrates the absence of charge
interactions and, at the same time, is indicative of the basic rote of hydrophobic interactions
for the d-2B4/d- b5 pair in 500 mM KP buffer.

RESPONSE
buffer
30 ~
20

iO

40 60 80
TIME,(S)
Figure 3. The binding of d- b5 or t-b5 to the immobilized d-284. The incubation mixture contained 500 mM
K-phosphate buffer pH 7.4 with 0.25 g Emulgen 913/1; T= 25° C. The d- or t-Fp (0.51JM) was added. Solid
line -fulllength protein, broken line - truncated protein.

a
RESPONSE buffer
1
,.
,rl'-"- --~~~
r'
30

20

10
....."....,..,
d-b5im
0
20 40 60 80 100
TIME,(S)

RESPONSE buffer
j
40 - --- - - ---'""1
I
20 I
\

80 100

Figure 4. The binding of d-ort- 284 to immobilized d- b5 (a) and the binding of d-ort- 284 to
immobilized t- b5 (b). The incubation mixture contained 500 mM K-phosphate buffer, pH 7.4, with 0.25 g
Emulgen 913/1; T= 25° C. The d- or t-284 (0.51JM) was added . Solid line -fulllength protein, broken line-
truncated protein. Solid line -full length protein, broken line - truncated protein.

180
 Table 3. Relation between complex formation and electron transfer constants for 2B4/b5
redox partners.

Pairs k.,. to' k.g; 't, Kq106M" 1 k., %

M·lsl s·I s s·I productive
coml!lexes
d-2B4+d-b5 1.2±0.5 0.4±0.2 2.5±1.3 3.0±2.0 1.00±0.03 100
d-2B4+t-b5 0 0
t-2B4;.,+d-b5 0
n.d.
d-b5;.,+t-2B4 4.3±1.3 0.5±0.1 2.0±0.4 8.6±7.1
t-2B4;.,+t-b5 0
0 0
t-b5;.,+t-2B4 2.8±1.2 0.5±0.1 2.0±0.4 5.6±3.1
d{Q-b5+d{Q-b5 0

To further elucidate the role of membrane tails of redox partners, we investigated the
binding between t-b5im and d-2B4 and, also, between d-2B4im and t-b5. For these pairs no
complex formation was registered. This means that the hydrophobic membraneaus tail of b5
plays an essential roJe in complex formation with d-2B4. The interaction between d- or t-b5
im and t-2B4 was also studied and with both these pairs the binding took place (Fig 3). One
can see that the kon and koff values obtained for d-or t-bS im and t-2B4 are close to the
appropriate values for the d-b5im/d-2B4 pair (Table 3). The binding of these pairs was also
found to be dependent on the partner being immobilized. As seen from Table 3, no
complexes of t-2B4;m with d- or t-bS were registered (the binding curves are not shown)-
unlike in the situation with immobilized d- or t-bS which did form complexes with t-2B4.
Thus for the interaction oft-2B4 with d- or t-b5 the positively charged groups oft-2B4 are
absolutely necessary, just as in the above-presented case of t-2B4 complexing with d- or t-
Fp. Thus the interactions of t-2B4 with d- and t-bS are due to electrostatic forces. Table 3
presents the koff values for these pairs and their electron transport constants ke. Comparing
the two constants, one can see that the life span of the d-2B4/db5 complex is higher than its
electron transfer time. Therefore the complexes formed by the full-length enzymes are I 00%
productive. Moreover, within the life span of the complex the transport of no Jess than 2
electrons is realized. If one of the interacting proteins is truncated, no productive complex
formation was observed. It appears therefore that the hydrophobic tails of d-b5 and d-2B4
play a dominant role in productive complex formation. It is of interest that no complexing of
d- or t-bS with one another was observed - nor were they seen in the case of d- and t-Fp;
however the complexes between d-or t-2b4 were indeed registered.
The Fp-bS interaction. We have also studied the possibility of complex formation
between d- or t- Fp and d- or t-b5 but failed to reveal any compexes, irrespective of which
ofthe partnerswas immobilized (the binding curves arenot shown). At the same time, with
all possible combinations of the full-length and truncated pairs, the rate constant for the
interpretein electron transfer remained unchanged --(0.4±0.1) s· 1. Thus the electron transfer
in d- or t-Fp/d-or t-b5 was realized not through complex formation but on random collision.
To elucidate the Jocalization of docking sites of d-2B4 complexes with d-Fp and d-bS,
we have also explored the competitive kinetics of complex formation between the full-length
d-Fp and d-bS with d-2B4 immobilized. At first d-Fp was added to the measuring cuvette up
to the saturation Ievel, then d-b5 was introduced (Figure 5). The comparison of the binding
curve for d-2B4im/d-b5 on the background of the flavoprotein-saturated 2B4, with the
binding curve of d-2B4imrnld-b5 in the absence of d-Fp revealed no distinctions between the
two curves. By analogaus procedure, the d-2B4/d-Fp pair (with d-2B4imm being saturated
with d-bS) was compared with the appropriate pair of proteins but without d-b5 saturation

181
 RESPONSE
120
b5
100

80
60 b5

40 d- 2
20

100 300 700

TIME,(S)

Figure 5. The binding of d- b5 to immobilized d- 284 in the presence of d-Fp and the binding of d- Fp to
immobilized d 284 in the presence of d- b5. The incubation mixture contained 500 mM K-phosphate buffer,
pH 7.4, with 0.25 g Emulgen 91311; T= 25° C. The d-Fp or d- b5 (1.5 ~)was added.

of d-2B4imm and again no significant distinctions between the binding curves were observed
(Figure 5). The data provide evidence for the absence of competition between d-Fp and d-b5
for the binding site at d-2B4 and allow us to conclude that cytochrome d-2B4 binding to d-
Fp and d-b5 occurs at different sites.
Based on our data on the interaction of the full-length forms of d-2B4, d-Fp and d-b5,
the electron transfer model for this complex was schematically represented (Figure 6).
Apparently, d-2B4 is able to form ternary compexes with d-Fp and d-b5 whereas with the d-
Fp/d-b5 pair no compex formation is possible. Thus, with the reconstituted microsomal
system in action, d-b5-can acquire one electron - through random collisions with d-Fp - and
to pass it over to d-2B4 during complex formation with this latter protein.

PLEX
FORMATION

Figure 6. The model of redox partners interaction in microsomal monooxygenase system.

182
Inßuence of ButTer Ionic Strength on Complex Formation Parameters for the 2b4-
Containing System

To elucidate the influence of buffer ionic strength on complex formation kinetic

constants in the P450 2B4 system, a comparative study was carried out of the kon and koff
values for the pairs 2B4/Fp and 2B4/b5 in high (500mM) and low (50 mM) ionic strength
KP buffer The binding curves for the full-length and truncated protein forms in 50 mM KP
buffer are presented in Figs. 7-8 and their kinetic constants, in Tables 4 and 5. Let us
consider in some detail the complex formation dependences for the pairs studied.
The 2B4 - Fp complex formation Decreasing KP buffer concentration from 500 to 50
mM did not Iead to any significant changes in binding parameters for the d-2B4irnrnld-Fp pair
(so the binding curve forthispair in 50 mM KP buffer is not shown). This factwas taken to
indicate - in accord with our earlier conclusion - the dominant roJe of hydrophobic
interactions in complex formation. Changing KP buffer concentration from 500 mM to 50
mM for the d-FPim/d-2B4 pair Ieads to substantial changes in complex formation rate
constants. The binding curve for the d-Fpimld-2B4 pair in 50mM phosphate buffer is shown
in Fig. 7a and its kon and kof!Values, in Table 4. It can be seen that the association reaction is
described by two constants, corresponding, respectively, to the fast phase and the slower
phase of the reaction process.

a
RESPONSE BUFFER
140
l
.. 1
I
120 'I
t -2ß4 1,.
100 I ...
BUFFER
!
I
80 )...
60
40
20 d-Fpim
0 ~--~----~----~----~--~----~-----
100 200 300 400 500 600
TIME (S)

b
RESPONSE
BUFFER BUFFER
400
------ ~ l
300
t -2B4 - ... - --
200 I

100 I
t - Fpim y d-2B4
Ot=~==~--~~--~~~~~
100 200 300 400 500 600
TIME (S)
Figure 7. The binding of d- or t-284 to the immobilized d- Fp (a) and to the immobilized t-Fp (b). The
incubation mixture contained 50 mM K-phosphate butfer pH 7.4 with 0.25 g Emulgen 913/1; T=25° C. The
d- ort- 284 (0.151!M) was added. Solid line -fulllength protein, broken line- truncated protein.

183
 Dissociation process is described by a koff constant that is one order lower than one
obtained in 500 KP buffer. The two affinity constants were calculated as K..q1= k orlkoffand
K.,q 2= kon'll'koff. The affinity increase associated with the presence of K.,ql for the d-FPim/d-
2B4 pair and the apparent ionic strength dependence of complex stability between d-Fp and
2B4 in low ionic strength buffer are explained by substantial contribution of electrostatic
interactions into this process. As noted by Nishimoto and Otsuka-Murakami (1988), the
interactions of d-2B4 with d-Fp may include an electrostatic component, affected by the
arnino groups of2B4 and the carboxyl groups ofFp. This finding is supported by the above-
mentioned buffer ionic strengh dependence ofcomplex formation in the case ofthe d-FPimld-
2B4 pair. At the same time our data on the absence of the buffer ionic strength dependence
for d-2B4m/d-Fp binding clearly demonstrates that after 2B4 immobilization via amino
groups the latter get blocked and hence are not able to electrostatically interact with
carboxyl groups of Fp. With such immobilization taking place, the complex formation
reaction can only proceed due to hydrophobic interactions. Thus for d-2B4/d-Fp there are
two variants of complex formation /ionic strength dependences and, accordingly, two
complex formation types. The first complex formation type is virtually unaffected by buffer
ionic strength. Lack of ionic strength dependence is taken to mean that interaction between
partners is deterrnined by hydrophobic interactions. The second type is characterized by a
decrease in the complex's dissociation rate with decreasing buffer ionic strength. Such a
dependence is indicative of ionic interactions significance for the complex stability. The
formation of the d-2B4/d-Fp complex may be realized through hydrophobic interactions and
the complex may be stabilized by electrostatic forces. It is of interest to compare the results
of our biosensor studies with Iiterature data. The kinetic data for the d-2B4/d-Fp protein pair
in HEPES buffer, as deterrnined by the fluorescence quench assay, are reported: (kon =
6.5*10 5 M" 1 s" 1 and koff= 0.025 s" 1) (Davydov et al., 1996). Thus our results obtained for the
d-Fpim/d-2B4 pair compare favourably with Iiterature data.

Table 4. Relation between complex formation and electron transfer constants for 2B4/Fp
redox partners in 50 mM KP buffer.

Pairs k ..J *106 kon2* 106 K.qt*10 6 Keq2*106

k~'{· M-t M-t
M-ts-t M·ls·l s
d-284im+d-Fp 0.33±0.21 0.20±0.10 1.7±0.9
d-Fpim+d-284 0.52±0.24 0.10±0.09 0.018±0.004 20±10 5.6±5.0
(0.10±0.01)* (0.90±0.09)**
d-284im+t-Fp 0

t-Fpim+d-284 0.56±0.26 0.04±0.02 0.016±0.004 14±8 2.5±1.3

(0.30±0.03)* (0.70±0.07)**
t-284im+d-Fp 0 0 0 0 0

d-Fpim+t-284 0.39±0.30 0.08±0.01 0.012±0.003 33±20 33±20

(0.36±0.04)* (0.63±0.06)**
t-284im+t-Fp 0 0

t-Fpim+t-284 0.48±0.07 0.02±0.01 0.019±0.008 25±15 6.7±18

(0.33±0.03)* (0.66±0.07)**

( ... )*., ( ... )** the portions offast and slower phases.

A comparative study of interactions of t- and d- 2B4 with their full-length and

truncated redox partnerswas undertaken with the use of 50 mM KP buffer (Table 4). The
binding curves are represented in Figure 7. No t-2B4im complexing with d-Fp (the binding

184
curve not shown) was observed - while their full-length protein partners were capable of
complexing. On the other hand, immobilized d-Fp interacted with t-284 with a koff constant
one order lower than in 500 ii E- buffer. Thus electrostatic interactions play an essential roJe
in complex stability but not in the association reaction. These findings confirm our previous
conclusion as to the electrostatic nature of these interactions: the charged groups of t-284
are absolutely necessary for complex stability of d-Fp with t-284. The same situation was
observed for d-284 complexing with t"FP and t-284 with t-Fp. Complex formation in 50
mM KP buffer occurred regardless of whether d- or t-Fp was immobilized with 284;
characteristically, koff values for the complexes thus formed were one order lower than in
500 KP buffer. This fact provides additional evidence for the dominant roJe of electrostatic
interactions for complex stability and at the same time points to the positively charged
groups oft-284 as an absolute requirement for its complexing with d- or t-Fp.
The 284-b5 complex formation. The binding curves for 284 im I d-b5 in 500 mM
(Figure 4) andin 50 mM KP buffers are similar (so the data obtained in 50 KP bufferare not
shown). As regards the d-284;mld-b5 pair (Tables 3 and 5), its kinetic rate constants and
affinity were unaffected by ionic strength - indicating the hydrophobic interaction as a
decisive factor for its complex formation. The binding curves for the d-b5im/d-284 pair in
50 mM and in 500 mM KP buffers proved to be different. The association reaction is
decribed by two constants: the fast phase rate constant and the slower phase one. The koff
value was substantially decreased with decreasing buffer ionic strength, Thus the Iack of
buffer ionic stength dependence for the d-284;m/d-b5 pair, along with the buffer ionic
strength dependence of affinity, connected with appearance of l<eqt for d-b5;m/ d-284,
indicate that in the latter case the electrostatic forces between the carboxyl groups of b5 and
amino groups of 284 are beginning to play an essential roJe in complex stability - with the
use of 50 mM KP buffer. This finding is in agreement with the Iiterature data (Gerber and
Sligar, 1994). Again one can see two types of interactions between d-284 and d-b5
molecules: the association reaction is determined by hydrophobic interactions and complex
stability, by electrostatic forces.
We investigated the binding between t-b5;ml d-284 and d-284im/t-b5. For these two
pairs no complex formation was registered in either 50 mM or 500 mM KP buffer This
means that the hydrophobic membraneaus tail of b5 plays an essential roJe in complex
formation.
The interactions between d-or t-b5 im and t-284 were also studied 500 and 50 mM KP
buffers (Figures 4 and 8). In both buffers, the binding of these pairs was found to be
dependent on the partner being immobilized. As seen from Table 5, no complexing of t-
284;m with d- or t-b5 was registered in either 50mM (the binding curves are not shown) or
500 mM KP buffer. One can see that the koff value obtained for d-b5;m/t-284 in 50 mM KP
buffer is one order lower than the one in 500 mM KP buffer; therefore this former value is
close to the appropriate value for the d-b5;mld-284 pair (Table 5). Thus the complex stability
of t-284/d-b5 is ensured by electrostatic forces and the positively charged groups of t-284
are absolutely necessary for complex stability.

P 450scc Containing System

Since the data on interprotein electron transfer in Jow ionic strength buffer are most
fully preented for just this system, P450scc was taken to study kinetic processes of complex
formation in 50 mM KP buffer.
To clarify the roJe of protein charged groups in complex formation, either of the
partners was alternatively immobilized through its arnino groups. The binding curves for
Ad/P450scc, Ad/AdR and P450scc/AdR are presented in Figures 9-11. For all these pairs,
we observe the dependence of complex formation kinetics on the partner immobilized; this
shows the crucial roJe of electrostatic interactions in the above pairs' complex formation.

185
 a
RESPONSE
BUFFER
j
~ \
300
- - --
/
I
200 I
I
BUFFER
t-2B4 / d-2B4
100 I 1
0 d-bsu.j:
100 200 300 400 500 600
TIME,(S)

b
RESPONSE
BUFFER
!
50 ,
--~.,.._...."#</"'.,....IV_.\

,,
~ .,.

40 t-284, ,;
'1~
30
I
....... __ .,..
20
10 t-b5·lDl
0 ~--~~------~--~------~~~~-
20 40 60 80 100 120 140
TIME,(S)

Figure 8. The binding of d-ort- 284 to immobilized d- b5 (a) and the binding of d-ort- 284 to
immobilized t- b5 (b). The incubation mixture contained 50 mM K-phosphate buffer, pH 7.4, with 0.25 g
Emulgen 913/1; T= 25° C. The d- or t-284 (0.15 J.LM) was added . Solid line -fulllength protein, broken line
- truncated protein. Solid line -fulllength protein, broken line - truncated protein.

Table 5. Relation between complex formation and electron transfer constants for 2B4/b5
redox partners in 50 mM KP buffer.

Pairs k •• , *106
M ' 1 s·•
k•• 2 *106
M·•,·• ,..
k.g, ~,,*10-6
M .J
~9 :*106
M·•
d-284im+d-b5 0.9±0.5 0.24±0.05 3.8±2.3 3.8±2.3

d-bSim+d-284 0.54±0.37 O.Ol3±0.003 0.007±0.002 77±46 1.9±07

(0.08±0.92)* (0.92±0.08)**
d-284+t-b5 0 0
t-284im+d-b5 0 0

d-bSim+t-284 0.11±0.01 0.027±0.020 0.019±0.004 5.8±3.5 1.4±l.l

(0.25±003)* (0.75±0.08)**
t-284im+t-b5 0 0

t-bSim+t-284 0.7±0.3 0.28±0.03 2.5± 1.5

( ... )*., ( ... )** the portians offast and slower phases.

186
 The Ad-P450scc complex formation. The binding curves for Adim/P450scc and
P450sccim/Ad are shown in Figures 9,10 and their kon and koffvalues, in Table 6. Clearly, the
affinity for the former pair is much higher than for the latter - at the expense of the lower
dissociation rate and the higher association rate. This was taken to mean that the positively
charged groups of P450scc play a decisive roJe in its complex formation. The kon value for
Adim/P450scc is in the order 0.21 M 1s" 1 i.e. it is close to the diffusion Iimit (27).
Comparison of the dissociation rate constant, 0.02s" 1, for the Adim/P450scc pair with the
interprotein electron transportrate value, 3s·t, reported in (Lambeth and Kriengsiri,1985)
(see Table 6) shows that the life-time ofthe complexes formed is about 50s, i.e. about 150
times higher than that required for the interprotein electron transport to occur; hence all the
complexes are productive and during formation of a single complex about 150 electrons can
be transferred.

Table 6. Relation between complex formation and electron transfer constants for redox
partners ofP450scc and P450cam systems.

Pairs kon 106 k~'{' 't, K.qt*106M 1 ke, The possible

M·•s·• s s·• quantity of
transferred
electrons in a
sing!e coml!lex
Adim+P450scc 0.21±0.02 0.02±0.01 50.5±25 10.5±5.3 31 150
P450sccim+Ad 0.05±0.02 0.4±0.1 2.5±0.6 0.13±0.7
Adim+AdR 0.018±0.004 0.02±0.01 50±25 0.9±0.5 4.6 2
230
A<IRUn+Ad 0
P450sccim+AdR 0.0039±0.0024 0.3±0.1 3.3±1.0 0.013±0.009 n.d.
A<~RUn+P450scc 0.0024±0.00 12 0.02±0.01 50±25 0.12±0.09
Pdim+P450cam 0.023±0.08 0.010±0.005 100±50 2.3±1.3 463 4600
P450camim+Pd 0
Pdim+PdR 0.12±0.02 0.02±0.01 50±25 6.0±3.2 264 1300
PdRim+Pd 0
P450camim+PdR 0.0049±0.037 0.07±0.02 14.3±4.3 0.07±0.02 n.d.
PdRim+P450cam 0.0073±0.007 0.57±0.39 1.8±1.2 0.013±0.009

1 Lambeth and Kriengsiri 1985

2 Lambeth and Kamin, 1977
3 Nakamura et al, 1994
4Roome and Peterson, 1988

Ad-AdR complex formation. The binding curve for Adi.JAdR is shown in Figure 9 and
its kon and koffi in Table 6. No binding for the AdRun/Ad pair was registered (Figure 11). This
dependence of complex formation on the protein immobilized is evidence for the decisive
roJe of AdR's positively charged groups in this process. The k,.. value for the Ad;",/AdR pair
is one order lower than for Adi,JP450scc, attesting to the presence of an thermodynamic
barrier in the complex formation reaction for the former pair. This means that electrostatic
interactions play an essential roJe in complex formation and decay. The dissociation rate
constant for Ad;",/AdR is much lower than the interprotein electron transport rate (Table 6).
Therefore the life-time of the complexes formed is about 50s, i.e. about 200 times higher
than that required for the interprotein electron transport to occur; hence all the complexes
are productive and during a single complex formation event more than 200 electrons can be
transferred.

187
 RESPONSE
DUFFER

400 t

300 P450scc

200

DUFFER
100
1

600 800 1000 1200 1400

TIME,(S)
Figure 9. The binding of P450scc and AdR to immobilized Ad. The incubation mixture contained 50 mM
K-phosphate buffer pH 7.4. T= 25° C. The P450scc or AdR (0.3 ~was added .

Thus more than 150 electrons may be transferred during the Ad-P450scc and Ad-AdR
complexes' life span. It is known that 6 electrons are necessary for the action of P450scc
monooxigenase system (Archakov and Bachmanova, 1990). Therefore during the life span
ofthe above complexes the P450scc system may realize more 25 tumovers.
The AdR-P450scc complex formation. The binding curves for AdRim/P450scc and
P450scci.JAdR are presented in Figure 10 and their kon and koff values, in Table 6. The kon
value for the P450scc/AdR pair is much lower than the diffusion Iimit (Janin 1995) - an
evidence for the presence of a thermodynamic barrier in reaction. It will be seen that the
dissociation rate constant for the formerpair is higher than for the latter.
This clearly demonstrates the decisive contribution of ionic interactions to complex
formation. AJI the complexes formed are unproductive because no direct electron transfer
from AdR to P450scc. is observed. The presence ofthese complexes may Iead to formation
of temary complexes of AdR/P450scc/Ad. Figure 11 represents the binding curve of Ad
with AdRim in the presence of P450scc. As was mentioned above, Ad would not complex
directly with AdRim but it is able to do so in the presence of -450scc. These data provide
evidence for the possible formation ofternary complexes in the P450scc system.

P 450cam-Containing System

The complex formation kinetics for the redox partners of this system was measured in
50 TRIS-HCI buffers with 200 mM KCI. To clarity the rote of protein charged groups in
complex formation, either of the partners was alternatively immobilized through its amino
groups. The binding curves for redox partners in the P450cam system are represented in
Figures 12, 13. F or all the pairs the dependence of complex formation kinetics on the partner

188
 RESPONSE BUFFER
I

240

220

200

180

160 P450scc

140

120
BUFFER
100
l
l
Ad
80
AdR
60 P4S0sCCiJn
BUFFER
40
l
20 P4S0sCCiJn

0
100 200 300 400 500
TIME ,(S)
Figure 10. The bindingofAd and AdR to immobilized P450scc and ofP450scc to immobilized AdR. The
incubation mixture contained 50 mM K-phosphate buffer, pH 7.4; T= 25° C. The Ad (45 !!M}, AdR ((1.2
).IM) and P450scc(l.2 !lM) were added.

189
 RESPONSE DUFFER

Ad
I
300

250

200 P4SOscc

150

100
AdRim
50 Ad
AdRim I
0 400 800 1200 1600
TIME,(S)

Figure 11. The bindingofAd with immobilized AdR in the absence and the presence ofP450scc. The
incubation mixture contained 50 mM K-phosphate buffer, pH 7.4; T= 25° C. The P450scc was added at 0.5
11M and Ad, at 45 (in the absence ofP450scc) or 6 ~(in the presence ofP450scc).

immobilized was observed, indicating the dominant roJe of electrostatic interactions in their
complex formation .
The Pd-P450cam complex formation . The binding curve for PdirnlP450cam is shown in
Figure 12 and its kon and k off, in Table 6. No complex formation for P450cam!Pd was
registered. The dependence of complex formation on the protein immobilized is evidence for
the decisive roJe ofthe positively charged groups ofP450cam in complex formation. The kon

RESPONSE DUFFER
90 1
80

70
BUFFER
60
l

40

20

10

0~~~~~~~~~~
200 400 600 800 1000 1200 1400
TIME,(S)

Figure 12. The binding ofP450cam and PdR to immobilized Pd and the binding ofPd to immobilized
P450cam and PdR . The incubation mixture contained 50 mM TRIS-HCI buffers with 200 mM KCI and 400
~M camphor, pH 7.4; T= 25° C. The P 450cam (4 ~. PdR (1.5 ~ or Pd (4 ~was added.

190
value for the Pdim!P450cam pair is in the order 0.02 M" 1s·•. -i .e. much lower than the
diffusion Iimit (Janin 1995) - an evidence for the presence of a thermodynamic barrier in the
complex formation reaction. This means that electrostatic interactions play an essential rote
in protein complex formation and decay. Comparison of the dissociation rate, 0.01 s·•, for
the PdimiP450cam pair with the interprotein electron transport rate in Table 6 - 46 s·• -
shows that the life-time of a complex is about I 00 s i. e. much high er (4600 tim es) than the
interprotein electron transport time. Therefore, all the complexes are productive and during
a single complex formation about 4600 electrons can be transferred.
The Pd-PdR complex formation. The binding curve for the Pdim/PdR pair is shown in
Figure 12 and its kon and koffi in Table 6. No binding for PdRim/Pd was seen. The
dependence of complex formation on the partner immobilized provides evidence for the
decisive rote of the positively charged groups of PdR. The kan value for the Pdim/PdR pair is
about 0.12 M-1s... which is close to the diffusion Iimit (Janin 1995). The charachteristic life

RESPONSE BUFFER
I
40

30
P450camim
BUFFER
20
l
lO

O L--~---:-:-:-------::-:------:-::-:--
200 400 600 800
TIME,(S)

Figure13. The binding ofPdR to immobilized P450camim and the binding ofP450cam to immobilized PdR.
The incubation mixture contained 50 mM TRIS-HCI buffers with 200 mM and 400 JJ.M camphor, pH 7.4; T=
25° C. The PdR or P450cam (1.5 JJ.M) was added.

span of the Pdim/PdR complex is about 50s i.e. about 1300 times higher than the
interprotein electron transport time (Table 6). Therefore all complexes formed are
productive and during a single complex formation about 1300 electrons can be transferred.
Thus more than 1300 electrons may be transferred during the life span of the system
composed of two complexes, Pd-P450cam and Pd-PdR. As known, 2 electrons are
necessary for for P450cam monooxigenase system (Archakov and Bachmanova, 1990).
Therefore during the combined life span of both these complexes the P450scc system may
realize more than 650 tumovers.
The PdR-P450cam complex formation. The binding curves for P450camim/PdR and
PdRimiP450cam are presented in Figure 13 and their kon and ko!J constants, in Table 6. The
kan for P450cam/PdR much lower than the diffusion Iimit (Janin 1995) It means that the
complex formation reaction has a thermodynamic barrier. Apparently, the dissociation rate
constants differ substantially, which points to the decisive contribution of ionic interactions
to these proteins' complex formation.

CONCLUSIONS

In this paper the possibility of complex formation between microsomal 2B4, Fp and bS
in the monomeric reconstituted system in real time was studied. The kinetic rate constants,
kon and koff, for these protein pairs, as weil as their affinities, were determined. The dominant

191
roJe of hydrophobic membranous tails of 2B4, Fp and b5 in productive complex formation
was established. The essential roJe of electrostatic interactions in complexes stability was
demonstrated. The interprotein electronic transfer may occur not only through complex
formation but also on random collision of protein partners - as was demonstrated in the case
of electron transfer between Fp and b5. The decisive significance of electrostatic interactions
for the P450scc and P450cam complexes with their partners was shown. All complexes
between redox partners within both systems, P450scc and P450cam, were found to be
productive.

ACKNOWLEDGMENTS

We thank Dr. J.J. Ramsden for valuable discussions, Drs. I.P. Kanaeva, G.P.
Kuznetzova and N.F. Samenkova for providing 2B4, Fp and b5 protein preparations, Drs.
M. Lehnerer, J.Schulze and P.Hiavica for providing t-2B4 preparation, Dr. S.A. Usanov for
providing P450scc, AdR and Ad protein preparations, Dr. Hui Bon Hoa for providing
P450cam, PdR and Pd protein preparations.
This work was supported by RFBR grant N 95 -04-12515a and by INCO-COPERNICUS
grant PL965070

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194
INDEX

Complex formation, 64, 173, 174, 177-179, 181-

Ab initio, I, 21, 140, 141, 144--146, 148, 149, 152, 153
Acetone, 97 188, 190, 191
Adrenalcortex,8,64,95,96,97, 175 Copper
Adrenodoxin (ADX), 4, 6, 8, 27, 52, 54--{)4, 66, 91, atoms, 149. 151, 156. 162-165, 169
93,94-99,173 ion, 146, 148, 149, 151, 155, 156, 158, 161
mutants, 57, 59 proteins, 139, 141. 161
Adrenodoxin reductase (ADR), 8, 52, 54, 57, 58, 64, sites, 161-163, 165, 167
91,93-99,173 Corticosterone, 52, 58--60, 62, 64
Air/water interface, 8, 9 Cortisol, 52, 64, 93
Antenna Coulomb
structure, 112 Blockade, 117,120
system, 103, 110 Gap, 118,119,135
Antibodies, 7, 96, 129 interaction, 142, 145
Ascorbate oxidase (AAO), 162-165, 167-169 Staircase, 118,119,135,136
ATP, 33,41-43,45-48, 79, 103, 107, 108 Crystallography, 2, 3, I 04, 166, 169, 170
electron transport, 33 CuS, 120, 128
formation, 47 CYP II AI: see Cytochrome P450scc
synthesis of, 33, 41,42 CYP II BI: see Cytochrome P450 II b
ATPase, 43, 78, 79 Cytochrome
Azurin, 162, 169 b5, 3, 4, 93, 100, 108, 173, 174, 177
b561• 106, 108--111
Bacteriochlorophylls (BChl), 104, 105, 107, 109, 112 c, 2, 14,22
Bacteriorhodopsin, 21 expression, 26
Bc 1 complex, 105, 106, 108--110 monolayer of, 9
BCH I: see Bacteriochlorophylls P420, 18
Bioelectronic devices, 91 P450, 1-4,8,9, II, 12, 14, 16--20,24,26,27,51,
Bioelectronics, I, 4, 27, 91 52, 54, 56, 59, 61', 63, 64, 91-94, 98--100,
Biosensor, 4, 26, 28, 173-175, 184 173, 174, 176, 183
Biosynthesis, 8, 26, 52, 91, 93, 107 P450llb,27,52,54-{)2,64
Biotechnology, 26--28 P4502B4,27,98, 173,174,176,183
Bragg reflections, 130 P450cam,4, 173-176,187,190-192
Brewster angle microscopy, 9 P450scc,2,8, 14,52,54-{)1,63,64
absorbance spectra, 16, 17
Cadmium arachidate, 120, 121, 125, 129 LB films, 8, 17-19
Carotenoids, 44, 45 monolayers, 8
CD: see Circular dichroism recombinant, 16
CdS, 120, 121, 125, 127-131, 133,137 reduction, 18
Ceruloplasmin (CP), 162-166, 168 spin state, 16, 17, 19
Chemiosmotic theory, 42 substrate, 24
Cholesterol, 4, 8, 17-19, 24, 52, 58, 60, 64, 75, 91- systems, 26, 27, 51, 52, 59, 64,94
93, 97, 98, 173 wild type, 12
Chromatography, 8, 16
Circular dichroism (CD), 10, II, 12, 55, 56 Density functional theory (DFT), 140, 141, 145, 146,
Close packing, 12-14 153, 155, 156, 158

195
Differential negativeresistance, 117-119, 124, 135, 136 Lipid, I, 22, 40,68-70, 72, 73, 75, 77-81, 86, 89, 91,
DNA technology, 26, 91 94,106,112,113,174
Drugs, I, 26--28,51,91,92, 173 Lowest unoccupied molecular orbital (LUMO), 153,
!56
Electron
diffraction, 7, 112 Mehler reaction, 46--48
microscopy, 82, I 04 Metalloproteins, I, 4, 8, 19, 21, 23, 91. 92, 139, 141,
spin resonance, 55 167
transfer, 1--4,8, 17, 19-21,23,26,27, 37, 40, 45, MgS. 128, 133
46,54-64,68,71, 79,80,91,93,94, 103, Microelectronic devices, 67, 75, 84. 87
105, 106, 108, 110, 114, 148, 152, 155, Microscopy interference, 8
161, 166--170,173, 176--179, 181-188, 192 Monolayer, 8, 9, 10, 12. 13. 22, 25, 69, 73, 74, 76--
transport, 8, 18, 33, 39,40--48, 93, 95, 174, 178, 78, 82, 84, 86, 125, 129, 131
181, 187, 191 Monooxygenase,26,93,94
E1ectronic Multicopper oxidases, 161-163. 167. 168
coupling, 21, 29, 82, 161, 168 Mutagenesis, 5, 20, 54. 58. 64, 140
devices, 68, 87, 133
Electrostatic NADP reduction, 38, 47,48
fields, 2 NADPH, 3, 8, 33, 47, 48, 52,93-97, 173, 174, 176. 177
interactions, 54, 141, 184, 185, 187, 190-192 Neutron reflectometry (NR), 71, 79
potential, 5, 6, 167 Nuclearmagneticresonance(NMR),2,55, 71,73,139
Ellipsometry, 10, 130, 133
Emerson effect, 39 Optical
EPR spectra, 55, 56, 163, 164 biosensor, 173, 175
spectroscopy, 166
FAD,41, 64 Oxidation, 19, 40, 41, 45--47, 52, 106, 108, 110, 148,
Fatty acids, 51, 130 161, 167, 173, 176
Field-eifeet transistor (FET), 68,81-86, 128 Oxyhemocyanin, 147, 148
Flavoprotein, 41, 52, 64, 181
Fluorescence,38,39,43--46,62, 71,173,184 Particles,22, 117-123,127-131,136,144
FTIR spectroscopy, 13, 71 PbS, 120, 127, 128, 131-133
Phenotype, 107, 108, II I, 113
Glucocorticoids, 52 Phosphorylation, 44
Granules, 117, 128, 131 Photochemical
activity, 38, 44, 46, 106, 110
Hartree-Fock (HF), 143-146 reaction, 33, 36, 37, 39,42--44, 46,47
Heat capacity, 62 Photosynthesis, 25, 33, 35, 37, 39--45, 47. 103
Heme, 2, 4, 5, 17, 19, 26, 41, 105, 106 Photosynthetic reaction center (RC), I, 3, 7, 24--26,
Hemocyanin, 141, 146, 150, 153, 155, 156 36--38,40,41,103-110,112-114
Highest occupied molecular orbital (HOMO), 153, 156 Photosystem, 37
Hydroxylation, 24, 51, 52, 57, 64, 98, 174, 177 Photovoltaic cells, 7
Pregnenolone, 8, 52, 64, 93, 98
Induced resonance, 37 Progesterone, 52
Interfacial architectures, 68, 71, 80 Protein recombinant, 8, 10, 12, 13, 16, 17, 98, 100
Puf operon, 107, 112
Junction, 85, 117-121, 124--128, 134, 135-137 PufX, 106--108, 110, 112-114

Kiessig fringes, 77, 78, 130 Quantum

Kinetic constants, 37, 173, 177, 178, 180, 183 chemistry, 140-143
yield ofphotosynthesis, 39,43
Laccase (LC), 162 Quinone
Langmuir-Blodgett (LB), 7, 8, 10-19, 22, 25, 120, QA, 105
121, 125, 127, 129, 130, 132, 133 Q 8 , 105, 106, 108, 110-114
Laser beam, 71
Lateral patteming, 87 Reaction center: see Photosynthetic reaction center
Light Redox
absorption, 35, 37, 43,47 partners, 54, 56, 61, 62, 91, 93,161,173,175,177,
harvesting 179, 181, 182, 184, 186--188, 192
antenna, 36 potential, 19, 45, 55, 56, 60, 61, 63, 64, 108, 109,
complex, I 04, I 08 111, 167, 170
LHC I, 44, 104--107, 110-114 Rhodobacter Sphaeroides, I 09
LHC 2, 44, 104, 105, 110-113 Rhodopsins, 7

196
S state, 41 Superlattices, 117, 132-134
Scanning tunneling microscopy (STM), 118, 119, Superoxide,46, 141,174,176
121-124, 126, 127, 131, 132 Surface
Self consistent field method (MCSCF), 144 coating, 83
SEM, 133 density, 12
Single electronjunctions, 122, 125, 127, 129 plasmon, 68, 70, 71, 73, 75, 77
Space self consistent field method (CASSCF), 144,
146, 149, 151-155 Tethered membranes, 68, 70, 75, 87
Steroid 4, 8, 24, 26, 27,51-54, 56, 64,91-93,99, 100 Thickness
STM: see Scanning tunneling microscopy average, 10, 131, 133
Structure film, 10, 125, 129, 131
primary, 27, 55, 112
quatemary, I 13 Vesicle fusion, 69, 79
secondary, 12, 14, 15, 16, 106, 112, 113, 146
Substrate, 3, 4, 8, 12, 14, 17-20, 24--26, 55, 63, 67- Xenobiotics, 51, 173
69, 73, 75-79, 81-84, 108, 125, 127, 129,
162, 166, 167, 176 Z scheme, 39, 40