PROTEINS: Structure, Function, and Genetics 27:385–394 (1997)
The Metal Site of Pseudomonas aeruginosa Azurin,
Revealed by a Crystal Structure Determination of the
Co(II) Derivative and Co-EPR Spectroscopy
Nicklas Bonander,1* Tore Vänngård,1 Li-Chu Tsai,2 Vratislav Langer,2 Herbert Nar,3 and Lennart Sjölin2
Departments of 1Biochemistry and Biophysics and 2Inorganic Chemistry, Göteborg University and Chalmers
University of Technology, Göteborg, Sweden; 3Max Planck Institut für Biochemie, Abteilung für Strukturforschung,
Martinsried bei München, Germany
ABSTRACT The crystal structure of co-
balt-substituted azurin from Pseudomonas ae-
ruginosa has been determined to final crystal-
lographic R value of 0.175 at 1.9 Å resolution.
There are four molecules in the asymmetric
unit in the structure, and these four molecules
are packed as a dimer of dimers. The dimer
packing is very similar to that of the wild-type
Pseudomonas aeruginosa azurin dimer. Re-
placement of the native copper by the cobalt
ion has only small effects on the metal binding
site presumably because of the existence of an
extensive network of hydrogen bonds in its
immediate neighborhood. Some differences are
obvious, however. In wild-type azurin the cop-
per atom occupies a distorted trigonal bipyra-
midal site, while cobalt similar to zinc and
nickel occupy a distorted tetrahedral site, in
which the distance to the Met121,Sd atom is
increased to 3.3–3.5 Å and the distance to the
carbonyl oxygen of Gly45 has decreased to
2.1–2.4 Å. The X-band EPR spectrum of the
high-spin Co(II) in azurin is well resolved (ap-
parent g values gx8 5 5.23; gy8 5 3.83; gz8 5 1.995,
and hyperfine splittings Ax8 5 31; Ay8 5 20–30;
Az8 5 53 G) and indicates that the ligand field is
close to axial. Proteins 27:385–394, 1997.
r 1997 Wiley-Liss, Inc.
Key words: azurin; cobalt; x-ray crystallogra-
phy; EPR; Pseudomonas aeruginosa
INTRODUCTION
The unusual spectroscopic properties of type 1
copper found in early investigations have been of key
interest, particularly to biophysicists and inorganic
chemists, through many years. The most striking
feature with the type 1 copper site is its intense blue
color resulting from an absorption at ,600 nm. The
molar absorption coefficient (e) of the band is located
in the range of 3000–7000 M21 cm21, much higher
than that of the visible band observed for ordinary
copper(II) complexes, which derive their color from
the d-d bands at 600–800 nm with e , 700 M21 cm21.
The EPR spectra of copper proteins containing type 1
copper are also unusual. Some proteins exhibit axial-
type signals with anomalously small hyperfine con-
r 1997 WILEY-LISS, INC.
stants in the z direction (Az), whereas other exhibit
rhombic signals with even smaller Az. Other charac-
teristics of type 1 copper include a high reduction
potential and unusual features of the Raman spec-
trum in the region of 200–500 cm21. Blue copper
proteins have also been investigated by using circu-
lar dichroism (CD) and magnetic dichroism (MCD).1,2
All these particular properties have triggered an
extensive amount of investigations directed toward
the elucidation of the metal site.
By the mid-1970s, extensive spectroscopic investi-
gations led to a consensus that type 1 copper had a
distorted tetrahedral coordination containing a cop-
per–cysteine thiolate bond. The most solid evidence
for the existence of the Cu}S bond was provided by
x-ray photoelectron spectroscopy (XPS).3,4 The Ra-
man lines observed in the region of 300–500 cm21
were suggested to be associated with the Cu}S bond,
although this complicated feature was not fully
understood.5–7 A few years later the puzzling physi-
cal properties of blue copper proteins began to be
understood, and the evidence came from numerous
spectroscopic investigations on stellacyanin, azurin,
laccase, and ceruloplasmin, and particularly the
preliminary x-ray structure of poplar plastocyanin to
2.7 Å resolution.8 From later crystallographic stud-
ies on plastocyanin,9 Alcaligenes denitrificans
azurin,10 and Pseudomonas aeruginosa azurin,11,12
however, a canonical copper site has evolved.
The P. aeruginosa azurin molecule consists of a
single polypeptide chain of 128 amino acid residues
and one copper atom. The Cu ion lies about 7 Å below
the surface and is coordinated by three equatorial
ligands, His46, Cys112, and His117, and has a weak
axial interaction with Gly45 and Met121 (Figs. 1 and
2).†13 The trigonal geometry defined by the equato-
Abbreviations: EPR, electron paramagnetic resonance;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
WT, wild-type.
*Correspondence to: Dr. Nicklas Bonander, Department of
Biochemistry and Biophysics, Lundberg Laboratory, Göteborg
University and Chalmers University of Technology, S-413
90 Göteborg, Sweden. E-mail: Nicklas.Bonander@BcBp.gu.se
Received 20 September 1996; accepted 30 September 1996.
†This was generated with coordinates 4azu.pdb (see Ref. 12),
by using the program MOLSCRIPT, Version 1.3.
386 N. BONANDER ET AL.

rial ligands is conserved in all known structures of

type 1 copper determined so far. The interaction
between copper and the axial methionine ligand has
been suggested as an important factor for the fine
tuning of the reduction potential.14
Metal substitutions in proteins have previously
been successfully used to probe structural and elec-
tronic states of metal sites in these proteins. Metal
ions, such as Ni(II) and Co(II), have then been used
as a replacement of the native metal ion in many
blue copper proteins.15,16 The substitution of ions
such as Co(II) for the spectroscopically silent Zn(II)
ion in zinc enzymes is a classic example of this
technique.17 The characterization of metal-replaced
derivatives has also provided information that has
been useful in evaluating the faithfulness of syn-
thetic analogues of the protein centers.18,19 Recently,
synthetic analogues of the type 1 copper site, which
mimic the optical and EPR parameters20 of this site
(reviewed in Ref.21), have been synthesized. Further-
more, in an early spectroscopic exploration on Co(II)-

Fig. 2. The copper site of azurin from Pseudomonas aerugi-

nosa.13 The strong ligands to the copper atom are His46 Nd1,
Cys112 Sg and His117 Nd1. In addition, there is a weaker axial
interaction with Gly45 CO and Met121 Sd.

substituted stellacyanin22 and plastocyanin,23 it was

Fig. 1. The three-dimensional structure of azurin from Pseudo-
monas aeruginosa.13
suggested that the cobalt ion is coordinated to a
thiolate sulfur in a tetrahedral ligand environment,
thus supporting the structure for the type 1 copper
site, proposed at that time.
Recently, 1H-NOE (nuclear Overhauser effect) ex-
periments on Co-azurin and Ni-azurin have allowed
partial assignments of the isotropically shifted pro-
ton resonances.24,25 Such assignments facilitate the
use of NMR to study the interaction of azurin with
its biological redox partners.
In previous work the substitution of Co(II) and
Ni(II) for Cu(II) has also proved to be helpful in
distinguishing the d-d bands from the charge trans-
fer bands of the blue copper centers.23,26 It was found
in these investigations that a metal ion variation
allowed a possibility to assess the extent to which
protein structural constraints govern conforma-
tional balance in blue copper centers.27 Moreover, it
has been suggested that replacement of the native
Cu(II) by other metal ions should have minimal
effects on the metal binding site because of the
existence of an extensive network of hydrogen bonds
and other interactions that make the copper site
 THE METAL SITE OF PSEUDOMONAS AERUGINOSA AZURIN
more rigid and thereby a more ordered part of the
structure.28–30 The optical absorbance bands of cop-
per, cobalt, and nickel plastocyanin and azurin have
been discussed in further detail by Gray et al.31 and
by Larsson et al.32
We have now determined the crystal structure of
Co(II)-azurin in order to find out the positions of this
metal in the metal site of azurin and thereby confirm
the assumptions made in numerous earlier spectro-
scopic investigations. The crystal structures of Zn(II)-
and Ni-azurin has previously been reported.33,34 In
addition, the crystal structures of two azurin mu-
tants are also included for comparison, namely the
Asn47Asp35 and Trp48Met36 with Zn(II) and Ni(II) in
the site, respectively. This now makes it possible to
correlate some structure–property relationships
among these metal-substituted azurins. One very
intriguing question we deal with in this paper is why
copper occupies an essentially distorted trigonal
bipyramidal site in azurin while the other metals,
Zn(II), Co(II), and Ni(II), adopt a distorted tetrahe-
dral position by using the carbonyl oxygen from
Gly45 as an additional ligand.
EXPERIMENTAL PROCEDURES
Expression and Purification
The cloning and expression of wild-type azurin
have been described previously.37 The azurin was
expressed in Escherichia coli strain RV308 and
fermented in 1-L cultures of LB media. The E. coli
was cultivated as a shake culture (16 hours) with
CuCl2 (100 µM), ampicillin (100 mg/L), and 0.5 mM
isopropyl-b-D-thiogalactopyranoside (IPTG) added
when the cultivation was started.
Wild-type azurin was purified essentially accord-
ing to Karlsson et al.38 applying a cation-exchange
step (CM-52) with the pH gradient 4.1 to 9. This was
followed by gel filtration in 100 mM phosphate
buffer, pH 7. The purity of the sample was checked
on a Pharmacia PhastSystem using Pharmacia SDS-
PAGE gradient 8-25 gel under reducing conditions
with b-mercaptoethanol, showing that the sample
only contained azurin. The copper was then removed
by dialysis against 0.1 M KCN in 20 mM Tris-HCl,
pH 7.2, for 14 days at room temperature. The
Co-azurin was formed by dialysis against 100 µM of
CoCl2 for 7 days at room temperature. Any residual
apo-, Cu-, and Zn-coordinated protein was removed
in an anionic exchange chromatography step on a
Waters Q-8 HR column. The sample was applied in
20 mM Tris-HCl, pH 8.3, to which 100 µM CoCl2 had
been added. The protein was eluted at room tempera-
ture with a gradient of 0–80 mM NaCl. Optical
spectra were recorded, and the fractions containing
the Co-azurin were pooled and concentrated, using
an ultrafiltration unit, and dialyzed against deion-
ized water for 48 hours at 4°C before the crystalliza-
tion experiment.
387
Spectroscopy
The X-band EPR spectrum was recorded on a
Bruker ER 200D-SRC spectrometer equipped with
an Oxford Instruments ESR-9 helium-flow cryostat.
The sample was about 1.1 mM in 100 mM Hepes pH
7.0, and the spectrum was recorded in the tempera-
ture range 8–56 K using a modulation amplitude of 2
mT. The simulations were performed with a local
program, kindly provided by Dr. Örjan Hansson.
Crystallization and Data Collection
The bluish well-formed prismatic crystals of the
Co-azurin were obtained by the vapor-diffusion hang-
ing-drop technique from a solution containing 3.6 M
ammonium sulfate, 0.5 M lithium nitrate, and 0.1 M
acetate buffer at pH 5.7 and at the temperature of
24–25°C in about 10 days. The largest crystal of this
form was about 0.4 3 0.2 3 0.1 mm.
The Co-azurin x-ray dataset was collected on an
Xentronix position-sensitive area detector system
using CuKa radiation from a RIGAKU RU200 BH
rotating anode operated at 40 kV and 80 mA with a
0.3 3 3.0 mm focal spot. A graphite monochromator
together with a 0.5 mm collimator was also used. The
area-detector chamber was mounted 10.5 cm from
the crystal, and the dataset was collected at room
temperature. The individual frames were contiguous
in that the beginning of each small oscillation range
(0.1°) coincided with the end of the previous range.
The determination of the unit cell parameters, crys-
tal orientation and the integration of reflection inten-
sities was performed with the XENGEN program
system.39
A second Co-azurin X-ray dataset was collected on
the novel SMART Siemens CCD based area detector
system using molybdenum radiation. X-rays were
generated using a regular sealed tube and an X-ray
generator operating at 55 kV and 50 mA with a 0.4 3
12 mm focal spot. The 9-cm-wide CCD area detector
was mounted 12.0 cm from the crystal, and the
dataset was collected at room temperature. A graph-
ite monochromator followed by a 0.5-mm collimator
was utilized. The individual frames were contiguous
with 0.3° oscillation ranges. The determination of
the unit cell parameters and the crystal orientation,
and the integration of reflection intensities were
performed with the SAINT program system.40 The
final dataset after internal scaling consisted of 31366
reflections to 1.9 Å resolution, and the complete data
collection took about 14 hours. The dataset repre-
sents 80% of the expected number of reflections to
this resolution. For the entire dataset 30,826 reflec-
tions had intensities greater than 1sI, 29,983 reflec-
tions had intensities greater than 2sI, and 27,413
reflections had intensities greater than 3sI. A de-
tailed presentation of the data collection statistics is
given in Table I. The two datasets for the cobalt-
azurin crystals were scaled together using a stan-
388 N. BONANDER ET AL.
dard least-squares procedure. The statistical Rscale
value became 0.065 based on F values larger than
3s(F) from the two datasets.
Structure Solution and Refinement
The structure of the cobalt-substituted azurin was
isomorphous with the wild-type structure previously
solved by Nar et al.12 Consequently, the primary
electron density maps were constructed by using
phases from the wild-type structure, including water
molecules and a nitrate molecule found in the wild-
type structure, but initially omitting the copper in
the metal site. Crystallographic refinement was car-
ried out with energy restraints using X-PLOR.41 The
cobalt site was refined without energy restraints.
After a conjugate-gradient, energy-restrained posi-
tional refinement, followed by a restrained tempera-
ture-factor refinement, electron density maps were
again calculated. These maps (2Fo–Fc and Fo–Fc)
were inspected, and the coordinates were manually
corrected on a graphics display (Evans and Suther-
land) by using the program FRODO.42 In particular,
the coordinates of the water molecules, initially
adopted from the wild-type azurin structure, were
inspected, and water molecules were added or de-
leted according to indications in the Fourier map and
the formation of reasonable hydrogen bonds. Finally,
the coordinates were subjected to additional posi-
tional and temperature refinement, and the R value
became 17.5% for the cobalt azurin structure. The
parameters from the final refinement are summa-
rized in Table I. Noncrystallographic symmetry re-
straints and free R factors were not utilized during
the refinement.
RESULTS
X-ray Crystallography
The final crystallographic models consist of 3872
atoms in the azurin structure and, in addition, 320
water molecules and one nitrate ion (Table I). The
four molecules in the asymmetric unit are packed as
a dimer of dimers, and the packing in this metal-
substituted azurin structure is almost identical to
the dimer packing in the wild-type crystals, grown
using the same conditions.
Most of the structure is well defined, especially the
b strands, the loops around the copper site and all
internal side chains. They have relatively low B
factors, 5–15 Å2. The 2Fo–Fc Fourier maps contoured
at the 1s level show continuous density for all
main-chain atoms (Fig. 3). A comparison of the four
monomers in the asymmetric unit suggests that the
deviation from the main-chain atom positions is
small. An inspection of the 2Fo–Fc electron density
Fourier maps at the metal site again reveals that
this site and its ligands are very well defined.
The copper sites in different azurins have previ-
ously been described and discussed in detail by
TABLE I. Data Collection Parameters and
Statistics, the Final Model and Refinement
Results for the Cobalt Azurin
Unit cell constant (Å)
a 57.4
b 80.4
c 110.3
Space group P212121
No.of molecules in the asymmetric unit 4
Crystal mosaicity (°) 0.17
Total no. of measurements 52176
Total no. of unique reflections 31366
Data completeness (%) 80
Reflection averaging (%) Rma 8.5
No. of atoms used in refinement 4193
protein atoms 3872
solvent 320
nitrate 1
No. of reflections in the resolution range 30862
No. of parameters 16773
Root means square diviation
bonds (Å) 0.014
angles (°) 1.88
R-value (%)b 17.5
aR
m 5 SH Si51 0 7I(H)8 2 I(H)i 0 /SH Si51 I(H)i where I(H)i is
N N
the ith measurement of reflection H, 7I(H)8 is its mean
value and the summation extends over all the reflections
measured more than once in the set.
bS\F 0 2 0 F \/S 0 F 0
0 c 0
Baker43 and by Nar et al.12 In other crystallographic
investigations the metal site has been investigated
when the copper has been replaced with zinc or
nickel.33–35 We also have determined the structure of
the nickel derivative, with results consistent with
those of Moratal et al.34 Table II contains a summary
of distances for the canonical metal site geometry for
different metals. For all cobalt and nickel structures
it was found that, similar to the zinc structures, the
ions have a distorted tetrahedral coordination. Thus,
Co and Ni are coordinated by four ligands, the atoms
of Gly45 CO, His46 Nd1, Cys112 Sg, and His117 Nd1
(Table II and Fig. 2). The distance between the
metals and the Sd of the Met121 is 3.1–3.5 Å, and the
interaction must be regarded as weak.
A complementary investigation utilizing the infor-
mation in the Cambridge Data Base (Version 5.08,
October 1995)44 has been performed. The database
was searched for Cu, Zn, Ni, and Co structures
having coordinating atoms as in the azurin struc-
ture. In the primary retrieval, the data base was
searched for all structures with the metal coordi-
nated to at least two N and one S (trigonal, tetrago-
nal, pentagonal, or octahedral). In a second search,
only structures having the coordinating atoms
NNSSO were accepted.
Table III summarizes the results from the primary
retrieval model where all structures having at least
the minimal NNS coordination were searched, and
the average bond distances and their statistical
 THE METAL SITE OF PSEUDOMONAS AERUGINOSA AZURIN
distribution are presented for typical metal–sulfur,
–nitrogen, and –oxygen bonds. Table III also con-
tains one column indicating the number of small
molecule structures having the five coordinating
atoms as in the metal azurin-derivative structures.
As can be seen from Table III the bond distances
found for the various metal–ligands in the azurin
389
Fig. 3 A and B. An omit map showing the electron density
for the cobalt site in azurin at 1s level.
structures closely relate to the averages found in the
Cambridge Data Base. This indicates that, although
the protein crystal structure of Co-azurin in this
work is of limited resolution (about 1.9 Å), the
distances in the metal site agree well with the
‘‘similar’’ small molecule complexes in the database,
mostly determined to atomic resolution (0.8 Å or
390 N. BONANDER ET AL.

TABLE II. The Metal Site Geometry in Cobalt Azurin Compared to Wild Type, Nickel and Zinc Azurin.
Also Included are the Structures of Nickel-Trp48Met and Zinc-Asn47Asp

Metal to ligand bond length (Å)a Co-WT Ni-WT34 Ni-Trp48Met36 Zn-WT33 Zn-Asn47Asp35 Cu-WT12
M—45 C/O 2.15 2.46 2.35 2.32 2.36 2.97
M—46 Nd1 2.32 2.23 2.15 2.07 2.09 2.11
M—112 Sg 2.20 2.39 2.49 2.30 2.27 2.25
M—117 Nd1 2.25 2.22 2.07 2.01 2.04 2.03
M—121 Sd 3.49 3.30 3.34 3.38 3.44 3.15
45 Ca—121 Ca 11.39 — 11.40 11.38 11.50 11.65
45 C/O—121 Ca 9.07 — 9.09 9.09 9.19 9.42
aThe presented distances are the average of the four molecules in the asymmetric unit of Pseudomonas aeruginosa, except for the
Ni-azurin34, where only molecules 1 and 3 were available.

TABLE III. Distances (Å) Between Metals and Liganding Groups, Collected From the Primary Search in the
Cambridge Data Base and Search for the Total Number of Exact Geometries

Sulfur Nitrogen Oxygen No. No. of

Metal min ave max min ave max min ave max of obs. exact geom.
Cu1 2.21 2.30 2.57 1.92 2.04 2.20 1.89 2.23 2.70 60 —
Cu21 2.26 2.28 2.62 1.93 2.02 2.26 1.91 2.21 2.64 68 16
Ni21 2.36 2.42 2.49 2.01 2.06 2.11 2.05 2.10 2.16 45 15
Co21 2.23 2.35 2.46 1.95 2.02 2.14 1.90 1.98 2.16 28 0
Zn21 2.29 2.40 2.53 2.06 2.17 2.34 1.94 2.19 2.52 37 0

better). There is no structure in the database having
the five coordinating atoms—NNSSO—for any Co
complex, making this metal site unique from a
structural point of view.
Spectroscopy
The recorded optical spectrum of cobalt-substi-
tuted wild-type azurin was in agreement with previ-
ously published spectra.45,46 Figure 4A shows the
X-band EPR spectrum of Co-azurin. It arises from
one of the two Kramers doublets in the S 5 3/2
ground multiplet of high-spin Co(II). The high-field
region is disturbed by a radical component at g 5
2.00, but it is obvious from the lineshape in this
region (the z direction ) and at the low-field end (x)
that Co hyperfine structure contributes to the width
(59Co has a nuclear spin I 5 7/2). In the region
around gy the hyperfine structure is unresolved.
However, the simulations revealed that Ay is in the
range 20–30 G.
The simulated Co(II) spectrum in Figure 4B was
generated with the effective g values (S8 5 1⁄2) gx8 5
5.23; gy8 5 3.83; gz8 5 1.995, the hyperfine splittings
Ax8 5 31; Ay8 5 28; Az8 5 53 G and the widths Wx 5
60, Wy 5 90, Wz 5 100 G, respectively. The angular
dependence of the width was that of unresolved
hyperfine structure. Using first-order perturbation
treatments and the relation between E/D and the
intrinsic g values (e.g., Werth et al.47), the param-
eters for the S 5 3/2 spin hamiltonian are estimated
to be E/D 5 0.11, gx 5 2.31, gy 5 2.27, gz 5 2.07, not
far from axial symmetry. The second Kramers dou-
blet is predicted to have one effective g value around
6 and the other two values ,1, which implies that
the transition probability is very low and its EPR
spectrum weak. This explains why no signal was
detected that could be associated with this doublet in
the temperature range 8–55 K.
The EPR spectrum of high-spin Co(II) usually is
strongly temperature-dependent due to efficient re-
laxation mechanisms. The Co-azurin spectrum was
studied in the range 8–55 K. Its shape was essen-
tially unchanged up to about 25 K, and it was
observable, although broadened, up to 55 K.
The EPR spectrum of Co-azurin has very recently
also been reported by Jiménez et al.48 Their spec-
trum of the protein at pH 7.5 differs from ours in g
values (2.01, 3.77, 5.91), is less resolved and appar-
ently lacks the hyperfine structure around g 5 2.
Furthermore, the spectrum is only clearly visible
below 20 K. Thus, their complex is different from the
one we observe, for reasons not understood. They
estimate that the zero-field splitting is only 7 cm21,
whereas our studies of the temperature dependence
suggest that the splitting is larger.
It has been demonstrated that it is not possible for
CN2 to enter the copper site of wild-type azurin.
However, in a recent report it has been shown that
CN2 can coordinate to the copper site of Met121X
(X 5 Gly, Ala, Val, Leu, and Asp) in the cavity that is
formed when Met121 is mutated.49,50 The movement
of the cobalt atom toward the carbonyl oxygen of
Gly45 has increased the distance to the axial Met121
and possibly introduced a cavity large enough for
CN2 to bind. To investigate this, Co-azurin was
subjected to 20 mM cyanide and the optical room
temperature spectrum was recorded. No change of
the optical bands could be observed, and it is con-
 THE METAL SITE OF PSEUDOMONAS AERUGINOSA AZURIN 391

Fig. 4. Recorded (A) and simulated (B) X-band EPR spectra of Co-substituted wild-type azurin
from Pseudomonas aeruginosa. Experimental conditions were, microwave frequency, 9.44 GHz,
temperature, 10 K, microwave power, 20 dB, and modulation amplitude, 20 G.

TABLE IV. Ionization Potentials for Various
Metals in the Azurian Metal Site57
Cu Zn Ni
1st 7.73 9.39 7.64
2nd 20.29 17.96 18.17
3rd 36.84 39.72 35.19
cluded that CN2 does not bind at room temperature
to the metal ion in Co(II)-azurin.
DISCUSSION
The Co, Ni, Cu, and Zn atoms belong to the first
transition series. The energies of the 3d and 4s
orbitals in the neutral atoms are quite similar, and
their configurations are 3dn4s2 except for Cu (3d104s1),
which is attributed to the stability of the filled d
shell. Since the d orbitals become stabilized relative
to the s orbital when the atoms are charged, the
predominant oxidation states in ionic compounds
and complexes are II or higher. Due to its electronic
structure, copper has a higher second ionization
potential than the other elements (Table IV) and
subsequently the Cu(I) state is also important. How-
ever, most cuprous compounds are fairly readily
oxidized to cupric compounds but further oxidation
to Cu(III) is difficult. The d9 configuration makes
Cu(II) subject to Jahn-Teller distortion if placed in
an environment of regular symmetry (i.e., octahe-
Co dral or tetrahedral), and this has a profound effect on
7.88 all its stereochemistry. The typical distortion found
17.08 in numerous small molecule compounds is a plane of
33.50 four short C ligand bonds and two trans long ones. In
the limit the elongation leads to a situation indistin-
guishable from square planar coordination, found in
many complexes of Cu(II).In azurin, the metal site,
defined by the protein matrix, is rather asymmetric
and thereby already acceptable for the copper atom,
and no further restraints are required on the ligands
from the copper atom point of view.
Divalent cobalt forms numerous complexes of vari-
ous stereochemical types in normal inorganic and
organometallic environments. Octahedral and tetra-
hedral ones are most common, but there is a fair
number of square planar complexes as well as some
which are five-coordinated.51 Cobalt(II) forms tetra-
hedral complexes more readily than most other
transition-metal ions. Co(II) is the only d7 ion of
common occurrence. The electronic absorption and
the magnetic circular dichroism spectra MCD of
Co-azurin have been argued to be indicative of Co(II)
in a distorted tetrahedral coordination geometry.52
The crystal structure demonstrates that the Co-
azurin is indeed in a distorted tetrahedral coordina-
tion. The long metal–Met121 Sd distance, 3.5 Å,
392 N. BONANDER ET AL.

shows that the interaction with Met121 must be very

weak, in spite of the sizable isotropic shift observed
in NMR.24
The observed g values of the EPR spectrum are
close to those of the pseudotetrahedral complex
described by Benchini et al,53 but neither the g
values54 nor the zero-field splitting are reliable indi-
cators of the geometry. However, the ligands in
azurin provide a ‘‘rack’’ that apparently is well
defined as seen from the electron density Fourier
maps and this may induce a strong ligand field
around the metal. In line with this, the EPR spec-
trum of Co-azurin is unusually well defined com-
pared to other cobalt substituted proteins, for ex-
ample, carbonic anhydrase,54 carboxypeptidase A,55
and alcohol dehydrogenase.47 Such a rigidity of the
site possibly reflects that the azurin metal site is
more buried and consequently in a more hydrophobic
environment. The buried nature of the azurin metal
site substituted with cobalt is also demonstrated by
the lack of interaction with exogenous cyanide.
Ni(II) forms a large number of complexes in an
inorganic or organometallic environment encompass-
ing coordination numbers 4, 5, and 6 and all the
main structural types. Moreover, it is characteristic
of Ni(II) complexes that complicated equilibria, which
are generally temperature-dependent and some-
times concentration-dependent, often exist between
these structural types. When small substituents are
present, planar or nearly planar complexes are
formed. Except for the [NiX4]22 species a rigorously
tetrahedral configuration is not expected. However, Fig. 5. Simplified one-dimensional representation of the metal
in some cases it can be found that there are marked sites in the azurin. Co (A) and Ni (B) in the wild-type protein34; Ni in
the Trp48Met mutant36 (C); Zn in the wild-type protein33 (D); Zn in
distortions from the highest symmetry possible, given the Asn47Asp mutant35 (E); and Cu in the wild-type protein (F).12
the inherent shapes of the ligands.
In general, the divalent zinc ion can be found in
small molecule structures with a variety of coordina-
tion numbers from 2 to 8. Since there is no ligand a simplified qualitative one-dimensional fashion.
field stabilization effect in the Zn(II) ion because of The Met121 sulfur and the Gly45 carbonyl oxygen
the completed d shell, its stereochemistry is deter- are positioned symmetrically around the zero level
mined solely by consideration of size, electrostatic such that the distance between them agrees with
forces, and covalent bonding forces. In most cases, that found in the crystal structures. Then, on the
the divalent zinc atom tends to assume the coordina- vector connecting these atoms, the metal ion has
tion number of 4. been projected. Finally, the plane defined by His46
From the reported crystal structure of the metal- Nd1,His117 Nd1, and Cys112 Sg is positioned at the
substituted Co-azurin, as well as the crystal struc- correct distance from the metal ion. One very clear
ture of the and Cu-, Ni-, and Zn-azurin, some struc- observation is that, while copper essentially occupies
ture–property relationships are now possible to a distorted trigonal bipyramidal site in azurin, the
explore. The coordination to the metal ions discussed other investigated metals (Zn, Co, and Ni) adopt a
here, which all are considered intermediate in the distorted tetrahedral position by using the carbonyl
soft-hard scale, is in gross terms quite similar, oxygen from Gly45 in the structure as an additional
illustrating that the protein provides a site that can ligand† (Fig. 5). In addition, it is clearly seen that
be modified only in relatively minor detail, presum-
ably because of the existence of an extensive network
of hydrogen bonds around the metal binding site. †The geometric center of gravity of the three equatorial
ligands and the carbonyl oxygen would be situated at a distance
Interesting differences exist, however, in the axial from the plane, which would be one-quarter of the distance
coordination. To further demonstrate the difference between the plane and the apical atom. This position could be
in ‘axial’ position between different metal sites deter- thought of as the ‘‘perfect’’ tetrahedral coordination. It should
be noted that, due to the linearization of the structure, the
mined so far, the position of the metal ion relative to distances between the metal ion and the axial ligands in Figure
its five nearest neighbors is presented in Figure 5 in 5 are shorter than the correct ones.
 THE METAL SITE OF PSEUDOMONAS AERUGINOSA AZURIN
when the metal–carbonyl oxygen distance decreases,
the equatorial plane of the three strong ligands also
gets closer to the apical oxygen.
The ions in question have all the same charge and
very similar ionic radii—Cu(II) 5 0.72 Å, Zn(II) 5
0.69 Å, Ni(II) 5 0.68 Å, and Co(II) 5 0.62 Å56—giving
no clue to the difference between their coordination
geometries. The data collected from the known struc-
tures (Table III) are, however, consistent with the
azurin structures. Thus, Co(II), Ni(II), and Zn(II) all
have significantly longer average distances to a
coordinating sulfur, and have comparatively short
average distances to a coordinating oxygen com-
pared to Cu(II). Lowrey and Solomon57 argue that
the position of the metal is a result of a delicate
balance between the coordination forces to the axial
ligands. For Zn(II), they suggest that the degree of
covalence of the binding to Met121 Sd decreases
relative to that of Cu(II), while the ionic interaction
with the Gly45 carbonyl oxygen increases. This
argument has shortcomings, however, as illustrated
by our finding that in the Met121Ala mutant, where
an empty space replaces the Met121, the Cu ion still
resides in the plane of the strong ligands (on the
average 0.03 Å from the plane on the 121Ala side).50
Zn(II) usually is considered to be a somewhat
softer ion than Cu(II) and thereby preferring sulfur
as a ligand. As shown by the statistics from small
complexes discussed above, this does not allow one to
conclude that Zn(II) would have a shorter metal–
sulfur distance. Such arguments are also not appli-
cable in the case of the various metal complexes with
azurin. What determines the unique geometry of the
Cu(II) azurin is the detailed orbital arrangement.
The bonding of the two imidazole nitrogens is of s
type, and the cysteine 3Spy orbital in the trigonal
plane plays the role of the two s orbitals from the two
missing ligands opposite to the nitrogens. In this
way the square planar coordination preferred by
Cu(II) is completed.32 This trigonal arrangement is
particularly stable if the third orbital is as large as
the cysteine 3Spy orbital. The profound covalency
induced by this arrangement, leads to the stable
arrangement, independent of smaller changes in the
axial ligation. Theoretical calculations and theoreti-
cal aspects on the electronic structure and orbital
arrangement are consequently essential components
to understand the influence of the protein on the
geometry of the metal coordination sphere and to
understand the metal coordination sphere itself.
The function of the copper in azurin is to allow
easy transfer of an electron in and out of the metal
ion. This is facilitated by the special geometry of the
Cu(II) protein. Its geometry is not very different from
that preferred by Cu(I), namely, tetrahedral. A small
displacement out of the trigonal plane as seen for the
other metals discussed here would be sufficient for a
switch to that geometry. Thus, the blue copper
proteins seem to have evolved from the requirement
of constructing a site that minimizes the electron-
393
transfer reorganization energy. For a better analysis
of this phenomenon, accurate data for the Cu(I)
protein would be required, not only for the wild-type
protein but preferably also for mutants such as
Met121Ala. Such work is in progress. The positions
of the various metal ions, found in crystallographic
determinations, in azurin from P. aeruginosa may
now serve as guidance for spectroscopic investiga-
tions particularly useful in the interpretation of the
fine details of obtained spectra. The established
positions can also be used in various theoretical
calculations where these metal sites are utilized as
model coordinates.
ACKNOWLEDGMENTS
We thank Dr. Roland Aasa for fruitful discussions
and assistance with the EPR measurements. This
work was supported by the Swedish Natural Science
Research Council and the BioVäst Foundation for
Biotechnology (Göteborg).
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