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Biochimica et Biophysica Acta 1757 (2006) 886 912

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Review
Chance and designProton transfer in water,
channels and bioenergetic proteins
Colin A. Wraight
Center for Biophysics and Computational Biology, MC-147, 607 South Mathews Street, University of Illinois, Urbana, IL 61801, USA
Received 3 May 2006; received in revised form 7 June 2006; accepted 13 June 2006
Available online 14 July 2006

Abstract

Proton transfer and transport in water, gramicidin and some selected channels and bioenergetic proteins are reviewed. An attempt is made to
draw some conclusions about how Nature designs long distance, proton transport functionality. The prevalence of water rather than amino acid
hydrogen bonded chains is noted, and the possible benefits of waters as the major component are discussed qualitatively.
2006 Elsevier B.V. All rights reserved.

1. Introduction mass. Consequently, for donoracceptor distances that are not


in close contact, so many possible paths are accessible to an
Although electron and proton transfer may appear to have electron that they effectively sample the average composition of
much common and, in certain regimes, have fundamental the medium; this comes down to atomic packing density, which
similarities, they differ greatly at a phenomenological level, is not very variable. Extensive surveys by Dutton and
especially as they are encountered in the context of protein coworkers have found no clear examples of proteins in which
function. With few exceptions, biological electron transfer (ET) the connectedness of redox centers is needed to account for the
is highly insensitive to the structural details of the protein as a kinetic competence of the electron transfer [1,6]. Furthermore,
transmission medium [1,2]. The protein plays a critical role in there is no distinction between the connectedness of (or packing
tuning the thermodynamic properties of redox active cofactors density between) two random points in any protein and that of
but, in all cases known so far, it otherwise serves only to two functional donoracceptor locations, e.g., cofactors. The
position them at distances that allow electron transfer rates that conclusion is very strong that structure, as in specific pathways,
are generally significantly faster than the rate limiting steps of is irrelevant to the vast majority of electron transfer reactions.
an overall process. This is not to say that the intervening The approach taken by evolutionary design has been to use
medium does not provide the mechanism of electron transfer protein ligands to place cofactors at distances short enough to
electrons are transmitted by virtue of the atomic and molecular ensure competent electron transfer rates. Often the rates are
orbital wavefunctions that set the excited state energies of the orders of magnitude faster than necessary, but in a few cases
medium [35]. However, this could be chicken fat for all the they are simply adequate. The latter may include the very fast
structure that is needed.1 kinetics of photosynthetic primary photochemical events, but
The justification for this cavalier attitude is the length scale these are achieved by near-contact of cofactors and the protein
over which electrons can tunnel, by virtue of their very tiny is actually excluded altogether!
The distances observed for intramolecular electron transfer
Abbreviations: ET, electron transfer; PT, proton transfer; gA, gramicidin A; in physiological reactions are almost universally less than 15 ,
GMO, glycerylmonoolein; PRG, proton release group (of bacteriorhodopsin); edge-to-edge.2 From Marcus theory of non-adiabatic electron
FTIR, Fourier-transform infrared; RC, (bacterial) reaction center
Tel.: +1 217 333 3245; fax: +1 217 244 6615.
2
E-mail address: cwraight@uiuc.edu. The exact meaning of this expression is the source of much of the debate in
1
This refers to one of Harry B. Gray's more colorful criticisms of the no- this area. In general, it means the distance between edge atoms that are strongly
pathway approach to biological electron transfer. coupled to the redox center, for example the conjugated macrocycle of a heme.

0005-2728/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2006.06.017
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 887

transfer, this distance limit ensures electron transfer times faster


than 100 s for reactions that are energetically favorable or even
slightly endergonic. At shorter distances, the rate increases by
10-fold for every 1.7 decrease in distance, allowing very rapid
rates to be achieved without direct contact [2].
In contrast to electron transfer, proton transfer (PT) is
exquisitely dependent on structure. Since tunneling is propor-
tional to (mass) 1/2, the distance over which a proton can
tunnel is less than a covalent bond length and the overriding
requirement for proton transfer is proximity, whether the
mechanism is non-adiabatic (tunneling) or adiabatic [7]. In
biology, there are few situations in which proton and hydrogen
atom tunneling appear to be kinetically important, involving
very weakly acidic groups, notably carbon acids [810], and
the general significance is still controversial [1116]. In the
case of hydrogen bonded normal acids,3 however, the
adiabatic process is generally so facile that tunneling is
irrelevant [18,19]. Nevertheless, because of the elementary
nature of PT, a relationship between energetics and kinetics is Scheme 1.
often apparent, and the formalisms for describing PT appear
similar to those used for ET, regardless of the underlying between H3O+ and H2O allows for a unique transport process
mechanism [2022]. known as the Grotthuss mechanism, which transfers protonic
Proton transfer in biology falls into two broadly useful, charge without diffusive movement of either an individual H+
but not fundamentally different, classesthe reactions of or oxygen atom (Scheme 1).
catalysis and chemistry, and the reactions of transport. In the This process is similar, in essence, to the proposal of von
former, the proton is transferred between two atoms in a Grotthuss, 200 years ago, for the migration of charge in aqueous
process of chemical conversion or acidbase catalysis, and electrolysis [26]. It is remarkable that this predates the
further transfers are absent or not kinetically important. In publication of the atomic theory [27] and that, at the time,
transport reactions, the purpose is to move protons over even afficionados did not know the correct composition of
significant distances, often on the order of a membrane water, which von Grotthuss wrote as OH. This transport
thickness. This necessarily involves many elementary proton mechanism is now celebrated with his name. Its true
transfer steps and implies a significant structural component, significance for H+ ion transport in water was not appreciated
or path. In principle the path may be permanent or transient, for 100 years, when Danneel first attempted a mechanistic
but there are substantial energetic constraints on forming one description of the elementary process [28]. This initiated
transiently. another 100 years of argument over the role of rotation (of
In trying to get a broader perspective on what features H3O+ or H2O) in the rate limiting step [2932].
characterize, or are essential for, biological PT, I have bitten off Hydrogen bonding between water molecules makes the
more than I can chew. Nevertheless, I hope that some of these Grotthuss process (also termed structural diffusion) fast and
observations will be useful to the bioenergetic community, at efficient, and it is responsible for the major part ( 85%) of the
large. apparent ionic mobility of H+ in water, which is about 67 times
that of Na+.4 The unique nature of this process has attracted
2. General features of H+ transport in water much interest, but the mechanism is only now becoming clear at
an atomic level [19,22,3235].
The electrostatic binding energy of the proton is so large that The current view of the Grotthuss mechanism in bulk water
it has no independent existence in condensed phases. In water, it is that proton transfer occurs in a step-wise manner, via the
is generally considered to be present as hydronium, H3O+, interconversion of an Eigen cation, H9O4+ = (H2O)3H3O+, and a
which is similar to Na+ in size and solvation characteristics. It Zundel cation, H5O2+ = H2OH+OH2 [32,33]. The activation
can therefore be expected to exhibit diffusive properties similar energy reflects the breakage of a more or less normal hydrogen
to Na+, with diffusion coefficient, DNa = 1.33 10 5 cm2 s 1 bond (approximately 2.5 kcal/mol) in the second solvation shell
[23], or perhaps water, with DH2O = 2.3 10 5 cm2 s 1 or (between H9O4+ and another H2O), allowing reorganization
0.23 2 ps 1 [24,25]. However, the rapid exchange of H+
4
Agmon [31] has argued that the solvated proton is hydrodynamically
3
Normal acids were defined by Eigen [17] as those that exhibit near immobile, so that all the measured mobility is by the Grotthuss mechanism.
diffusion controlled PT rates when thermodynamically favorable (pKa > 0), This seems extreme and unwarranted, since the hydrogen bond strengths
with the reverse rate smaller by a factor 10pK. These are also what are defined between the solvent water and the first solvation shell of Na+ and H3O+ are
as acids (or bases) in common experience, involving electronegative atoms, similar. Thus, somewhat equivalent mobilities of Na+(aq) and H3O+(aq) (i.e.,
especially oxygen and nitrogen. H9O+4) should be expected.
888 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

around the hydronium to produce the Zundel ion configuration, can be extensive, as first noted by Zundel, and the charge
within which the excess proton is shared equally between two distribution is highly polarizable and is very sensitive to local
oxygen centers. Reformation of a hydrogen bond with another perturbations [42,43]. Depending on the extent of the
water molecule localizes the excess proton on a new Eigen ion. delocalization, polarizable protons give rise to varying degrees
These events allow transfer of the proton charge across the of continuum absorbance in the infrared spectrum. In extreme
OO distance of an Eigen cation (approximately 0.255 nm) in cases, which are nevertheless easily created in hydrated films of
the lifetime of proton exchange in water (1.33 ps from NMR weak acidbase mixtures, the continuum extends throughout
[36]). This yields a 3-dimensional diffusion coefficient of the mid-IR, from the OH stretch of water (at about 3600 cm 1)
(0.255 nm)2/6(1.33 ps) 8.1 10 9 m2 s 1 (0.81 2 ps 1) To to the far-IR region. In less extensive delocalizations, the IR
this should be added the normal (molecular or vehicle) signature is contracted, and can be distinctive. Calculations
diffusion of H3O+ (similar to that of Na+, 0.13 2 ps 1), show clear differences between solvated H3O+ and H5O2+ in the
yielding a total in good, but perhaps fortuitous, agreement with mid-IR between 1800 and 2600 cm 1 [4345], with some
the experimental value (0.93 2 ps 1 [37]). An important experimental support [4648].
conclusion from this correspondence is that proton transfer in The PT step rate within a hydrogen-bonded chain can be as
water is an incoherent process. fast as for the Grotthuss mechanism in bulk water, with
The elementary PT rate in bulk water may benefit somewhat elementary transfer times between sites on the order of 1 ps.
from the fact that the free energy of the Zundel ion (essentially a However, the transfer of a proton along the chain reorients the
transition intermediate) lies quite close to that of the Eigen ion hydrogen bond connectivity, which must be restored in order
[38]. However, the pairwise jump time is expected to be fast for to conduct a second proton in the same direction. This led to
any energetically favorable or near-neutral proton transfer, e.g., much attention being spent on both the hopping action that
for normal acids with similar pKa values [7,17]. Thus, a transports the proton and the turn motion that regenerates the
Grotthuss-like mechanism can be envisioned for any chain of starting configuration of the chain for steady state turnover
hydrogen bonded acidbase groups with an appropriate [49]. In contrast, in bulk water, the high coordination number
geometry for continuous hydrogen bondingeach hydrogen- of water yields a multiplicity of hydrogen-bonded paths in 3
bonded atom must act simultaneously as donor and acceptor. dimensions, and the turn motions restoring a prior arrange-
This could be fulfilled by several O and N functional groups, ment are not important in the steady state transfer of protons
and especially OH. The potential for hydrogen-bonded chains (conductivity).
to facilitate fast proton transfer in biology was developed by Thus, the question arises whether the potential for rapid
Nagle and Morowitz and coworkers following a suggestion by proton transfer in hydrogen bonded chains is realized in
Onsager ([39], and see [40]), and gave rise to the term proton proteins. Furthermore, the highly directed PT in a one-
wire [40,41] (Scheme 2). dimensional path can be controlled and/or limited by catalytic,
electrostatic and conformational events, which may be either
3. Long distance proton transport incidental or designed.

Each step of a long distance proton transport requires 4. Ion channels and other transport devices
rearrangement of charge. Inside the low dielectric of a protein,
this implies substantial electrostatic and reorganization costs at If we look for inspiration in the rapid ion transport by K+
every step. Furthermore, if the proton originates in the bulk and Na+ channels, the electrostatic and reorganization costs of
phase, there is an additional desolvation penalty that is also bringing charge into a protein and membrane interior have
largely electrostatic (Born energy). Proton wires would be a been addressed directly by preorganized structures that
uniquely suitable way of transporting H+ in a structured compensate for the desolvation penalty in the selectivity filter,
medium, because the covalent association of the H+ with the and resolvate and electrostatically stabilize the cations as they
carrier groups and the hydrogen bonding between them enter a large, internal, aqueous cavity. The selectivity filter
substantially diminish the electrostatic cost of bringing the necessarily binds the desolvated cations, which is counter-
charge into the lower dielectric medium of the protein. The productive for rapid transport. This is overcome by ionion
delocalization of the charge through a hydrogen bond network interactions that help displace the ions as they proceed in
single file through a sequence of 5 or 6 weak binding sites.
This was proposed half a century ago on the basis of
electrophysiological studies of channel properties [50], and it
was stunningly confirmed by the K+ channel X-ray structures
[51,52].
For less specific cation-selective channels, such as the
acetylcholine receptor, structures are not yet available that can
support specific mechanistic models, but binding is certainly
weak and the multiple-ion conduction mode is not critical for
high conductances. Among channels of low specificity, the
Scheme 2. gramicidins are especially relevant because they have been
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 889

widely studied for both their alkali metal cation and proton complications from ion binding, which depresses the con-
conductance properties. ductance and requires multiple-ion mechanisms to overcome it
[67,72,73]. It should also be determined at low potentials, to
4.1. Gramicidin avoid saturation and interfacial polarization effects [66]. From
the large literature on cation conductances, for example in
The ionophorous gramicidin antibiotics are 15-residue linear membrane bilayers of glycerol monoolein (GMO), I extract the
peptides of alternating D- and L-amino acids that form ion following. The maximum single channel conductance for H+ in
channels in membranes [5355]. There are many minor gA (gH,max) is at least 2 nS [55,59,64]. From this, the diffusion
sequence variations, but the best known is gramicidin A constant for H+ in gA can be obtained [59]:
(gA) 5 -HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-
Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-NH- DH L2p :gH;max =ekB T
(CH2)2OHnote the charge-neutralizing N and C terminal 25  108 2 2  109 =1:6  1019  0:025
modifications. The gramicidins associate strongly with mem-
3  105 cm2 s1
branes and adopt a wide-bore, right-handed, 6 helix6 structure
that spans approximately one leaflet of a membrane bilayer, where Lp is the length of the gramicidin pore ( 25 ). This is a
with the formyl-N-terminus buried. Two molecules, in op- significant fraction of the value in water (9.3 10 5 cm2 s 1). In
posite leaflets, can associate by hydrogen bonding between fact, because the proton conductance of gA is so high, it is
their N-termini to form a complete, but transient, transmem- possible that gH,max is determined by the bulk phase mobility,
brane channel (Fig. 1). The lifetime of these head-to-head which saturates at similar acid concentrations (> 3 M HCl),
dimers is on the order of 0.11 s, depending on pH, tem- due to changes in water structure at high ionic strength
perature, and membrane lipid type. Covalently linked dimers, [55,60,74,75]. In this case, the mobility within the gA channel
exhibiting long-lived conductances, have proven very useful could be as large as that in water, or even greater.
for studying cation conductances, especially at the single The self-diffusion constant for bulk water is 2.3 10 5 cm2
1
channel level [5560]. s [25]. Hydraulic permeability measurements [71] show that
The gramicidin channel is readily permeable to monovalent the net diffusion of water through the gA channel in GMO
cations, notably the alkali metal cations, ammonium and bilayers is about 10 times smaller, DH2O 0.2 10 5 cm2 s 1.
protons. The tryptophan residues form a collar located in the Since net transport of water requires the coordinated diffusion
lipid headgroup-interfacial region of the membrane, and play an of the whole file of 89 waters in the channel, the local diffusive
important role in the energetics of cation entry into the channel. mobility of the individual waters is 89 times that seen in net
The substitution of one or more of the four tryptophans by flux, and is therefore almost indistinguishable from the bulk
phenylalanine or tyrosine in gramicidins B, C, and M, has phase value. This is supported by computational (Brownian
distinct effects on the conductance properties [6164]. dynamics) methods [76]. This indicates relatively weak inter-
Gramicidin has long been considered the poster-child for actions between the waters and the channel walls, i.e., no
the Grotthuss mechanism at work, and there is a tendency to stronger than waterwater.
impute abnormally high proton conductance to it. The The mobility of the water column as a whole is expected to
quantitative basis for the latter inference is shaky, however, provide an upper limit for alkali cation diffusion in the channel,
due to a number of technical difficulties, including surface as they move in single file with the waters, and conductance
potentials, surface polarizability, ion binding and access to the measurements yield somewhat similar values. For cesium, Cs+,
channel, that conspire to obscure the true channel conductance the most permeable of the alkali metal cations, gCs,max = 50
(rather than the total conductance, including entrance and exit 100 pS [67,73], and DCs 0.15 10 5 cm2 s 1 within the
events) [6567]. However, it is clear from electroosmotic and channel. This is close to the limit expected from the diffusive
streaming potential measurements that alkali metal cations permeability (Pd) of water in the channel, but there is a factor of
pass through the pore in single file with channel waters, while 23 uncertainty in the value of Pd, which has not yet been
protons pass without significant water flux [6872]. This resolved [53,68,70,77]. In bulk solution, DCs 2.05 10 5 cm2
strongly implicates the Grotthuss mechanism. s 1, so Cs+ mobility is decreased at least 14-fold (compared to
Conductance is a concentration dependent parameter, but the 3-fold, or less, for H+). Nevertheless, the Cs+ mobility is still
intrinsic mobility of most ions in a channel is related to the substantial and implies that interactions of Cs+ with the channel
maximum conductance [59,67]. Unfortunately, this is not a walls are weak. Measured dissociation constants support weak
straightforward measurement, due to conflicting requirements. binding for all alkali metal cations, although reported values are
It should be determined at high ionic concentration to override very variable [64,72, 73,7880].
the external resistance from bulk diffusion, but this introduces Thus, although protonic charge movement may be slightly
depressed relative to bulk, the effective diffusion coefficient for
5
H+ is at least 20 times larger than for any other ionic species, or
The commercial preparation, gramicidin D, is a mixture of gramicidins A for net flux of water. The large protonic conductance of
(the major component), B and C. The latter two have Tyr and Phe, respectively,
substituted for Trp at position 11. Gramicidin M, which lacks all Trp residues,
gramicidin is a consequence of a Grotthuss-type mechanism
is synthetic. that proceeds independently of molecular diffusion, at a rate
6
Also referred to as a (L,D) and 6.3 helix. quite similar to that in bulk water.
890 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

Fig. 1. Gramicidin A. Left: longitudinal view of the head-to-head dimer, with water molecules (green) modeled in. The membrane plane is vertical. Right: view down
the central pore (no waters). Note the four tryptophan residues at the C-terminal end of each monomer. The structure file, with waters placed according to a molecular
dynamics equilibration, was kindly provided by Rgis Poms (Toronto, Canada). All figures were prepared in VMD [277].

4.2. Rate limiting events in gramicidin permeation hydrogen bond reorientation is modulated by interactions
between the interfacial dipoles of the gramicidin tryptophan
Because of the known speed of pairwise PT rates, turning residues and those of the channel water column, which
motions were considered, at first, to be the likely rate-limiting kinetically control the entrance of a defect necessary for the
step for steady state proton conduction in a one-dimensional turning motion, starting at the channel exit. However, de Godoy
hydrogen bonded chain [40,49]. Initially, some microscopic and Cukierman found a counter-influence of the intrinsic dipole
calculations seemed to support this by showing that the turn potential of different lipids [88]. Warshel has argued strongly
motions in single files of water have significantly larger that the limiting barrier to most transport phenomena is the
activation energies than the proton hopping steps [81,82]. unfavorable free energy of transfer of charged or polar species
However, experimental results [83,84] and further computa- to the channel interior [87,8991]. This comprises a direct
tional studies [64,8587] of gramicidin A indicate that neither electrostatic component and a desolvation energy, both of
process is rate limiting in the net transfer of protons through the which must be sufficiently compensated or minimized to allow
channel. This is also true for the permeation of water through gA. rapid, net transport. However, without more specific develop-
Brownian dynamics simulations show that the major limitation ment, it is not at all clear which are kinetic and which are
to net transfer is an access resistance to entry of water energetic constraints in any of these models.
molecules [76]. By far the largest contribution to the access
resistance is associated with the desolvation process (removal of 4.3. Hydrophobic channels
approximately two water molecules) as bulk water becomes
single file channel water. Proton transfer in the polar gramicidin channel seems to be
A very similar picture has emerged for the conduction of entirely consistent with the Grotthuss mechanism in water, but
alkali metal cations through gA. For example, NMR and some unexpected behaviors have come to light in computational
computational studies show that the desolvation of Na+ occurs studies of hydrophobic channels.
in discrete steps as the ion enters the gA channel [80].
Ultimately, the cation is solvated by only two waters in a 4.3.1. Water transport in hydrophobic channels
single file of waters through the channel, and this configura- In long (> 50 ns) molecular dynamics simulations of
tion is able to maintain about half of the total solvation energy. carbon nanotubes (diameter 8.1 ), Hummer et al. [92] found
The energy cost of desolvation is partly compensated by water to spontaneously enter and to exhibit a diffusion
dipole interactions with the four tryptophan residues that form coefficient at least as great as in bulk phase. However, the
a collar in the membrane headgroup region of the channel occupancy was very sensitive to the precise interaction energy
[64,80]. The reverse sequence of events would accompany the between water and the channel wall. In pores constructed of
exit of the cation from the other end of the channel. NMR methane-like pseudoatoms, Beckstein and Sansom [93],
studies also show that Na+ is loosely bound at discrete sites found that channels narrower than 1213 diameter were
through the channel, with a significant amount of positional substantially closed to small ions, while channels of diameter
disorder. This minimizes the entropy loss upon uptake from <89 were empty of water. This, too, was very sensitive to
the bulk solution and is likely to be a contributing factor in the polarity of the pore, i.e., the waterwall interactions. At
allowing the high mobility of ions in the channel. intermediate diameters, the water oscillates between liquid
Experimental and simulation approaches tend to place the and vapor states; the average density is less than bulk and
rate limiting step for proton conduction by gA outside the presents a significant electrostatic barrier to ion passage
channel proper, but the molecular nature of the event is still [94,95]. The exact cut-off diameters are clearly sensitive to
unclear. Schumaker [86] suggested that exit of the proton is rate details of the interaction energy between water and the pore
limiting. Phillips et al. [84] proposed that the rate of initiation of wall, but the general features are robust. Some influence of a
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 891

hydrophobic pore can be seen even at diameters as large as models used for water or on the composition or nature of the
20 ; this is not reproduced in continuum electrostatic hydrophobic material (see [94]). When proton transfer is
calculations [95]. involved, however, results must be considered more critically.
The large diameter pore effects arise from interactions with
the second solvation shell and the development of structured 4.3.2. Proton transport in hydrophobic channels
water layers at the hydrophobic surface of the pore [96]. Ion Several computational studies have been carried out on
passage is restricted due to the energy cost of desolvation and proton transfer along water chains in hydrophobic channels,
the development of a layered solvent structure (at least 3 with largely consistent results, as first reported by Poms and
molecular layers thick 6 ) of the water adjacent to the Roux [82]. Waters placed in a hydrophobic pore may be able to
hydrophobic wall. The cost of depleting even the second conduct a protonic excess charge extremely rapidly by the
solvation shell (radius 5.5 for Na+), plus the layered water Grotthuss mechanismas much as 40 times faster than in bulk
structure at the wall, restrains ions from entering a pore of less water [102,103]. Following proton transfer, the orientation of
than 13 diameter, while the energy necessary to strip any of the water chain is restored by migration of either of the two
the first solvation layer (radius 3.5 for Na+) completely types of hydrogen bonding defect (D or L). As suspected by
prohibits entrance to a channel less than 7 in diameter. Nagle and Onsager and colleagues, this turn motion is slower,
Evidently, channel hydration can be modulated by small but it is still fast by bulk phase diffusion standards [103].
changes in diameter or in polarity, and it was suggested that However, the relevance of any of these determinations to net
channel conductance gating could be based on such changes in proton transport is questionable, as they do not address the rate
a suitably constructed channel pore [93,96]. of entry and exit of protons from the bulk phase and, therefore,
Such a model may be applicable to the acetylcholine the energetics of occupancy. Burykin and Warshel [89] found a
receptor channel, where the closed state appears, from prohibitively high barrier for proton occupancy of a 8
cryoelectron microscopy, to be a 6.5 diameter hydrophobic diameter hydrophobic pore, and the simulations of Sansom and
constriction in the pore formed by the M2 transmembrane coworkers would agree with this [96].
helices of the five subunits (2) [93,97]. Similar results Brewer et al. [102], using a water-filled, smooth-walled,
have been reported for simulations on the small mechan- hydrophobic cylinder, found that the diffusion coefficient of
osensitive channel, MscS [98]. In addition, hydration of MscS waters placed in the cylinder was close to the bulk value for
was found to be stimulated by an applied voltage, suggesting a channel diameters greater than 5 . However, as the diameter
possible, intrinsic mechanism of voltage gating [99]. decreased from 5 to 4 , the diffusion coefficient of the channel
These results illustrate the control that the Born and water went almost to zero, as previously found by Lynden-Bell
desolvation energies have on ion transport, and underscore and Rasaiah [104], while the mobility of an excess proton
the necessity for a narrow channel to provide adequate charge increased dramatically (> 40-fold). This is an interesting
compensation for any desolvation of an ion. Clearly gramicidin and unexpected result, but it is of uncertain significance to real
achieves a fine balance between providing enough polarity to proton transfer systems, as the simulation times were insuffi-
allow the channel to be occupied by water, and a minimum of cient to establish the stability of the water in the column. Similar
interaction that would slow down the diffusive movement for results were obtained by Dellago et al. [103], using carbon
water and monovalent cations. nanotubes of 8.1 , which Hummer et al. [92] previously found
However, the wall interactions in a polar pore are not to be stably hydrated.7 However, the long-time simulations of
negligible, as they appear to be in a hydrophobic pore. Sansom and coworkers, discussed above, indicate rather
Simulations on carbon nanotubes [100] and hydrophobic strongly that no water would reside in channels as narrow as
pores [93,96] show that as the diameter of the pore is decreased 5 , and even at 913 diameter the water density was lower
below 20 , the average density of water decreases due to than bulk, and underwent rapid oscillations between liquid and
increasing frequency of transition to the vapor phase. At the vapor states [94]. This would break any hydrogen bonded
same time, the diffusion coefficient increasesby as much as connectivity necessary for rapid proton transfer.
3-fold in the narrowest channels to have significant water Nevertheless, such large enhancements of proton charge
density. Contributing factors to this effect include reduction in conduction, as calculated in these studies, seem to fulfill the
dimensionality, transition to vapor phase behavior, and the hopes and aspirations of the early proposals for proton wires,
lack of polar interactions with the wall. A recent experimental and understanding why the PT rate is dramatically enhanced in
study of fluid transport through slightly wider bore (1420
diameter) nanotubes, found dramatic enhancements over 7
In fact, it is unclear how diameter is defined in these works (refs.
macroscopic predictions [101]. Gas flow was more than an [92,103]). If it is the center-to-center distance between carbons on opposite
order of magnitude greater than the Knudson limit, and water sides of the tube, then the lumen diameter is substantially smaller than this,
flux was 23 orders of magnitude greater than predicted by approx. 4.5 . This would be comparable to the narrowest, smooth-walled
Poiseuille's law for hydrodynamic flow. hydrophobic tube in which Brewer et al. also found greatly enhanced proton
Given the still common problem (at least for the non-expert) transfer [102]. It would also be very much smaller than the narrowest
hydrophobic pore that is stably hydrated in the simulations of Sansom and
of comparing results from different computational schemes, it is coworkers (who clearly define their diameters as the lumen). Thus, although the
worth noting that the main features of the simulations on PT results may be reasonably consistent, the physical reality of a suitably
hydrophobic channels results do not depend significantly on the narrow, stably hydrated, hydrophobic pore is uncertain.
892 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

the narrow tubes may be instructive for how fast PT could be also move rapidly through the channel. The necessary design
engineered. The simulations of Brewer et al. [102] showed that features for this also allow H+ transfer by a one-dimensional
the narrow (4 ) diameter and lack of polar interactions with the Grotthuss mechanism, achieving H+ passage rates comparable
wall made the Eigen- and Zundel-like ion configurations almost to those in bulk water. However, the H+ contribution to the net
equal in energy, facilitating the interconversion of Eigen and current is negligible at physiological pH and it seems
Zundel ions and stimulating the Grotthuss-type transfer of reasonable to suppose that the high PT capability is a purely
excess protonic charge. incidental property of a chain of waters with high mobility
In contrast, simulations on a synthetic (but real) proton resulting from incomplete coordination.
channel, with a similar average diameter but some polar This view is supported by the behavior and design of
attributes, showed normal water mobility [105]. The channels aquaporins. Here, the channels are intended to pass water at
were formed from synthetic amphiphilic helix bundles, with a high rates, but with very low proton permeability. Water is
repeating leucine-serine motif, and exhibited high proton transported rapidly in single file with high specificity, as in
conductance [106]. However, depending on channel width gramicidin, but some specific design features prevent proton
and interactions between channel water and serine hydroxyl conduction (Fig. 2). A rough two-fold symmetry axis in the
groups at the channel wall, either Eigen- or Zundel-like ion plane of the membrane introduces an interesting structural
configurations were relatively stabilized. The presence of a transition around two conserved NPA motifs in the middle of
strongly stabilized Eigen ion at one location was expected to the channel. At this location the water chain changes polarity,
limit proton conductivity in the channel. presenting an apparent block to proton conduction by a
Experimentally, the synthetic peptide (LSLLLSL)3 has Grotthuss-type mechanism. On this basis, the NPA motif was
> 40-fold greater permeability for H+ (in 0.5 M HCl) than for originally suggested to be the structural basis for the low H+
any alkali metal cation at similarly high concentrationsnone mobility [110113]. However, molecular dynamics simulations
was detectable for Li+, Na+ or K+ at 2.510 M [106]. In the and electrostatic calculations showed that the channel dynamics
same study, a slightly different peptide (LSSLLSL)3 was are very fast and the limiting factor is dominated by the
readily permeable to all monovalent cations (like the acetylcho- energetics of channel occupancy, which are determined by the
line receptor), with H+ permeability five-fold greater than Na+. desolvation penalty and a positive electrical potential near the
This is very close to the ratio of mobilities in water, indicating center of the channel [89,114,115].
both free diffusion and Grotthuss transfer in the channel. The In AQP1, a water-specific channel, the barrier height for a
significantly greater selectivity of the first peptide for H+ proton is on the order of 25 kcal/mol [89,113,116], while the
therefore reflects the fact that diffusive mobility was blocked for
alkali cations while the Grotthuss mechanism for H+ remained
active, implying full occupancy of the channel by water.
Voth's studies [102,105] suggest that Eigen-like structures
create effective charge traps along a conduction pathway, which
is expected to inhibit the Grotthuss mechanism for proton
transfer. Conversely, interactions that stabilize the Zundel-like
configuration will generally enhance PT by the Grotthuss
mechanism. This could conceivably lead to net gain if Eigen-
like configurations are avoided elsewhere in the path. The
greater charge delocalization (and polarizability) of the Zundel
ion, relative to the Eigen cation, may contribute to minimizing
the energetic penalty of a charge entering such a pathway.

4.4. Aquaporinswater vs. H+ transport

Gramicidin seems to represent a structure almost ideally


designed for rapid proton transfer. The mode of bacteriocidal
action of gramicidin is still under debate,8 but, if the toxic
intent of this antibiotic lies in its membrane permeation
properties, this is actually served by the conduction of alkali
metal cations, with the primary goal of depolarizing a target Fig. 2. Aquaporin. Only one monomer is shown, although aquaporins and
cell membrane. To achieve fast cation transport, water must aquaglyceroporins are homotetramers, each monomer apparently acting
independently. Note the two half helices and re-entrant loops positioned
8
There is strong evidence that gramicidins act on transcription, with a quasi-symmetrically across the midpoint of the structure. The membrane plane is
specific effect on a factor associated with sporulation [107109] and it is horizontal. Waters shown (green) are crystallographic. The asparagines, N78
very likely that one important role is in the regulation of sporulation of the host/ and N194, of the two NPA sequences at the end of each half-helix, are in orange.
source species, Bacillus brevis. There are several gramicidins, some linear (A, Histidine H182 and arginine R197 provide some of the electrostatic barrier to
B, C) and some cyclic (S, J). Both types are reported to affect transcription, but cation passage through the channel (see text). Structure file: 1j4n.pdb, for bovine
only the linear ones transport cations effectively. red blood cell AQP1 at 2.2 [110].
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 893

barrier in aquaglyceroporin, GlpF, is about 15 kcal/mol occupancy, or will serve a master process that can be
[114,117], perhaps reflecting a wider bottle-neck and therefore substantially slower.
less stringent desolvation penalty. Various mutational mani- In other cases, however, especially those associated with
pulations, carried out in silico, seem to support this. Turning off active pumping, the speed of proton delivery may be important
the positive potential (from an arginine and histidine, and the to prevent slippage and de-coupling of the energy conserving
positive end of the macrodipole of a short, reentrant -helix PT path from the coupled reaction which, again, could be ET or
from each side of the membrane) substantially lowers the conformational changes. The speed of proton transfer at critical
H+ barrier, probably to as little as 8 kcal/mol, although the steps in the coupled reaction pathway will then contribute to the
calculated values differ widely [89,113,114,116]. This is still pumping stoichiometry (yield), and will become increasingly
significantly higher than for gramicidin. The remaining barrier influential as the transmembrane proton gradient builds up. If
is due to the loss of solvation of the ion (H3O+) in the single the rates of PT needed for this are beyond what can be achieved
file of waters [118]. by incoherent or unconcerted proton hopping, we can expect to
Experimental mutation studies also support the important encounter them in such reactionsin proton pumping ATPases,
role of electrostatics in the aquaporin proton block. In AQP1, a cytochrome oxidase, bacteriorhodopsin, etc.
double mutant of the key arginine and nearby histidine exhibits
electrical conductance (the single mutants also have a very low 6. Proton availability and supply
but measurable level) [119]. This is prematurely identified as
proton conductance, but it does illustrate successful modifica- The overriding impact of electrostatic and desolvation
tion for ionic conductance of some sort, and the calculated processes in controlling net rates of ion transfer is especially
barrier heights for a monovalent test charge are consistent with relevant for protons, since H+ and OH are rarely, if ever,
this [116]. The substantial impact of desolvation, beyond direct present at significant concentrations. Thus, proton availability is
electrostatics, is strongly supported by a neutral mutation in a key limiting factor. Indeed, this has been a major focus of
AQP6 (Asn60 Gly), which inhibits its native function as an attention over the last 25 years, and crucial roles of proton
anion channel but enhances its water permeability [120,121]. buffers, carriers, reservoirs and antennae, etc. have become
In contrast to aquaporin, gramicidin assists the desolvation of evident [124128].
ions as they enter the mouth of the pore. The four Trp residues at
each end of the channel each provide about 1 kT of dipole 6.1. Proton conduction in the Fo domain of H+-ATPase
interaction with the ion, as it loses more than half of its first
solvation shell [80]. A net barrier to charge permeation remains, Perhaps the most striking example of proton transport that
which accounts for the activation energy of 45 kcal/mol [122]. exceeds the apparent supply rate is the Fo component of F-type,
H+-translocating ATPases. The proton conductance of Fo
5. Coupled proton transfer channels (if that is what they are) has been studied by Junge
and coworkers [129], and it is remarkable in both its magnitude
While ultrafast PT might be possible in certain structures, and its pH-(in)dependence. The maximum is 10 fS, at pH 8, but
such as a narrow hydrophobic channel, the structural and it also varies only 3-fold over the range of pH 6 to 10. The
energetic costs of doing so are large. It is also evident that proton conductance at pH 8 corresponds to 6.103 s 1 at 100 mV
mobile waters in a weakly polar environment can support PT at [129]. This is much faster than needed for ATP synthesis, but
a pretty good lick. Diffusion is notoriously unproductive when also exceeds the expected rate of delivery of H+ by free
unbiassed, but can readily yield results when a driving force is diffusion from the bulk solution at a concentration of 10 8 M.
applied. In simulations of the D-channel in cytochrome oxidase, With the typical second order rate constant used for H+
with a free energy span of 1 eV, proton transfer proceeded at a reactions, kH = 4 1010 M 1 s 1, the delivery rate should not
rate equivalent to 25 in less than 100 ps [123]. However, such exceed 4 102 s 1.
speeds may not even be generally necessary, as net proton The problem of diffusive supply was originally considered in
translocation involves uptake and/or release of a proton from the the context of ion channels, perhaps first by Luger [130], but
bulk phase, and these are often rate limiting, as discussed above the approach is inherent in the most basic formulation of a
for gramicidin. second order rate constant by the steady state Smoluchowski
In catalytic systems, including bioenergetic complexes, equation:
chemical events are likely to be limiting. For many proton-
coupled reactions, the key event is a single H+ delivery followed NA
by other rate-limiting processes (further ET, or a conformational k 4p R1 R2 D1 D2 1
1000
change). Here, the important thing is rapid equilibration with the
surface pH. Even if turn motions were truly slow, the proton R1, R2 and D1, D2 are the radii and diffusion coefficients for
moves back and forth along the same orientation of the path, two reactive species (particles) in solution. If one is significantly
and reorientations necessary for the next H+ delivery can take more mobile than the other, then (D1 + D2) is well approximated
place at relative leisure during the subsequent events. In such by the faster one, and this applies well to reactions between small
cases, fast diffusion within a channel or pathway will be slaved molecules and proteins or membranes. The sum of the radii
to rate limiting entry and exit or by energetically unfavorable represents an encounter radius, i.e., a distance of close approach,
894 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

below which reaction is assured. However, in reactions between For protons, with a bulk phase diffusion coefficient of
small and large molecules, or with membranes, the capture 10 4 cm2 s 1, we can estimate the capture radius necessary to
radius is not defined by molecular dimensions but requires achieve the observed conductance Fo at pH 8, i.e., = (10 fS)/
specification of the reactive surface, which is often not well (10 8 M) = 10 6 S/M. This requires R0 to be about 40 . This is
known. (R1 + R2) therefore becomes an empirical parameter, the large and cannot be simply interpreted as a hemispherical
capture radius, R0. For H+, with DH 10 4 cm2 s 1, the capture volume, as it would extend much too far into the bulk
commonly used empirical value of kH = 4 1010 M 1 s 1 phase to correspond with the Coulomb length at any likely ionic
corresponds to an encounter radius of about 5 , not strength. However, as suggested by Eigen's analysis of
unreasonable for a soluble base or buffer. dimensional effects on diffusion [134], it could be indicative
The encounter rate described by the simple Smoluchowski of a surface area of similar proportions, comprising the
equation is also modified by any forces between the particles overlapping Coulomb cages of many groups covering the
[131]. As expected, attractive forces, e.g., oppositely charged protein and the surrounding membrane.
particles, increase the rate of encounter while repulsive forces Transposing this result to a near-surface domain assumes
decrease it. The effect of interaction can be addressed explicitly, that the diffusion coefficient of the proton remains similar to
if the form of the interaction is known analytically, or implicitly the bulk phase. In general, this is not to be expected as a
as a modified encounter or capture radius. For electrostatic retentive Coulomb cage would be mostly generated by ionized
interactions, the equation can be modified according to Debye carboxylates residues with pKa 5 and a dissociation rate
[37,126,128,132]: constant of about 106 s 1. This is slow enough to substantially
retard the average progress of a proton as it hops, via water,
NA d
k 4p R1 R2 D1 D2  d from one site to another. On the other hand, if the acids are
1000 e 1 close enough to transfer protons directly, or with only a single
  2
jR0 intervening water molecule, the PT rate is much faster, i.e.,
exp d
1 jR0 between more or less matched pKa groups, and the process
becomes Grotthuss-like.
The characteristic parameter, here, is , the signed ratio of Also, after much controversy, there seems to be some
the Bjerrum length, RB, which is the distance at which the consensus on the lateral diffusion coefficient for protons, along
electrostatic interaction between two ions becomes equal to the lipid/water interface [135,136]. A recent determination on a
kBT, at zero ionic strength, and the unmodified encounter phosphatidylcholine (zwitterionic) lipid bilayer yielded
distance, R0: 5.8 10 5 cm2 s 1 [136], which is only twofold lower than in
the bulk (9.3 10 5 cm2 s 1), and greater than for any other ion
z1 z2 RB jz1 z2 je2 in the bulk phase. It is considered compatible with a Grotthuss-
d d where RB 3
jz1 z2 j R0 4per e0 kB T mechanism involving surface water.
At the present time, the information on Fo is of insufficient
The third factor in Eq. (2) modifies the interaction according resolution to say whether the requisite surface charges are
to the ionic strength of the medium 1 is the Debye length. present to make the large capture radius a possibility. In terms of
The resulting, effective capture radius is often referred to as the protein alone, it is probably unlikely, but with lipid headgroups
Coulomb length, and the associated volume as the Coulomb included, especially considering the apparent propensity for
cage. negatively charged lipids, like cardiolipin, to associate with
For membrane systems especially, an important situation bioenergetic proteins, it is possible. However, there is now
arises when charged species are close enough for their Coulomb growing belief in the existence of water structure at membrane
cages to overlap and merge. Under these circumstances, a interfaces that can restrain ion movement in the absence of net
proton or other mobile charge can diffuse a considerable surface charge. This is possibly related to the layering effects at
distance within the cage before escaping to the bulk phase hydrophobic surfaces seen in the long molecular dynamics
[126]. For protons, this may be further facilitated by dynamic simulations of Sansom and coworkers [94,95]. The evidence for
structuring of water in the presence of the charges [133]. an interfacial barrier is discussed at length by Mulkidjanian et al,
In the case of membrane channels, Luger [130] considered in this volume [137].
the influence of the capture radius on single channel A number of other mechanisms exist that can relieve the
conductances by conceptually adding a hemisphere, of radius limitation of proton availability. These have been widely
R0, at each end of the pore, and computing the diffusive flux. discussed [126,128,138] and will only be briefly summarized
The convergence permeability (reciprocal of the access here. They include non-specific effects like transport by soluble
resistance) then becomes: buffers and the related phenomenon of water hydrolysis
[17,124,125,139]. Soluble buffers represent sources of labile
Pa 2pR0 D cm3 s1 4 protons at generally much higher concentrations (mM) than
free protons (< M), and their diffusion rates are usually within
and the corresponding molar ionic conductance: an order of magnitude of the proton. A limiting feature may be
that the most prevalent physiological buffers, including
K 103 FPa F=RT S=M 5 phosphates, are negatively charged, which diminishes their
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 895

local concentration at and accessibility to negative surfaces, The relative selectivity of the gramicidin channel for H+, for
like natural membranes. example, lies in the lower diffusive mobility of alkali metal
The action of water as a proton donor (hydrolysis) is a cations compared to the Grotthuss-conducted protons. Even
special case of this buffering activity. It was first brought to greater selectivity is seen in the (LSLLLSL)3 peptide helical
prominence in biological systems by Kasianowicz et al. bundle of Lear et al. [106], which exhibits a proton
[139] who were forced to invoke it to account for the high conductance of 120 pS in 0.5 M HCl, but no detectable
rates of proton transport effected by S-13, a very potent (5 pS) conductance for alkali metal cations at much higher
uncoupling agent. Although water is an extremely poor concentrations 10 M in the case of Li+.
donor, with a pKa of 15.74 on a molar scale, it is of course Quite remarkable selectivity for protons is manifested by
present at very high concentration (55 M) and is therefore some transport devices, such as the Fo component of H+-
effective in the last resort. The effective rate constant translocating F-type ATPases. In addition to the very high
depends on the pKa of the acceptor, and is given approx- proton conductance in the face of very low proton concentra-
imately by 3.1010 55 10pK 15.74 = 3 1010 + pK 14 s 1 where tions, the conductance to any other ion is negligible. The
3.1010 M 1 s 1 is the second order rate constant for reaction of selectivity (H+/Na+ permeability ratio) of Fo is in excess of 107
OH with the neutral acid: [129]. This likely reflects chemically specific steps in the
transfer, involving protonation and deprotonation of at least one
H2 O A OH AH carboxylic acid residue near the center of the membrane
[150,151]. Additional buffer groups are needed to account for
Water was also suggested to be the dominant proton donor to the remarkably small pH dependence of the proton conductance
the secondary quinone, QB, in reaction centers in native [129]. It should be noted that the powerful selectivity of Fo is
membranes [140], although this did not appear to be the case not required for its activity, per se, as the closely related Na+-
for isolated RCs [141]. However, the new thinking concerning a translocating F-type ATPases, which have a very similar Fo
dielectric barrier at membrane surfaces may bring these two sequence and structure, function well with Na+ and a much
experimental results in to concordance [137]. lower selectivity against H+ [150].
A more specific, structural solution is local proton reservoirs Such a high degree of selectivity may well require one or
or buffers. These are amino acid residues, notably histidine, that more chemical steps of this sort, i.e., if a simple water filled
are significantly protonated at physiological pH and can act as channel cannot be so discriminating. This is evidently true for
immediate proton sources for donation to the protein [142]. A gramicidin A. In contrast, a modified gramicidin, with a
role for histidines has often been correlated with sensitivity to CH2OH group protruding into the lumen, is almost perfectly
Zn2+ ions and other divalent transition metals, and has H+-selective, while maintaining H+ conductance that is within
implicated histidines at the input sites of pathways in several a factor of 3 of the unmodified channel (S. Cukierman,
proton transfer proteins, including bioenergetic proteins and personal communication). This latter result also suggests a role
proton channels [128,143146]. The diagnostic value of for a serine sidechain in the H+ selectivity of the synthetic
divalent metal ion sensitivity has been given substantial (LSLLLSL)3 peptide channel [106], although experimentally
credence by the recent identification and cloning of the long- the selectivity of this peptide channel is only known as a lower
sought voltage-gated proton channel [147,148], which is limit (> 40). The simulations of Wu and Voth [105] suggested
inhibited by Zn2+ [146]. Site specific mutagenesis identified that the water column was continuous in this channel, but
two histidines as the source of the Zn-sensitivity [148]. these were carried out on a model structure since it is not
A related motif is the combination of histidines and acidic known experimentally. At the present time, therefore, these
residues, which is found in cytochrome c oxidase and in and other molecular dynamics studies of hydrophobic channels
bacterial photosynthetic reaction centers (see Sections 7.1, 7.4 that imply the possibility of high proton conduction with
and 7.5). This arrangement may simultaneously attract protons negligible water diffusion [102,103] should be viewed with
from the bulk phase, through the negatively charged carbo- caution.
xylates, and provide immediate proton injection from the
protonated histidines [149]. 7. Proton coupled electron transfer and conformationally
coupled proton transfer
6.2. Proton selectivity
7.1. Cytochrome oxidasethe D-pathway
In discussing proton transfer in aquaporin and gramicidin,
there is some tendency to equate the issue of the Grotthuss A water-filled channel, the D-pathway, is responsible for
mechanism with whether structural reorientation is kinetically delivery of at least 6 and possibly 7 of the 8 protons consumed or
determining, e.g., [87]. Warshel's view is that electrostatic and transferred in the full turnover of cytochrome oxidase [152154]:
desolvation energies exert an over-riding influence in con-
trolling the rate of proton transfer and, indeed, most processes.
4e 8H in O2 2H2 O 4H out
It is important, however, not to let this obscure the crucial role
that the Grotthuss mechanism plays in both the mechanism and The D-pathway is marked by an eponymous aspartic acid
the remarkable specificity of many proton transport processes. (D) at the entrance to the channel (Asp-91 in bovine
896 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

mitochondria) and ends at a highly conserved glutamic acid


(Glu-242 in bovine mitochondria) near the center of the
membrane span.9 Mutation of either the Asp or Glu residue
essentially shuts down proton delivery [155]. They seem,
therefore, to act as effective and proton specific gates.
X-ray structures of cytochrome (c) oxidase show several
well-defined, crystallographic water molecules in the D
channel (Fig. 3), but even at the best available resolution
(< 2 ) the chain is incomplete in the last 7 between Ser-157
and Glu-242 [156158]. Computational methods have been
used to place additional waters to effectively fill the channel, up
to the entrance to the central cavity between heme a and the
binuclear center (heme a3-CuB). The glutamic acid, Glu-242, is
highly, but not rigorously, conserved. The main part of the
channel is lined by several polar amino acid residues, but these
are not generally critical to either PT or proton pumping [159],
and simulations indicate them to have only a small role, in
stabilizing the water column [123].
Accepting the computed water placements, the D pathway
has a substantially complete hydrogen bonded chain of water
molecules, extending about 16 from a ring of three
Fig. 3. The D-pathway of cytochrome oxidase. The path begins with an aspartic
asparagines, near the entrance, up to Glu-242. In the central
acid (Asp-91) at the mitochondrial matrix (or bacterial cell cytoplasm) side and
cavity, the PT path must branch in order to deliver protons ends at a glutamic acid (Glu-242), close to heme a and the binuclear center
alternately to the oxygen reduction chemistry at the binuclear (heme a3 and CuB). Waters shown are crystallographic; none are present in the
center, on the one hand, and the proton pumping mechanism, last 67 before Glu-242. The binuclear center is also empty of waters in the
on the other. Neither of these paths is defined at the current fully oxidized and reduced resting states of the enzyme. Structure file: 1v54.pdb,
for fully oxidized bovine cytochrome c oxidase at 1.80 [158].
level of resolution (< 2 ) and it seems likely that highly
mobile water molecules are involved. These have been
computationally modeled and utilized in MD simulations to not be very fast (approximately 1 H2O per 2 ms) and it has been
illustrate possible scenarios for proposed proton pumping suggested that water movement can be rate limiting for turnover
mechanisms [90,123,160165]. It is interesting to speculate of the enzyme [166].
that this region, and possibly the top third ( top as drawn in A high degree of connectivity in the D channel water chain
Fig. 3) of the D channel, may be at least partially dry in the could be important for providing sufficiently rapid PT to Glu-
resting state of the enzyme, and could be primed by water 242 to facilitate the pumping process. Molecular dynamics
molecules generated by O2 reduction in the active site. simulations of PT within the D channel show it to be very fast
Once fully occupied, the water chain in the D channel of (ignoring entry) when Glu-242 is ionized, with transfer over a
cytochrome oxidase provides a well-defined PT structure. In distance of 13 in about 25 ps, under a substantial driving
gramicidin, where the water chain is continuous, 10 the force of about 1 eV [123]. This corresponds, very roughly, to a
associated, fast PT activity may be an incidental gain of diffusion coefficient of about 0.5 2/ps, in good agreement
function, as suggested above, with the intention being for rapid with our expectations of PT by a non-concerted Grotthuss
water movement and alkali metal cation diffusion. However, for mechanism. The PT exhibits a brief pause in a proton trap
cytochrome oxidase, proton delivery is clearly of primary region that is wider and allows an Eigen ion-like coordination
importance, although the D channel would also seem to be well structure to be formed with Ser-156 and 157 [123]. If these
designed to function as an exit path for water molecules residues are mutated to Ala, in silico, the computed PT is 5
generated by the catalytic reduction of oxygen. The glutamic 10 times slowera water molecule takes the place of the Ser
acid residue at the top of the channel provides one proton- OH but is less effective at maintaining the continuity of the
selective feature, and the entrance to the channel provides water chain, which breaks more readily, and the charge
another. Whether these can be breeched by water molecules on remains in the trap for significantly longer. In corresponding
the time scale of active site turnover will determine if the (real) mutants of cytochrome oxidase (Ser-201 in Rb.
channel could function in both these roles. However, this need sphaeroides, or Ser-193 in P. denitrificans), the steady state
rate and pumping efficiency (H+/e) are minimally affected
9
Where necessary, the bovine mitochondrial numbering will be used, and [159,167]. However, it should be borne in mind that H+
Fig. 3 is of bovine cytochrome c oxidase. Other common designations are: for pumping stoichiometries are determined when the protonmo-
Asp-91 Asp-132 in Rb. sphaeroides, Asp-124 in P. denitrificans, and Asp-
tive force is negligible and slip is not expected to be a
135 in E. coli bo3 quinol oxidase; for Glu-242 Glu-286 in Rb. sphaeroides,
Glu-278 in P. denitrificans, and Glu-286 in E. coli bo3 quinol oxidase. significant factor.
10
However, it is defined only by computational analysis and not by When Glu-242 is already protonated, the excess protonic
experimental structural methods. charge may remain in the region of the serine residues [123],
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 897

perhaps indicating a capacity for storing a proton as H3O+ (or 7.3. Bacteriorhodopsin
H5O2+) ready for the next reductive step of the binuclear center.
The kinetic basis of active pumping does provide motivation Transient modifications of water chains and networks are
for fast proton transfer on demand, in order to maximize the essential to the H+ pumping mechanism of the bacteriorho-
yield of pumping and minimize side reactions or unproductive dopsin photocycle (Fig. 4), and are well documented by an
back reactions (slip). In this context, an interesting suggestion exceptional collection of X-ray structures of intermediates
is that the water column of the D channel could collapse [174176], by FTIR [177], and by electrostatics calculations
transiently after PT to the terminal Glu, breaking contact at [178,179] and molecular dynamics simulations [180182]. The
this point and retarding back transfer or proton slip ([157], action is partitioned between two domainsthe extracellular
and Martyn Sharpe (Michigan State University), personal and cytoplasmic domains, separated by the photoactive retinal-
communication). lysine Schiff base. Following light absorption, the all-trans
retinal isomerizes to a 13-cis, 15-anti configuration, which is
7.2. Transient vs. static PT paths held in a highly twisted form by constraints of the protein.
Subsequent relaxation of the 13-cis form drives protein
The D channel in cytochrome oxidase and gramicidin seem conformational changes and coupled pKa shifts that lead to
to provide examples of well defined and highly connected PT proton release from the extracellular space and proton uptake
pathways, but functional connectivity can also be achieved from the cytoplasmic side.
transiently with highly mobile water molecules. This could also In the extracellular domain, a proton is transferred from the
provide a mechanistic component of gating or catalysis. The protonated Schiff base of retinal-Lys216 to Asp85 within about
idea that a PT pathway of water molecules can be transiently 500 s. Coupled to this, Arg82 moves down and away from
organized at a specific place and point in time is inherent to Asp85, and a proton is transferred to the protein surface from
proposed gating mechanisms of proton pumping in the central the proton release group, formed by Glu194, Glu204 and about
cavity of cytochrome oxidase [162165], and in bacteriorho- 4 water molecules. This proton escapes to the bulk phase on a
dopsin (see below). It is also of possible significance in acid much slower time scale, which roughly corresponds to
base catalysisfor example, the transient assembly or or- reprotonation of the Schiff base from the cytoplasmic side.
ganization of a proton conducting water chain has been The reprotonation of the Schiff base occurs via proton transfer
proposed as a prerequisite for rate limiting PT in the active from Asp96 over a distance of about 11 , and involves the
site turnover of carbonic anhydrase [168171]. However, this is structuring of water molecules to act as a bridge between donor
controversial [172,173]. and acceptor. As the reprotonated Schiff base returns to its all-

Fig. 4. Bacteriorhodopsin (left) and its photocycle (right). The light-adapted all-trans retinal (mauve) is covalently linked to Lys216, forming a protonated Schiff base
(HC = NH+). The retinal chromophore isomerizes in less than 1 ps, and the resulting strains on the protein give rise to several early spectroscopic intermediate, at
least one of which (J) is not shown. Relaxation of these strains via spectroscopic intermediates K and L cause deprotonation of the Schiff base to yield M1. The proton
is released to the extracellular phase (see text). Further relaxation completes the bond rotations in M2, which switches accessibility of the Schiff base from the
extracellular to the cytoplasmic side. The Schiff base is then reprotonated by proton uptake from the cytoplasmic side. This facilitates the reisomerization back to the
all-trans state. Structure file: 1c3w.pdb, at 1.55 [278].
898 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

trans configuration, Asp85 transfers its proton to the proton configuration has relaxed and the nitrogen lone pair is
release group, Arg82 recovers its original position, and the comfortably facing the cytoplasmic domain. When the Schiff
resting state is restored. base is reprotonated from the cytoplasmic side and begins to
reisomerize back to the all-trans form, Asp85 protonates the
7.3.1. Proton release from bacteriorhodopsin PRG and Arg 82 returns to its up position.
The nature of the proton release group (PRG) was initially
problematical, as FTIR studies could not identify appropriate 7.3.2. Proton uptake by bacteriorhodopsin
changes in the protonation state of any relevant amino acid At the opposite extreme from the well defined and connected
side chain. This has now been resolved by the recognition of a water chains of gramicidin, the D channel of cytochrome
delocalized proton, in the form of H5O2+, associated with oxidase, and the networks of the proton release complex of
Glu194 and Glu204, both of which are ionized, Arg82, and at bacteriorhodopsin, is the proton uptake pathway at the
least two additional water molecules [178,183]. Delocalized cytoplasmic side of bacteriorhodopsin. Here the proton
protons, of which H5O2+ is the simplest, are characterized by conducting structure linking the cytoplasm to the Schiff base
broad IR absorption bands [43], and time resolved changes in is assembled from scratch by structural changes that draw in
the 18001900 cm 1 region have been identified and shown to water from the aqueous phase. This allows transfer of a proton
fit the expected behavior of the PRG [45,183]. With bulk from Asp96, which is protonated in the ground state; it is
phase pKa values of 4.4 for glutamic acid and approximately subsequently reprotonated from the cytoplasmic medium.
1.7 for H5O2+ , this would appear to be an unusual These events are initiated by relaxation of the 13-cis
arrangement, perhaps requiring a delicate balance of electro- retinylidene, which forces movement of helices F and G that
static forces. Indeed, it is abolished when either Glu194 or opens a channel and draws water in from the bulk phase. A PT
Glu204 are replaced by glutamine, but it is partly retained pathway is established between Asp96 and the deprotonated
when aspartatic acid substitutes for Glu204 and in many other Schiff base and, at the same time, structural changes around
mutants in the general vicinity [45]. It is, therefore, at least Asp96, including water movements, lower the pKa of Asp96
minimally robust to small perturbations of local structure and thereby driving the proton transfer to the Schiff base. The water
electric potential. chain from Asp96 to the protein surface, which allows
In fact, the dominant electrostatic contributions are likely to reprotonation of the acid, is completed as the inner water
be quite local and the stabilization of the ionized species by chain to the Schiff base is collapsing.
mutual interaction could be a general phenomenon. Spassov et This sequence of events in the cytoplasmic domain rapidly
al. [178] modeled the electrostatic interactions in this region and builds and destroys two short water chains, employing one or
found that, in the absence of a protonatable model for water, two waters that are already sequestered within the protein in the
Glu194 and Glu204 interacted so strongly that one was always ground state, but importing at least 2 or 3 others. This is an
ionized (pKa < 0) and the other was always protonated expensive business, as the creation of water-sized cavities costs
(pKa > 15). When H5O2+ was permitted in the calculations, it on the order of 5 kcal/mol [189]. Bacteriorhodopsin can afford to
became the preferred protonation site, with both Glu194 and do this as it has a great deal of excess free energy derived from
Glu204 ionized. The net interaction energy among these three the photochemical isomerization of the retinal (12 kcal/mol),
groups (Glu194, Glu204 and H5O2+) is sufficiently favorable but it is unlikely to be a generally applicable design strategy. In a
to oppose the intrinsic propensity for H5O2+ to transfer its proton recent description of events on the extracellular side, Gerwert
to Glu. [190] has proposed that the remodeling of the hydrogen-bonded
This balance is upset when the retinal isomerizes and network in the PRG constitutes a significant fraction of the
disrupts the interaction between the protonated Schiff base and energy stored in the protein, transferring it from the photo-
ionized Asp85, which is mediated by a water molecule.11 The chemical product as the distorted 13-cis retinal relaxes.
Schiff base proton is transferred to Asp85, causing Arg82 to Similarly, the cavity formation on the cytoplasmic side could
move away and down towards the PRG, and this drives the readily represent a similar magnitude of free energy storage, and
proton from the PRG to the surface, where it is delayed by would reflect the transfer of the strains from the proton release to
surface forces for up to a millisecond [185188]. Molecular the proton uptake theater of operations.
dynamics simulations suggest that as the Arg82 moves down,
the water cluster near the Schiff base can merge with the water 7.3.3. Is the presence of a Zundel ion (H5O2+) significant?
cluster of the PRG [181,182]. It was suggested that this could It is clear that water plays crucial dynamic and structural
allow rapid, Grotthuss-type, proton transfer, but for what reason roles in the proton transfers leading to proton release in
is not clear. bacteriorhodopsin. Not only does it provide much of the
The movement of Arg82 and the coupled pKa change for pathway for transfer but it is also the repository of the labile
Asp85, effectively inhibit back transfer of a proton from the proton, H5O2+, in the ground state. This is the first unequivocal
extracellular side to the Schiff base, at least until the 13-cis identification of a delocalized proton in a functional biological
role, but it may turn out to be a widespread phenomenon, e.g., in
11
Interestingly, the correct placement of this water (wat402) in computational RCs (see below), and recently proposed for the D channel of
analyses is one of the few examples where quantum mechanical refinements, or cytochrome oxidase (Martyn Sharpe, personal communication).
an unusual parametrization of the polarizability, are required [184]. The question arises whether there is any particular advantage to
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 899

it or is it just one of many possible structural solutions to a given membrane dielectric represents only about 15% of the
functional requirement? average transmembrane charge transfer per electron turnover
Strong electrostatic interactions, leading to marked pKa [140,193195].
shifts, are widely encountered in proteins and underlie many of The reactions involving PT are summarized in the acceptor
their unique attributes. The operational pKa of an acidbase quinone cycle shown in Fig. 5 (left). Reducing equivalents are
group is determined by the summed interactions with its charge stored in the double reduction of the secondary quinone, QB
environment and by its intrinsic pKa, where the usual suspects (ubiquinone in Rhodobacter (Rb.) sphaeroides), via the primary
for protein groups range from about 4.5 (for Asp and Glu) to quinone, QA, and quinol is released into the membrane after two
12.5 (for Arg). Water adds to this a significantly stronger acidic light-activated turnovers of the RC [196,197]. The oxidized
pKa of 1.7, identified with H3O+. The Zundel cation is not primary donor, P+, is reduced by a secondary donor (cytochrome
expected to have a significantly different intrinsic pKa, but it c2 in vivo) after each photoactivation, and only the quinone
does represent a more delocalized charge, which will help in events are shown in the cycle [149,198].
sequestering it in a lower dielectric environment. Sub-stoichiometric (H+/e < 1) proton uptake occurs on the
An assortment of amino acid side chains could very readily first flash, due to the electrostatic influence of the semiquinone

provide the same charge balance as the native PRG (net 0 or anions, QA and QB , on the pKas of nearby ionizable amino
1), from among the protonated Schiff base (SBH+), Asp85, acid residues [149,199202]. Super-stoichiometric proton
Arg82+, Glu194, Glu204, and possibly Asp212. In fact, uptake occurs on the second flash, as QB is reduced to quinol
several mutants do just this, but they tend to be functionally and released into the membrane. The average stoichiometry is
impaired, like E204Q and E194Q, and the continuum band is H+/e = 1, as stipulated by the overall chemical reaction.
lost. The inclusion of protonated water, with low intrinsic The RC quinones are well buried in the protein, and proton
pKa, in the native PRG may allow more rapid proton release transfer to QB must extend over a distance of about 15 . Site
when the charge balance is upset. When H5O2+ snaps back to directed mutagenesis studies have implicated several members
H3O+, the low pKa is manifested with a proton dissociation of a large cluster of ionizable residues (predominantly acidic)
rate of > 1011 s 1, and a correspondingly fast pairwise PT as being involved in the proton delivery pathway (reviewed in
rate to the nearest acceptor. This may be important in [149,198,203]). However, it is barely possible, from simple
directing the proton transfer to the out-going path, before kinetic studies, to make the distinction between actual proton
structural changes allow back transfer to the Schiff base. This carriers and those having influence, for example, by electro-
could be an effective device in any situation where rapid, static interaction. Direct spectroscopic evidence of a proton
intermittent proton delivery must compete with other pro- carrying role has been presented only for Asp-L210 and Glu-
cesses, i.e., pumps. L212, and possibly a surface histidine, on the basis of static
and time resolved FTIR studies of the first turnover [204
7.4. Bacterial photosynthetic reaction centers 209]. However, compelling evidence comes from radical
changes in the mechanism of the second electron transfer upon
At the present time, there appears to be no evidence for mutation of AspL213, SerL223, HisH126 and HisH128,
specialized, ultrafast PT in proton pumping bioenergetic and AspM17 (together with AspL210) [210220].
systems, beyond the incoherent hopping of Grotthuss-type The pathway is outlined in Fig. 5 (right). The path from the
mechanisms. Some might consider this to be specialized, but it entry point near HisH126 and HisH128 at least up to Asp
is a natural manifestation of acidbase features common to L210 is shared by both the first and second H+ taken up from
many chemical functionalities. If this is so, then non-vectorial the aqueous medium. The H+ taken up on the first flash settles
(in the sense of non-transmembrane) PT is likely to arise with predominantly on GluL212, at neutral pH, although it is
the same natural designs as encountered in proton pumps, and probably distributed over other residues, too. The H+ taken up
the two classes can be considered together in terms of on the second flash is delivered directly to the C1O group of
identifying underlying principles. Where pumping mechanisms QB , via AspL213 and SerL223. This is followed by

might have additional kinetic requirements is in the gating electron transfer from QA to form QBH. The quinol is fully
mechanisms required to prevent slippage. protonated by transfer from GluL212, possibly as it unbinds
The bacterial photosynthetic reaction center (RC) provides [221224].
a good and experimentally amenable system for studying In wild type RCs, the second reduction of QB to QBH is rate
long distance PT, although it is, for the most part, non- limited by electron transfer, i.e., proton transfer from the
vectorial. In reaction centers, the transfer of electrons in the aqueous phase is very fast [225]:
primary photochemical events generates most of the electrical
fast PT slow ET
component, while electron-coupled proton uptake and release Q 
A QB X Q
A QB H Y QA QB H

accompanying the redox reactions of secondary acceptors
and donors is largely responsible for the proton concentration The net rate of ET is given by kobs = kET[QBH] [226]. The ET
gradient (pH). The reduction of quinone (Q) to quinol rate constant, kET, has been estimated at 106 s 1, which places
(QH2) represents the initial step of a chemiosmotic proton the PT equilibration rate at > 107 s 1 [149,227]. The rate of
pumping redox loop [191,192]. However, this process is equilibration is the sum of forward and back transfer rates,
only minimally electrogenic, and the charge moved into the keq = kH[H+] + kH. This is dominated by the deprotonation rate
900 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

Fig. 5. The bacterial photosynthetic reaction center acceptor quinone cycle (left) and proton pathway (right). Following the first flash, an electron is transferred to the
acceptor quinones, and the photooxidized primary donor, P+, is re-reduced by secondary donor, D. After the first flash, the anionic semiquinone charge induces pKa
changes in some ionizable amino acid residues and H+ are taken upespecially to Glu-L212. (The precise stoichiometry of H+ uptake depends on whether the electron
is on QA or QB). This subsequently provides the 2nd proton, H+(2), transferred to QB, after the second flash. After a second flash, the transfer of the second electron
from QA only takes place after QB has been protonated. Protonation is fast but unfavorable as the pKa of QB/QBH is low, but transfer of the second electron forms the
quinol with high pKas for both protons. The two protons taken up to reduce QB to QBH2 follow much the same path, with initial proton transfer from HisH126. The
C1O site is the first to be protonated (H+(1)), via AspL213 and SerL223. The C4O group is protonated second (H+(2)), via GluL212. The bifurcation of the
pathway occurs at or after AspL210.

of the semiquinone, which has a pKa of about 4.5 and, therefore, causes a dramatic change in the mechanism of the reduction of
a deprotonation rate to water of about kH10pK. Using the bulk QB to QBH, from ET-limited to PT-limited. These include
solution value of kH 4.1010 M 1 s 1, the computed off-rate AspL213 Asn (mutant L213DN), SerL223 Ala
would be inadequate: kH = 4.1010 10 4.5 106 s 1. However, (L223SA), the double mutant AspL210 Asn + Asp
measurements of the rate of H+ uptake to RCs in the absence of M17 Asn (L210DN/M17DN), and the double mutant His

ET, i.e., uptake to the QA state, show a substantially larger H126 Ala + HisH128 Ala (2xHis) (reviewed in
second order rate constant [141], which increases with pH [149,198,203,229]). In the case of mutant L213DN, the second
above the isoelectric point of the protein (pI 6.1). This electron transfer rate was inhibited by at least 104 fold at pH > 7
probably reflects the influence of surface potential on the local, [211]. However, because PT is now rate limiting [220], the
surface pH. In spite of the different binding site locations of QA proton transfer rate was much more strongly inhibited

and QB, proton binding in response to uptake by the QA state of estimated as inhibited by 107-fold in the mutant. Interes-
the protein involves most of the same residues [202,228] and tingly, this mutant could be partially reactivated (rescued) by
occurs via the same pathway as for the QB state [218]. small weak acid anions, such as azide (N3) and nitrite [212].
Related to this, the pKa of QB is pH dependent because it is In RCs with the mutation GluH173 Gln (mutant
influenced by the local potential in the QB binding pocket, and H173EQ), the 2nd electron transfer was also strongly inhibited,
this is determined by the ionization state of amino acid residues by more than 102 fold, and could be fully rescued by azide
around it [149,202,228]. The pKa 4.5 was estimated for pH 7.5 [230]. However, in this case, the reaction remained ET-limited
[227]. At lower pH, the pKa decreases, indicating a faster off rate, [220] and the effect on the PT rate is indeterminate, although it
and the on rate, kH[H+], also increases. At higher pH, the pKa could be substantially impaired. The effect of this, and other
increases, but the apparent rate constant, kH, also increases, so impaired mutations in which ET is still rate limiting, is
the deprotonation rate constant changes little. Thus, the rate of consistent with an alteration in the pKa of QB a decrease in
proton equilibration to QB /QBH remains fast compared to the ET pKa will decrease the equilibrium population of QBH, and
rate over most or all of the accessible pH range (pH 3.511.5). therefore decrease the net rate of ET. In such circumstances, full
Several amino acid residues have been strongly implicated restoration of the 2nd electron transfer rate by azide must
as proton carriers because mutation to non-ionizable forms include recovery of the native pKa value for QB . This could be
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 901

achieved by binding of the azide anion, N3, restoring the to setting the pKas of other proton transfer groups. Function can
negative electrostatic potential lost upon mutation of ionized be restored to mutant RCs by small buffers but, unlike the 2xHis
GluH173 to Gln [230]. Activity of the protonated species, mutant, proton transfer is only substantially restored by weak,
N3H, as a proton carrier could also supplement the proton neutral acids, (AH) [247]. Rescue activity seems to be mainly
delivery function of the protein [212]. limited by steric and electrostatic factors restricting access
Several primary mutations of acidic residues are partially through a narrow channel. The acid forms were active at low
recovered by second site revertants, some at considerable concentrations, but, because of their generally low pKa values,
distance, which can be understood as restoring negativity to the the total concentration of salt required near neutral pH was high.
QB domaineither by adding a new acidic residue or removing A Brnsted plot of activity vs. pKa of the rescuing acids was
a wild type basic residue [231235]. This is an attractive linear, with a slope of 1, and extrapolated to diffusion-limited
general explanation but the molecular mechanism in some behavior at pKaapp 1. However, the maximum rate achieved
cases, at least, involves significant structural changes, including under saturating concentrations of acid (salt) did not correlate
relocation or introduction of water molecules [236238]. with pKa. This behavior suggested a simple model in which the
acid and anion species compete for binding, both with weak
7.4.1. Proton entry to bacterial reaction centers affinity. The model predicts that the intercept at the diffusion
The proton entrance site was first indicated by the striking limit corresponds to a true pKa = 45, again consistent with a
inhibition of the 2nd electron transfer by divalent transition carboxylic acid or even QB , itself.
metal cations, especially Zn2+ and Cd2+ [145,213,239241], The binding affinities for both acid and anion forms was very
and a lesser effect on the 1st electron transfer [145,242]. weak (1 M) for all active species, and the efficacy and
Both effects follow from the inhibition of H+ uptake, and properties of the rescue relied on very rapid on and off rates
identify the proton entry site as the same for both protons the on-rates were near the diffusion limit. However, the
[213,219]. stringent size limitation indicates a need to access a small
The metal binding site was shown by X-ray crystallography internal volume, which we consider to be the proton channel to
[243] and by EPR [244,245] to be a surface patch, comprising the QB domain. Such rapid, diffusive access to a buried site may
histidines H126 and H128, and AspM17, with AspL210 just seem a tall order, but there are several clear precedents.
below the surface. Mutation of the two histidines to alanine Dramatic examples include acetylcholinesterase [248,249] and
(mutant 2xHis) inhibited both the 1st and the 2nd electron catalase [250,251], both of which support diffusion limited
transfer at pH > 8, and the 2nd electron transfer switched from entry to active sites at the end of long (2030 ), narrow
being ET to PT limited [219]. channels. Perhaps this should not be too much of a surprise after
This strongly supports the notion that the histidine residues our consideration of transmembrane ion channels, except that
function as a surface buffer or reservoir of protons for local the RC pathway to QB was not designed to accept small weak
injection into the proton pathway [128,142]. This design is acids and, as discussed below, may well be designed to exclude
adequate to keep proton delivery non-rate limiting in the wild small, potentially redox-active species.
type at pH values well above the functional pKas of the
histidines (pKa = 6.36.8 [241]), while the 2xHis mutant is 7.4.3. The proton path in bacterial reaction centers
proton limited [219,246], because even a small degree of The entry site and path for proton delivery to QB in wild type,
protonation of the histidines will provide much greater, effective and the probable access route for weak acids in the L210DN/
proton availability than the nanomolar concentrations of free H+ M17DN mutant RCs is shown in Fig. 6. Water molecules are
at alkaline pH. largely absent from this view at the present resolution of RC X-
Both 1st and 2nd electron transfer reactions were restored in ray structures (< 2 ), and modeling adds none with clear
the 2xHis by addition of imidazole [219], and Paddock et al. consensus among different structures. Water molecules are
[246] found that the 1st electron transfer in 2xHis mutant RCs present elsewhere, in the QB domain, however, and void
could be rescued by a variety of cationic buffers (BH+) in a volumes are available in the X-ray structures to accommodate
manner that was proportional to the buffer pKa over a wide others in the putative pathway. It seems likely therefore that
range of values (Brnsted plot). Analysis in terms of rate water molecules are present but highly mobile.
limiting proton transfer from the protein surface revealed the A functional role for water in the protonation reactions of the
involvement of a low pKa intermediate (pKa 4) in the transfer quinone states is supported by infrared spectroscopy, through
pathway, on the way to the final destination, GluL212, which the characteristic IR spectra (Zundel bands) of polarizable
has a higher pKa. This would be consistent with the presence of protons [43]. Breton and Nabedryk observed a broad and

several carboxylates in the QB domain. featureless component in the QA /QA FTIR difference spectrum
of Rps. viridis and Rb, sphaeroides, which they assigned to a
7.4.2. Proton carriers in bacterial reaction centers Zundel band [206]. This is an indicator of water in a hydrogen
In the double mutant, AspL210 Asn + AspM17 Asn bond network containing an excess charge.
(L210DN/M17DN), the 2nd electron transfer is severely PT Remy and Gerwert have made time resolved FTIR
limited over a wide pH range, but can also be rescued by added measurements of the 1st electron transfer, by the step-scan
buffers [247]. Thus, one or both of these aspartic acids is a method, and see direct evidence for transient proton transfer
functional proton carrier, and not just an electrostatic contributor from a surface histidine to Glu-L212, via Asp-L210 [207]. The
902 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

Fig. 6. The proton entry site, proton pathway and weak acid route in reaction centers from Rb. sphaeroides. Left: view from the inside looking towards the protein
surface. Right: view looking down on the entrance from the outside. The white contoured surface encloses surface residues that define a hole sufficient for weak acid
entryAspH124, HisH126 and AspM17 (also shown in bond mode), and ProH172 and GlyM19 (in contour mode only). All other residues, except HisH128,
are buried. The bound cadmium (Cd2+) is an inhibitor of proton entryit lowers the pKas and deprotonates the histidine residues. Its presence does not impair access
by the smaller weak acids, but slightly impedes the largest (formate) [247]. Residues are sized to indicate depth of field. Structure file: 1dv3.pdb [243].

latter residue undergoes transient protonation in about 15 s, preceding the 2nd electron transfer [209,228,253,254]. Muta-
and deprotonation in 150 s. tion of SerL223 to threonine or aspartic acid retains activity,
but asparagine and alanine do not [215,216]. Remarkably,
7.4.4. Proton delivery in bacterial reaction centers replacement with glycine does yield activity, suggesting that
Mutation of Asp-L213 to a non-ionizable residue, e.g., the absence of a side chain in Gly allows room for a water
mutant L213DN, causes a dramatic, 104-fold inhibition of the molecule, which functions in the same way as the serine
2nd electron transfer and a switch to PT limitation [220,252]. hydroxyl [254].
The extent of PT inhibition is on the order of 107 fold. This Even without taking into account local electrostatics and
mutant is partially rescued by some of the same small weak pKa perturbations, the relevant donor species to QB-(apparent
acids as are active in L210DN/M17DN [212], but the pKa 4.5) must almost certainly be SerOH2+ (solution
recovery is very weak and shows no sign of saturating at pKa 2), rather than SerOH (solution pKa 16), because
the highest concentrations available (E. Takahashi and C. A the pKas in the latter case are much more unfavorable.
Wraight, unpublished observations). Examination of the X-ray SerOH2+ could be formed at very low occupancy by proton
structures indicates that there is little volume available on the transfer from AspL213COOH (pKa 4), which may also
other side of Asp-L213, where it transfers a proton to Ser- be at low occupancy depending on its functional pKa.
L223 and thence to QB , and no water molecules have shown (Functional pKa values in the QB domain acid cluster are
up there. This would clearly inhibit the action of a weak acid, very hard to determine and to specify because of the large
which would have to surmount the steric hindrance of the number of ionizable groups and the strength of interactions
mutation site (Asn-L213, for example) to access Ser-L223, at [202,228,255257]). However, the transfer from SerOH2+ to
least momentarily. QB will be very favorable.
Interestingly, the 2nd electron transfer in AspL213 Glu After the 2nd electron transfer and protonation of the C1O
mutant RCs (L213DE) is also substantially inhibited (approxi- group, the C4O group is protonated and the quinol unbinds
mately 102-fold) but it is not PT-limited [252]. A likely and leaves the QB site. The proton source is GluL212, which
explanation is that changes in the network of electrostatic appears to have complex titration behavior, and is partially
interactions, due to the longer side chain or slightly higher ionized over a wide pH range [202,204,208,228,258261]. It
intrinsic pKa of glutamic acid, lead to GluL213 being neutral, becomes fully protonated after the first flash and functions with
whereas AspL213 is ionized. The local change in electrostatic a high effective pKa 9. It is unlikely that GluL212 donates
potential (less negative) would downshift the pKa of QB , as in directly to the quinol, but perhaps via one or more water
many other mutations of acidic sites. The L213DE mutant is molecules that could move into position as the quinol anion
similar to the GluH173 Gln mutation (H173EQ) in the extent attempts to detach from a hydrogen bond with HisL190. In
of inhibition, while maintaining PT faster than ET [220,230]. mutants where the second proton transfer is inhibited, quinol
Finally, SerL223 is implicated both as a hydrogen bond unbinding is greatly slowed down and follows the slow proton
donor to the semiquinone and as the immediate proton donor, uptake kinetics [221].
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 903

7.5. Selectivity revisited common salts in the 100 mM range do not detectably alter the
proton transferring activity of any of these proteins, with H+ at
The ability for small weak acids to penetrate the proton submicromolar levels. It would seem, therefore, that achieving
transfer pathway of reaction centers, apparently with ease, considerable selectivity is not a difficult engineering challenge,
raises the question of selectivity again. For the reaction center, and that ionizable amino acids can perform this function well.
this may not be a critical issue, since the proton path dead- Although the resolution of the available X-ray crystal
ends at QB and is not part of a transmembrane, pumping structures for most of these examples is not definitive, it
activity. Nevertheless, there is no indication that the wild-type seems likely that the ionizable sidechains interrupt any water
structure permits entry of small ions, i.e., proton delivery is column present, and impart chemical specificity by necessita-
not inhibited by high concentrations of salts, and is only ting protonationdeprotonation of the acidic group. For such
affected at all by virtue of well-understood ionic strength a mechanism, rapid proton throughput requires fast deproto-
effects [141,247]. nation rates. This implies low pKa functional groups, and the
It is possible that the path is sufficiently flexible that it could common occurrence of carboxylic acids in these roles is
accommodate some occupancy by other ions without impairing entirely consistent with this.
proton delivery, but the terminal transfer step from AspL213
to QB- appears to chemically specific. Some (but not all) of the
weak acids that were effective in rescuing the L210DN/M17DN 8. Why are there long proton paths for non-vectorial
mutant could also restore low levels of activity in L213DN reactions?
mutant RCs, but were more than two orders of magnitude less
effective. This suggests that circumventing AsnL213 is For transmembrane-vectorial proton transfer, as in proton
extremely difficultor, possibly, that the native aspartates at pumping systems, the ultimate goal is to move protons across
positions L210 and M17 act with the surface histidines as an the significant dimensions of the membrane and this may
effective proton-specific filter. motivate evolutionary design to maximize the distances covered
For proton pumps, the issue of selectivity is potentially more by any one process. However, it is not obvious why essentially
serious. The D channel of cytochrome oxidase, for example, non-vectorial proton uptake and release reactions should be
might accommodate an alkali metal cation, but this would long range.
almost certainly have a highly deleterious effect on proton In the L210DN/M17DN double mutant, we observed that
translocation. No such inhibition has been observed. Inhibition H3O+ was not as active in rescuing function as its pKa would
by Zn2+ ions does occur, but this is associated with binding to imply, and that NH4+ was also weaker than predicted from a
surface histidine residues, impairing their function as local linear Brnsted plot [247]. This suggests that cationic donors
proton sources [144,262,263]. It is also worth noting that are less effective than neutral acids in this interior environment,
movement of a cation in the D channel would require flux of contrary to their action at the surface as seen in the rescue of
water molecules, as in gramicidin. But this would be in the the 2xHis mutant. Of course, the mutations themselves
opposite direction from any likely water movement and would introduce some change in net and surface charge, and
deposit unwanted waters in the catalytic center. Thus, it seems L210DN/M17DN mutant RCs might be expected to have
that the entry point of the D channel is well served by the diminished affinity for cationic species compared to wild type
structure, including the key aspartic acid and surface histidines, RCs, and the 2xHis mutant RCs relatively more. Nevertheless,
to be proton specific. Also, the water continuity is broken at the the native QB-site engineering appears to be primarily directed
ring of asparagines just inside the channel, which would at stabilizing the anionic semiquinone, achieved by specific
presumably necessitate the complete desolvation of the cation in hydrogen bonding interactions and by a positive electrostatic
an energetically prohibitive step. potential [149,228,257]. As pointed out by Rongey et al. [232],
For bacteriorhodopsin, proton specificity is not due to the a positive potential may favor electron transfer reactions at QB
Schiff base alone, as indicated by the remarkable similarity but it is incompatible with the equally important function of
between bacteriorhodopsin and halorhodopsin [264]. However, delivering H+ ions to QB for full reduction. Thus, to the extent
the local electrostatics and proton acceptor activity provided by that the QB site is net positive, simple accessibility to H3O+ is
Asp85 (which is threonine in halorhodopsin) ensure that not a good solution for proton delivery, in spite of its strong
bacteriorhodopsin acts as a proton pump. The covalent pro- proton donor potential. This, of course, is in addition to its very
tonation of the Schiff base ensures that no other cation is low concentration at physiological pH. Consequently, a
translocated. Whether other ions freely enter the extracellular designed proton conduction path, mostly via neutral acid
cavity seems very unlikely, given the balance of electrostatic groups, is necessary for kinetic competence.
interactions necessary to stabilize the protonated water ion in the However, since the proton carrying activity of the aspartic
proton release group. Furthermore, none are evident in the high- acids L210 and M17 can be restored by soluble neutral buffers,
resolution X-ray structures, so they would have to be highly albeit small ones, and the proton injector function of the surface
mobile. The excluding factor is presumably electrostatics histidines is fully rescued by soluble cationic buffers, perhaps
including the ubiquitous desolvation costs. we should askwhy does the QB site need a specially
The limits on selectivity for these systems have not be tested constructed proton delivery path? Why is it not just accessible
in the same systematic way as for the H+-selective Fo, but to the numerous physiological buffers?
904 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

8.1. Long-lived states simplest forms of electron transfer theory are completely
adequate [272]:
The proton pathway of the QB domain clearly has some
attributes in common with those of proton pumps like k k0 expbRexpDG*=kB T 7
bacteriorhodopsin and cytochrome oxidase, but the RC does
not pump. Furthermore, the thermodynamic properties of the As Dutton and coworkers have effectively shown, for
one-electron redox couples of QB appear to derive from quite purposes of understanding evolution and natural design of
simple principles that need not require elaborately obscured intramolecular electron transfer proteins, the electronic cou-
binding sites [149,257]. However, QB acts to accumulate two pling parameter, , is essentially constant with a value of
electrons from sequential photochemical events, and it must 1.4 1 [1,2,273]. Thus, ET rates are determined by the
be able to store one electron in a metastable formQB is distance, R, and the free energy of reaction, which modifies the
stable for many minutes. This makes it susceptible to non- activation free energy G* according to the usual Marcus
specific reactions, which would partially short circuit the formula [274]:
energy storing reactions of light driven electron transfer and  
proton uptake. We have previously shown the propensity for k DG- 2
DG* 1 8
QA to be reoxidized even by the highly charged ion, 4 k
ferricyanide [265]. However, electron exchanges between
one-electron agents may be more readily reversible than The reorganization energy, , is a measure of how much
charge accumulating reactions. The long lifetime of QB the molecular framework and surroundings change when the
requires it to be sufficiently buried to protect it from even the electron is transferred. Since electronic and electrostatic
most pedestrian scavenging reactions with cytoplasmic energies are large, is usually large, too. If both reactants
electron acceptors and donors, some of which are quite are in protein and therefore somewhat shielded from the
small and might be able to approach QB if it were not well solvent, is on the order of 1 eV, or slightly less. On the
sequestered. other hand, free energies of biological reactions are
Similar considerations may apply to other examples where commonly small. With these guidelines, it is apparent that
one-electron and two-(or more) electron redox chemistries physiologically meaningful reactions, generally faster than
meet. For example, Complex II (succinate dehydrogenase), 1 ms, are limited to distances less than 15 (edge-to-edge)
which is also a non-proton-pumping bioenergetic membrane [1]. This is a sufficiently strong statement that reactions
complex, has a long and highly connected chain of water purported to occur over distances greater than this should be
molecules that links the aqueous-membrane interface to the examined for possible alternative mechanisms, and even
deeply buried quinone site [266]. Like the D-channel in errors.
cytochrome oxidase, it is terminated at each end by ionizable For intermolecular electron transfer, binding of reaction
side chains. The Qo site of the cytochrome bc1 complex has a partners must be taken into account. Otherwise the same
particularly tightly coordinated two-electron chemistry to principles apply, although has not been so well sampled and
perform, and just the assembly of reactive components could be somewhat larger. For a physiological reaction,
cytochrome bL, quinol, and the Rieske ironsulfur protein molecular recognition and binding produce a productive
creates a fully encased quinone site that has no direct access to association in which the electron transfer distance is minimized
solvent. It, too, has a chain of water molecules that link the and binding is of sufficient duration to allow transfer. However,
active site to the periplasmic or intermembrane space, which is the affinity should be no stronger than necessary, since
considered a likely conduit for proton release [267]. Like disassociation is an equally important part of turnover.
reaction center proton uptake, the bc1 proton release is Functional proteinprotein dissociation constants are com-
essentially a non-vectorial reaction and is generally considered monly on the order of Kd 1 M. For a diffusion limited
non-electrogenic (but see [268]). reaction between medium sized, soluble proteins (50 kDa, 50
Hydrogenases are also noteworthy for their deeply buried diameter), the bimolecular rate constant is about 5 108 M 1
active sites. Hydrogen gas is able to move through the protein s 1 even in the absence of any attractive forces. Thus, the
by hopping between small, transiently formed, non-polar dissociation rate constant, koff, is about 5 102 s 1 and the
cavities[269]. Protons, however, are thought to access the lifetime of a complex is about 1 ms. This is long enough for a
active site via water-filled channels [270,271]. reaction with G 0 to occur over a distance of about 15 .
It may therefore be generalizable that redox cofactors with Shorter complex lifetimes could service reactions at shorter
metastable reduction states will be deeply buried, and, if they distances (determined by the location of the redox cofactors in
are also protonation sites, they will require proton transfer each protein).
paths. Conversely, an undesirable reaction might involve a small
redox-active molecule binding non-specifically to the protein
8.2. How buried is that? with a low affinity. If non-specific binding typically has
Kd 10100 mM, this corresponds to koff 107 s 1, or a
This is quite readily answered with the principles that govern lifetime of about 0.1 s. To limit the chance of the redox
non-adiabatic electron transfer in natural systems. For this, the reaction occurring in the lifetime of the complex, also with
C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912 905

G 0, the distance to the protein cofactor should be 9.1. Why water?


10 .12 Clearly a compromise has to be reached for a
protein that is involved in physiological bimolecular asso- An obvious difference between water and, say, serine is the
ciations because the cofactors cannot be buried that deep in mobility that water has in situ. A serine hydroxyl is restrained to
both partners without violating the productive reaction distance rotate about the CC bond, which means that the geometric
limit of 15 . In fact, exchange with one-electron centers may constraints for productive proton transfer are much greater. If
not be especially at risk in this way, as the reactions are more the OH is also hydrogen bonded, then the group is essentially
freely reversible. However, for charge accumulating reactions, fixed in space. In contrast, Hummer et al. noted that, despite the
in which one-electron transfers communicate with n-electron quasi-one-dimensional order of the single-file waters in
centers, e.g., quinone (n = 2), with metastable intermediate nanotubes, the water chain retained considerable entropy due
states, such parasitic reactions are potentially highly dele- to their free rotation about their aligned hydrogen bonds,
terious. The intermediate states can also be very long-lived, resulting in a degenerate energetic ground state [92]. Similarly,
making them vulnerable to even very rare events and very Tian and Cross noted that cations inside gramicidin maintained
feeble reagents. a relatively large positional entropy in the channel sites, and
For a redox center not involved in intermolecular ET, there suggested that this was important for their high transport
is no such constraint, except modesty of cost. Thus, to find mobility [80].
the QB site sequestered 16 inside the protein is not The local mobility of water provides a much greater
unreasonable. The semiquinone lifetime is tens of minutes in the dielectric response than would otherwise be seen, and this
dark, although the relevant lifetime should be taken from low will contribute significantly to the energetics of ion/proton
light conditionsthe lowest at which the organism can grow entry. For protons, the potential for charge delocalization could
phototrophically. also be a significant factor in the energetics. These considera-
The result of such a defensive design for electron transfer is tions also underlie the computational truth that unless water is
that physiological proton transfer must be facilitated over handled with full dynamics, rather than discrete sampling of
similar distances and in such a way that it does not open up the conformations, it is usually better to throw them out and use
redox center to small redox active species, i.e., a proton specific = 80 in the voids.
pathway is required. A unique advantage of water as a proton carrier component is
that it is resistant to mutationcavities capable of accommodat-
9. Implications for intraprotein proton transfer pathway ing water are more robust to mutations than specific amino acids,
design i.e., mutational substitutions of nearby residues, including those
that define the cavity, will often have similar molecular volumes
Unlike electron transfer, the necessary pathways for proton and will not change the volume of the cavity or may only
transfer are highly dependent on structural details, and yet displace it slightly. There are some suggestions, from computa-
robustness remains an important and inevitable criterion for tional studies, that a water-filled channel could be highly proton
natural, evolutionarily selected design. specific, but this would require fine engineering to hold the water
It is evident that water is the major constituent in many of rather rigidly in a limited range of coordinations, and it would
the long distance PT paths actually encountered in proteins. In then be susceptible to mutational breakage. A simpler solution is
contrast, Onsager's original concept of hydrogen bonded to utilize as much mobile water as possible and add selectivity
chains in protein function emphasized a role for amino acid only where needed, in the form of ionizable residues, ideally at
side chains [39,40]. In their further development of this idea, the entrance. This is the basic design of the D channel of
Nagle and Morowitz certainly recognized that water was a cytochrome oxidase. Far greater sophistication is present
candidate [41,49], but they initially focussed on protein elsewhere, at the top of the channel and in the central cavity,
functional groups. As the idea matured, however, Nagle to handle the gating requirements of the pump.
recognized that water was likely to be more than a candidate In reaction centers, the requirements are less stringent, and
and was an important, major component of proton conducting the path functions very effectively with a mixed bag of acids and
hydrogen bonded chains [275]. His motivation was the relative waters. It is clear from this example that specific pKa values are
dearth of hydrogen-bonding residues in the membrane- not very important. Provided the main players are intact, the
spanning regions of bacteriorhodopsin that were being revealed local electrostatics, and hence the functional pKas, are not critical
by electron diffraction studies at that time [276]. This was [229]the PT rate stays fast compared to ET. The pKa of QB, of
confirmed by high resolution X-ray crystallography, and has course, is an exception, because that alters the population of the
subsequently been shown for some other proteins, too, notably active form (QBH) in the rate determining reaction.
cytochrome oxidase. Nagle speculated briefly, but presciently,
on the possible properties of water-wires as proton conducting 10. Conclusions
pathways [275].
Unlike electron transfer, proton transfer has a great deal of
12
For a small molecule bound at the surface and therefore essentially in structural requirement, but still less than might be expected. Fast
water is likely to be larger than 1 eV, which will slow down the rate of proton transfer depends on close proximity, to the point of
reaction and shorten its range. hydrogen bond formation. The constraints that this could
906 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912

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