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

Although electron and proton transfer may appear to have
much common and, in certain regimes, have fundamental
similarities, they differ greatly at a phenomenological level,
especially as they are encountered in the context of protein
function. With few exceptions, biological electron transfer (ET)
is highly insensitive to the structural details of the protein as a
transmission medium [1,2]. The protein plays a critical role in
tuning the thermodynamic properties of redox active cofactors
but, in all cases known so far, it otherwise serves only to
position them at distances that allow electron transfer rates that
are generally significantly faster than the rate limiting steps of
an overall process. This is not to say that the intervening
medium does not provide the mechanism of electron transfer
electrons are transmitted by virtue of the atomic and molecular
orbital wavefunctions that set the excited state energies of the
medium [35]. However, this could be chicken fat for all the
structure that is needed.1
The justification for this cavalier attitude is the length scale
over which electrons can tunnel, by virtue of their very tiny
Abbreviations: ET, electron transfer; PT, proton transfer; gA, gramicidin A;
GMO, glycerylmonoolein; PRG, proton release group (of bacteriorhodopsin);
FTIR, Fourier-transform infrared; RC, (bacterial) reaction center
Tel.: +1 217 333 3245; fax: +1 217 244 6615.
E-mail address: cwraight@uiuc.edu.
1
This refers to one of Harry B. Gray's more colorful criticisms of the no-
pathway approach to biological electron transfer.
in close contact, so many possible paths are accessible to an
electron that they effectively sample the average composition of
the medium; this comes down to atomic packing density, which
is not very variable. Extensive surveys by Dutton and
coworkers have found no clear examples of proteins in which
the connectedness of redox centers is needed to account for the
kinetic competence of the electron transfer [1,6]. Furthermore,
there is no distinction between the connectedness of (or packing
density between) two random points in any protein and that of
two functional donoracceptor locations, e.g., cofactors. The
conclusion is very strong that structure, as in specific pathways,
is irrelevant to the vast majority of electron transfer reactions.
The approach taken by evolutionary design has been to use
protein ligands to place cofactors at distances short enough to
ensure competent electron transfer rates. Often the rates are
orders of magnitude faster than necessary, but in a few cases
they are simply adequate. The latter may include the very fast
kinetics of photosynthetic primary photochemical events, but
these are achieved by near-contact of cofactors and the protein
is actually excluded altogether!
The distances observed for intramolecular electron transfer
in physiological reactions are almost universally less than 15 ,
edge-to-edge.2 From Marcus theory of non-adiabatic electron
2
The exact meaning of this expression is the source of much of the debate in
this area. In general, it means the distance between edge atoms that are strongly
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
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
often apparent, and the formalisms for describing PT appear
similar to those used for ET, regardless of the underlying
mechanism [2022].
Proton transfer in biology falls into two broadly useful,
but not fundamentally different, classesthe reactions of
catalysis and chemistry, and the reactions of transport. In the
former, the proton is transferred between two atoms in a
process of chemical conversion or acidbase catalysis, and
further transfers are absent or not kinetically important. In
transport reactions, the purpose is to move protons over
significant distances, often on the order of a membrane
thickness. This necessarily involves many elementary proton
transfer steps and implies a significant structural component,
or path. In principle the path may be permanent or transient,
but there are substantial energetic constraints on forming one
transiently.
In trying to get a broader perspective on what features
characterize, or are essential for, biological PT, I have bitten off
more than I can chew. Nevertheless, I hope that some of these
observations will be useful to the bioenergetic community, at
large.
2. General features of H+ transport in water
The electrostatic binding energy of the proton is so large that
it has no independent existence in condensed phases. In water, it
is generally considered to be present as hydronium, H3O+,
which is similar to Na+ in size and solvation characteristics. It
can therefore be expected to exhibit diffusive properties similar
to Na+, with diffusion coefficient, DNa = 1.33 10 5 cm2 s 1
[23], or perhaps water, with DH2O = 2.3 10 5 cm2 s 1 or
0.23 2 ps 1 [24,25]. However, the rapid exchange of H+
3
Normal acids were defined by Eigen [17] as those that exhibit near
diffusion controlled PT rates when thermodynamically favorable (pKa > 0),
with the reverse rate smaller by a factor 10pK. These are also what are defined
as acids (or bases) in common experience, involving electronegative atoms,
especially oxygen and nitrogen.
887
Scheme 1.
between H3O+ and H2O allows for a unique transport process
known as the Grotthuss mechanism, which transfers protonic
charge without diffusive movement of either an individual H+
or oxygen atom (Scheme 1).
This process is similar, in essence, to the proposal of von
Grotthuss, 200 years ago, for the migration of charge in aqueous
electrolysis [26]. It is remarkable that this predates the
publication of the atomic theory [27] and that, at the time,
even afficionados did not know the correct composition of
water, which von Grotthuss wrote as OH. This transport
mechanism is now celebrated with his name. Its true
significance for H+ ion transport in water was not appreciated
for 100 years, when Danneel first attempted a mechanistic
description of the elementary process [28]. This initiated
another 100 years of argument over the role of rotation (of
H3O+ or H2O) in the rate limiting step [2932].
Hydrogen bonding between water molecules makes the
Grotthuss process (also termed structural diffusion) fast and
efficient, and it is responsible for the major part ( 85%) of the
apparent ionic mobility of H+ in water, which is about 67 times
that of Na+.4 The unique nature of this process has attracted
much interest, but the mechanism is only now becoming clear at
an atomic level [19,22,3235].
The current view of the Grotthuss mechanism in bulk water
is that proton transfer occurs in a step-wise manner, via the
interconversion of an Eigen cation, H9O4+ = (H2O)3H3O+, and a
Zundel cation, H5O2+ = H2OH+OH2 [32,33]. The activation
energy reflects the breakage of a more or less normal hydrogen
bond (approximately 2.5 kcal/mol) in the second solvation shell
(between H9O4+ and another H2O), allowing reorganization
4
Agmon [31] has argued that the solvated proton is hydrodynamically
immobile, so that all the measured mobility is by the Grotthuss mechanism.
This seems extreme and unwarranted, since the hydrogen bond strengths
between the solvent water and the first solvation shell of Na+ and H3O+ are
similar. Thus, somewhat equivalent mobilities of Na+(aq) and H3O+(aq) (i.e.,
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,
within which the excess proton is shared equally between two
oxygen centers. Reformation of a hydrogen bond with another
water molecule localizes the excess proton on a new Eigen ion.
These events allow transfer of the proton charge across the
OO distance of an Eigen cation (approximately 0.255 nm) in
the lifetime of proton exchange in water (1.33 ps from NMR
[36]). This yields a 3-dimensional diffusion coefficient of
(0.255 nm)2/6(1.33 ps) 8.1 10 9 m2 s 1 (0.81 2 ps 1) To
this should be added the normal (molecular or vehicle)
diffusion of H3O+ (similar to that of Na+, 0.13 2 ps 1),
yielding a total in good, but perhaps fortuitous, agreement with
the experimental value (0.93 2 ps 1 [37]). An important
conclusion from this correspondence is that proton transfer in
water is an incoherent process.
The elementary PT rate in bulk water may benefit somewhat
from the fact that the free energy of the Zundel ion (essentially a
transition intermediate) lies quite close to that of the Eigen ion
[38]. However, the pairwise jump time is expected to be fast for
any energetically favorable or near-neutral proton transfer, e.g.,
for normal acids with similar pKa values [7,17]. Thus, a
Grotthuss-like mechanism can be envisioned for any chain of
hydrogen bonded acidbase groups with an appropriate
geometry for continuous hydrogen bondingeach hydrogen-
bonded atom must act simultaneously as donor and acceptor.
This could be fulfilled by several O and N functional groups,
and especially OH. The potential for hydrogen-bonded chains
to facilitate fast proton transfer in biology was developed by
Nagle and Morowitz and coworkers following a suggestion by
Onsager ([39], and see [40]), and gave rise to the term proton
wire [40,41] (Scheme 2).
3. Long distance proton transport
Each step of a long distance proton transport requires
rearrangement of charge. Inside the low dielectric of a protein,
this implies substantial electrostatic and reorganization costs at
every step. Furthermore, if the proton originates in the bulk
phase, there is an additional desolvation penalty that is also
largely electrostatic (Born energy). Proton wires would be a
uniquely suitable way of transporting H+ in a structured
medium, because the covalent association of the H+ with the
carrier groups and the hydrogen bonding between them
substantially diminish the electrostatic cost of bringing the
charge into the lower dielectric medium of the protein. The
delocalization of the charge through a hydrogen bond network
Scheme 2.
can be extensive, as first noted by Zundel, and the charge
distribution is highly polarizable and is very sensitive to local
perturbations [42,43]. Depending on the extent of the
delocalization, polarizable protons give rise to varying degrees
of continuum absorbance in the infrared spectrum. In extreme
cases, which are nevertheless easily created in hydrated films of
weak acidbase mixtures, the continuum extends throughout
the mid-IR, from the OH stretch of water (at about 3600 cm 1)
to the far-IR region. In less extensive delocalizations, the IR
signature is contracted, and can be distinctive. Calculations
show clear differences between solvated H3O+ and H5O2+ in the
mid-IR between 1800 and 2600 cm 1 [4345], with some
experimental support [4648].
The PT step rate within a hydrogen-bonded chain can be as
fast as for the Grotthuss mechanism in bulk water, with
elementary transfer times between sites on the order of 1 ps.
However, the transfer of a proton along the chain reorients the
hydrogen bond connectivity, which must be restored in order
to conduct a second proton in the same direction. This led to
much attention being spent on both the hopping action that
transports the proton and the turn motion that regenerates the
starting configuration of the chain for steady state turnover
[49]. In contrast, in bulk water, the high coordination number
of water yields a multiplicity of hydrogen-bonded paths in 3
dimensions, and the turn motions restoring a prior arrange-
ment are not important in the steady state transfer of protons
(conductivity).
Thus, the question arises whether the potential for rapid
proton transfer in hydrogen bonded chains is realized in
proteins. Furthermore, the highly directed PT in a one-
dimensional path can be controlled and/or limited by catalytic,
electrostatic and conformational events, which may be either
incidental or designed.
4. Ion channels and other transport devices
If we look for inspiration in the rapid ion transport by K+
and Na+ channels, the electrostatic and reorganization costs of
bringing charge into a protein and membrane interior have
been addressed directly by preorganized structures that
compensate for the desolvation penalty in the selectivity filter,
and resolvate and electrostatically stabilize the cations as they
enter a large, internal, aqueous cavity. The selectivity filter
necessarily binds the desolvated cations, which is counter-
productive for rapid transport. This is overcome by ionion
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
gramicidins are especially relevant because they have been
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
widely studied for both their alkali metal cation and proton
conductance properties.
4.1. Gramicidin
The ionophorous gramicidin antibiotics are 15-residue linear
peptides of alternating D- and L-amino acids that form ion
channels in membranes [5355]. There are many minor
sequence variations, but the best known is gramicidin A
(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-
(CH2)2OHnote the charge-neutralizing N and C terminal
modifications. The gramicidins associate strongly with mem-
branes and adopt a wide-bore, right-handed, 6 helix6 structure
that spans approximately one leaflet of a membrane bilayer,
with the formyl-N-terminus buried. Two molecules, in op-
posite leaflets, can associate by hydrogen bonding between
their N-termini to form a complete, but transient, transmem-
brane channel (Fig. 1). The lifetime of these head-to-head
dimers is on the order of 0.11 s, depending on pH, tem-
perature, and membrane lipid type. Covalently linked dimers,
exhibiting long-lived conductances, have proven very useful
for studying cation conductances, especially at the single
channel level [5560].
The gramicidin channel is readily permeable to monovalent
cations, notably the alkali metal cations, ammonium and
protons. The tryptophan residues form a collar located in the
lipid headgroup-interfacial region of the membrane, and play an
important role in the energetics of cation entry into the channel.
The substitution of one or more of the four tryptophans by
phenylalanine or tyrosine in gramicidins B, C, and M, has
distinct effects on the conductance properties [6164].
Gramicidin has long been considered the poster-child for
the Grotthuss mechanism at work, and there is a tendency to
impute abnormally high proton conductance to it. The
quantitative basis for the latter inference is shaky, however,
due to a number of technical difficulties, including surface
potentials, surface polarizability, ion binding and access to the
channel, that conspire to obscure the true channel conductance
(rather than the total conductance, including entrance and exit
events) [6567]. However, it is clear from electroosmotic and
streaming potential measurements that alkali metal cations
pass through the pore in single file with channel waters, while
protons pass without significant water flux [6872]. This
strongly implicates the Grotthuss mechanism.
Conductance is a concentration dependent parameter, but the
intrinsic mobility of most ions in a channel is related to the
maximum conductance [59,67]. Unfortunately, this is not a
straightforward measurement, due to conflicting requirements.
It should be determined at high ionic concentration to override
the external resistance from bulk diffusion, but this introduces
5
The commercial preparation, gramicidin D, is a mixture of gramicidins A
(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,
is synthetic.
6
Also referred to as a (L,D) and 6.3 helix.
889
complications from ion binding, which depresses the con-
ductance and requires multiple-ion mechanisms to overcome it
[67,72,73]. It should also be determined at low potentials, to
avoid saturation and interfacial polarization effects [66]. From
the large literature on cation conductances, for example in
membrane bilayers of glycerol monoolein (GMO), I extract the
following. The maximum single channel conductance for H+ in
gA (gH,max) is at least 2 nS [55,59,64]. From this, the diffusion
constant for H+ in gA can be obtained [59]:
DH L2p :gH;max =ekB T
25  108 2 2  109 =1:6  1019  0:025
3  105 cm2 s1
where Lp is the length of the gramicidin pore ( 25 ). This is a
significant fraction of the value in water (9.3 10 5 cm2 s 1). In
fact, because the proton conductance of gA is so high, it is
possible that gH,max is determined by the bulk phase mobility,
which saturates at similar acid concentrations (> 3 M HCl),
due to changes in water structure at high ionic strength
[55,60,74,75]. In this case, the mobility within the gA channel
could be as large as that in water, or even greater.
The self-diffusion constant for bulk water is 2.3 10 5 cm2
1
s [25]. Hydraulic permeability measurements [71] show that
the net diffusion of water through the gA channel in GMO
bilayers is about 10 times smaller, DH2O 0.2 10 5 cm2 s 1.
Since net transport of water requires the coordinated diffusion
of the whole file of 89 waters in the channel, the local diffusive
mobility of the individual waters is 89 times that seen in net
flux, and is therefore almost indistinguishable from the bulk
phase value. This is supported by computational (Brownian
dynamics) methods [76]. This indicates relatively weak inter-
actions between the waters and the channel walls, i.e., no
stronger than waterwater.
The mobility of the water column as a whole is expected to
provide an upper limit for alkali cation diffusion in the channel,
as they move in single file with the waters, and conductance
measurements yield somewhat similar values. For cesium, Cs+,
the most permeable of the alkali metal cations, gCs,max = 50
100 pS [67,73], and DCs 0.15 10 5 cm2 s 1 within the
channel. This is close to the limit expected from the diffusive
permeability (Pd) of water in the channel, but there is a factor of
23 uncertainty in the value of Pd, which has not yet been
resolved [53,68,70,77]. In bulk solution, DCs 2.05 10 5 cm2
s 1, so Cs+ mobility is decreased at least 14-fold (compared to
3-fold, or less, for H+). Nevertheless, the Cs+ mobility is still
substantial and implies that interactions of Cs+ with the channel
walls are weak. Measured dissociation constants support weak
binding for all alkali metal cations, although reported values are
very variable [64,72, 73,7880].
Thus, although protonic charge movement may be slightly
depressed relative to bulk, the effective diffusion coefficient for
H+ is at least 20 times larger than for any other ionic species, or
for net flux of water. The large protonic conductance of
gramicidin is a consequence of a Grotthuss-type mechanism
that proceeds independently of molecular diffusion, at a rate
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
Because of the known speed of pairwise PT rates, turning
motions were considered, at first, to be the likely rate-limiting
step for steady state proton conduction in a one-dimensional
hydrogen bonded chain [40,49]. Initially, some microscopic
calculations seemed to support this by showing that the turn
motions in single files of water have significantly larger
activation energies than the proton hopping steps [81,82].
However, experimental results [83,84] and further computa-
tional studies [64,8587] of gramicidin A indicate that neither
process is rate limiting in the net transfer of protons through the
channel. This is also true for the permeation of water through gA.
Brownian dynamics simulations show that the major limitation
to net transfer is an access resistance to entry of water
molecules [76]. By far the largest contribution to the access
resistance is associated with the desolvation process (removal of
approximately two water molecules) as bulk water becomes
single file channel water.
A very similar picture has emerged for the conduction of
alkali metal cations through gA. For example, NMR and
computational studies show that the desolvation of Na+ occurs
in discrete steps as the ion enters the gA channel [80].
Ultimately, the cation is solvated by only two waters in a
single file of waters through the channel, and this configura-
tion is able to maintain about half of the total solvation energy.
The energy cost of desolvation is partly compensated by
dipole interactions with the four tryptophan residues that form
a collar in the membrane headgroup region of the channel
[64,80]. The reverse sequence of events would accompany the
exit of the cation from the other end of the channel. NMR
studies also show that Na+ is loosely bound at discrete sites
through the channel, with a significant amount of positional
disorder. This minimizes the entropy loss upon uptake from
the bulk solution and is likely to be a contributing factor in
allowing the high mobility of ions in the channel.
Experimental and simulation approaches tend to place the
rate limiting step for proton conduction by gA outside the
channel proper, but the molecular nature of the event is still
unclear. Schumaker [86] suggested that exit of the proton is rate
limiting. Phillips et al. [84] proposed that the rate of initiation of
hydrogen bond reorientation is modulated by interactions
between the interfacial dipoles of the gramicidin tryptophan
residues and those of the channel water column, which
kinetically control the entrance of a defect necessary for the
turning motion, starting at the channel exit. However, de Godoy
and Cukierman found a counter-influence of the intrinsic dipole
potential of different lipids [88]. Warshel has argued strongly
that the limiting barrier to most transport phenomena is the
unfavorable free energy of transfer of charged or polar species
to the channel interior [87,8991]. This comprises a direct
electrostatic component and a desolvation energy, both of
which must be sufficiently compensated or minimized to allow
rapid, net transport. However, without more specific develop-
ment, it is not at all clear which are kinetic and which are
energetic constraints in any of these models.
4.3. Hydrophobic channels
Proton transfer in the polar gramicidin channel seems to be
entirely consistent with the Grotthuss mechanism in water, but
some unexpected behaviors have come to light in computational
studies of hydrophobic channels.
4.3.1. Water transport in hydrophobic channels
In long (> 50 ns) molecular dynamics simulations of
carbon nanotubes (diameter 8.1 ), Hummer et al. [92] found
water to spontaneously enter and to exhibit a diffusion
coefficient at least as great as in bulk phase. However, the
occupancy was very sensitive to the precise interaction energy
between water and the channel wall. In pores constructed of
methane-like pseudoatoms, Beckstein and Sansom [93],
found that channels narrower than 1213 diameter were
substantially closed to small ions, while channels of diameter
<89 were empty of water. This, too, was very sensitive to
the polarity of the pore, i.e., the waterwall interactions. At
intermediate diameters, the water oscillates between liquid
and vapor states; the average density is less than bulk and
presents a significant electrostatic barrier to ion passage
[94,95]. The exact cut-off diameters are clearly sensitive to
details of the interaction energy between water and the pore
wall, but the general features are robust. Some influence of a
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
hydrophobic pore can be seen even at diameters as large as
20 ; this is not reproduced in continuum electrostatic
calculations [95].
The large diameter pore effects arise from interactions with
the second solvation shell and the development of structured
water layers at the hydrophobic surface of the pore [96]. Ion
passage is restricted due to the energy cost of desolvation and
the development of a layered solvent structure (at least 3
molecular layers thick 6 ) of the water adjacent to the
hydrophobic wall. The cost of depleting even the second
solvation shell (radius 5.5 for Na+), plus the layered water
structure at the wall, restrains ions from entering a pore of less
than 13 diameter, while the energy necessary to strip any of
the first solvation layer (radius 3.5 for Na+) completely
prohibits entrance to a channel less than 7 in diameter.
Evidently, channel hydration can be modulated by small
changes in diameter or in polarity, and it was suggested that
channel conductance gating could be based on such changes in
a suitably constructed channel pore [93,96].
Such a model may be applicable to the acetylcholine
receptor channel, where the closed state appears, from
cryoelectron microscopy, to be a 6.5 diameter hydrophobic
constriction in the pore formed by the M2 transmembrane
helices of the five subunits (2) [93,97]. Similar results
have been reported for simulations on the small mechan-
osensitive channel, MscS [98]. In addition, hydration of MscS
was found to be stimulated by an applied voltage, suggesting a
possible, intrinsic mechanism of voltage gating [99].
These results illustrate the control that the Born and
desolvation energies have on ion transport, and underscore
the necessity for a narrow channel to provide adequate
compensation for any desolvation of an ion. Clearly gramicidin
achieves a fine balance between providing enough polarity to
allow the channel to be occupied by water, and a minimum of
interaction that would slow down the diffusive movement for
water and monovalent cations.
However, the wall interactions in a polar pore are not
negligible, as they appear to be in a hydrophobic pore.
Simulations on carbon nanotubes [100] and hydrophobic
pores [93,96] show that as the diameter of the pore is decreased
below 20 , the average density of water decreases due to
increasing frequency of transition to the vapor phase. At the
same time, the diffusion coefficient increasesby as much as
3-fold in the narrowest channels to have significant water
density. Contributing factors to this effect include reduction in
dimensionality, transition to vapor phase behavior, and the
lack of polar interactions with the wall. A recent experimental
study of fluid transport through slightly wider bore (1420
diameter) nanotubes, found dramatic enhancements over
macroscopic predictions [101]. Gas flow was more than an
order of magnitude greater than the Knudson limit, and water
flux was 23 orders of magnitude greater than predicted by
Poiseuille's law for hydrodynamic flow.
Given the still common problem (at least for the non-expert)
of comparing results from different computational schemes, it is
worth noting that the main features of the simulations on
hydrophobic channels results do not depend significantly on the
891
models used for water or on the composition or nature of the
hydrophobic material (see [94]). When proton transfer is
involved, however, results must be considered more critically.
4.3.2. Proton transport in hydrophobic channels
Several computational studies have been carried out on
proton transfer along water chains in hydrophobic channels,
with largely consistent results, as first reported by Poms and
Roux [82]. Waters placed in a hydrophobic pore may be able to
conduct a protonic excess charge extremely rapidly by the
Grotthuss mechanismas much as 40 times faster than in bulk
water [102,103]. Following proton transfer, the orientation of
the water chain is restored by migration of either of the two
types of hydrogen bonding defect (D or L). As suspected by
Nagle and Onsager and colleagues, this turn motion is slower,
but it is still fast by bulk phase diffusion standards [103].
However, the relevance of any of these determinations to net
proton transport is questionable, as they do not address the rate
of entry and exit of protons from the bulk phase and, therefore,
the energetics of occupancy. Burykin and Warshel [89] found a
prohibitively high barrier for proton occupancy of a 8
diameter hydrophobic pore, and the simulations of Sansom and
coworkers would agree with this [96].
Brewer et al. [102], using a water-filled, smooth-walled,
hydrophobic cylinder, found that the diffusion coefficient of
waters placed in the cylinder was close to the bulk value for
channel diameters greater than 5 . However, as the diameter
decreased from 5 to 4 , the diffusion coefficient of the channel
water went almost to zero, as previously found by Lynden-Bell
and Rasaiah [104], while the mobility of an excess proton
charge increased dramatically (> 40-fold). This is an interesting
and unexpected result, but it is of uncertain significance to real
proton transfer systems, as the simulation times were insuffi-
cient to establish the stability of the water in the column. Similar
results were obtained by Dellago et al. [103], using carbon
nanotubes of 8.1 , which Hummer et al. [92] previously found
to be stably hydrated.7 However, the long-time simulations of
Sansom and coworkers, discussed above, indicate rather
strongly that no water would reside in channels as narrow as
5 , and even at 913 diameter the water density was lower
than bulk, and underwent rapid oscillations between liquid and
vapor states [94]. This would break any hydrogen bonded
connectivity necessary for rapid proton transfer.
Nevertheless, such large enhancements of proton charge
conduction, as calculated in these studies, seem to fulfill the
hopes and aspirations of the early proposals for proton wires,
and understanding why the PT rate is dramatically enhanced in
7
In fact, it is unclear how diameter is defined in these works (refs.
[92,103]). If it is the center-to-center distance between carbons on opposite
sides of the tube, then the lumen diameter is substantially smaller than this,
approx. 4.5 . This would be comparable to the narrowest, smooth-walled
hydrophobic tube in which Brewer et al. also found greatly enhanced proton
transfer [102]. It would also be very much smaller than the narrowest
hydrophobic pore that is stably hydrated in the simulations of Sansom and
coworkers (who clearly define their diameters as the lumen). Thus, although the
PT results may be reasonably consistent, the physical reality of a suitably
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
engineered. The simulations of Brewer et al. [102] showed that
the narrow (4 ) diameter and lack of polar interactions with the
wall made the Eigen- and Zundel-like ion configurations almost
equal in energy, facilitating the interconversion of Eigen and
Zundel ions and stimulating the Grotthuss-type transfer of
excess protonic charge.
In contrast, simulations on a synthetic (but real) proton
channel, with a similar average diameter but some polar
attributes, showed normal water mobility [105]. The channels
were formed from synthetic amphiphilic helix bundles, with a
repeating leucine-serine motif, and exhibited high proton
conductance [106]. However, depending on channel width
and interactions between channel water and serine hydroxyl
groups at the channel wall, either Eigen- or Zundel-like ion
configurations were relatively stabilized. The presence of a
strongly stabilized Eigen ion at one location was expected to
limit proton conductivity in the channel.
Experimentally, the synthetic peptide (LSLLLSL)3 has
> 40-fold greater permeability for H+ (in 0.5 M HCl) than for
any alkali metal cation at similarly high concentrationsnone
was detectable for Li+, Na+ or K+ at 2.510 M [106]. In the
same study, a slightly different peptide (LSSLLSL)3 was
readily permeable to all monovalent cations (like the acetylcho-
line receptor), with H+ permeability five-fold greater than Na+.
This is very close to the ratio of mobilities in water, indicating
both free diffusion and Grotthuss transfer in the channel. The
significantly greater selectivity of the first peptide for H+
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
cell membrane. To achieve fast cation transport, water must
8
There is strong evidence that gramicidins act on transcription, with a
specific effect on a factor associated with sporulation [107109] and it is
very likely that one important role is in the regulation of sporulation of the host/
source species, Bacillus brevis. There are several gramicidins, some linear (A,
B, C) and some cyclic (S, J). Both types are reported to affect transcription, but
only the linear ones transport cations effectively.
also move rapidly through the channel. The necessary design
features for this also allow H+ transfer by a one-dimensional
Grotthuss mechanism, achieving H+ passage rates comparable
to those in bulk water. However, the H+ contribution to the net
current is negligible at physiological pH and it seems
reasonable to suppose that the high PT capability is a purely
incidental property of a chain of waters with high mobility
resulting from incomplete coordination.
This view is supported by the behavior and design of
aquaporins. Here, the channels are intended to pass water at
high rates, but with very low proton permeability. Water is
transported rapidly in single file with high specificity, as in
gramicidin, but some specific design features prevent proton
conduction (Fig. 2). A rough two-fold symmetry axis in the
plane of the membrane introduces an interesting structural
transition around two conserved NPA motifs in the middle of
the channel. At this location the water chain changes polarity,
presenting an apparent block to proton conduction by a
Grotthuss-type mechanism. On this basis, the NPA motif was
originally suggested to be the structural basis for the low H+
mobility [110113]. However, molecular dynamics simulations
and electrostatic calculations showed that the channel dynamics
are very fast and the limiting factor is dominated by the
energetics of channel occupancy, which are determined by the
desolvation penalty and a positive electrical potential near the
center of the channel [89,114,115].
In AQP1, a water-specific channel, the barrier height for a
proton is on the order of 25 kcal/mol [89,113,116], while the
Fig. 2. Aquaporin. Only one monomer is shown, although aquaporins and
aquaglyceroporins are homotetramers, each monomer apparently acting
independently. Note the two half helices and re-entrant loops positioned
quasi-symmetrically across the midpoint of the structure. The membrane plane is
horizontal. Waters shown (green) are crystallographic. The asparagines, N78
and N194, of the two NPA sequences at the end of each half-helix, are in orange.
Histidine H182 and arginine R197 provide some of the electrostatic barrier to
cation passage through the channel (see text). Structure file: 1j4n.pdb, for bovine
red blood cell AQP1 at 2.2 [110].
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
barrier in aquaglyceroporin, GlpF, is about 15 kcal/mol
[114,117], perhaps reflecting a wider bottle-neck and therefore
less stringent desolvation penalty. Various mutational mani-
pulations, carried out in silico, seem to support this. Turning off
the positive potential (from an arginine and histidine, and the
positive end of the macrodipole of a short, reentrant -helix
from each side of the membrane) substantially lowers the
H+ barrier, probably to as little as 8 kcal/mol, although the
calculated values differ widely [89,113,114,116]. This is still
significantly higher than for gramicidin. The remaining barrier
is due to the loss of solvation of the ion (H3O+) in the single
file of waters [118].
Experimental mutation studies also support the important
role of electrostatics in the aquaporin proton block. In AQP1, a
double mutant of the key arginine and nearby histidine exhibits
electrical conductance (the single mutants also have a very low
but measurable level) [119]. This is prematurely identified as
proton conductance, but it does illustrate successful modifica-
tion for ionic conductance of some sort, and the calculated
barrier heights for a monovalent test charge are consistent with
this [116]. The substantial impact of desolvation, beyond direct
electrostatics, is strongly supported by a neutral mutation in
AQP6 (Asn60 Gly), which inhibits its native function as an
anion channel but enhances its water permeability [120,121].
In contrast to aquaporin, gramicidin assists the desolvation of
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
interaction with the ion, as it loses more than half of its first
solvation shell [80]. A net barrier to charge permeation remains,
which accounts for the activation energy of 45 kcal/mol [122].
5. Coupled proton transfer
While ultrafast PT might be possible in certain structures,
such as a narrow hydrophobic channel, the structural and
energetic costs of doing so are large. It is also evident that
mobile waters in a weakly polar environment can support PT at
a pretty good lick. Diffusion is notoriously unproductive when
unbiassed, but can readily yield results when a driving force is
applied. In simulations of the D-channel in cytochrome oxidase,
with a free energy span of 1 eV, proton transfer proceeded at a
rate equivalent to 25 in less than 100 ps [123]. However, such
speeds may not even be generally necessary, as net proton
translocation involves uptake and/or release of a proton from the
bulk phase, and these are often rate limiting, as discussed above
for gramicidin.
In catalytic systems, including bioenergetic complexes,
chemical events are likely to be limiting. For many proton-
coupled reactions, the key event is a single H+ delivery followed
by other rate-limiting processes (further ET, or a conformational
change). Here, the important thing is rapid equilibration with the
surface pH. Even if turn motions were truly slow, the proton
moves back and forth along the same orientation of the path,
and reorientations necessary for the next H+ delivery can take
place at relative leisure during the subsequent events. In such
cases, fast diffusion within a channel or pathway will be slaved
to rate limiting entry and exit or by energetically unfavorable
893
occupancy, or will serve a master process that can be
substantially slower.
In other cases, however, especially those associated with
active pumping, the speed of proton delivery may be important
to prevent slippage and de-coupling of the energy conserving
PT path from the coupled reaction which, again, could be ET or
conformational changes. The speed of proton transfer at critical
steps in the coupled reaction pathway will then contribute to the
pumping stoichiometry (yield), and will become increasingly
influential as the transmembrane proton gradient builds up. If
the rates of PT needed for this are beyond what can be achieved
by incoherent or unconcerted proton hopping, we can expect to
encounter them in such reactionsin proton pumping ATPases,
cytochrome oxidase, bacteriorhodopsin, etc.
6. Proton availability and supply
The overriding impact of electrostatic and desolvation
processes in controlling net rates of ion transfer is especially
relevant for protons, since H+ and OH are rarely, if ever,
present at significant concentrations. Thus, proton availability is
a key limiting factor. Indeed, this has been a major focus of
attention over the last 25 years, and crucial roles of proton
buffers, carriers, reservoirs and antennae, etc. have become
evident [124128].
6.1. Proton conduction in the Fo domain of H+-ATPase
Perhaps the most striking example of proton transport that
exceeds the apparent supply rate is the Fo component of F-type,
H+-translocating ATPases. The proton conductance of Fo
channels (if that is what they are) has been studied by Junge
and coworkers [129], and it is remarkable in both its magnitude
and its pH-(in)dependence. The maximum is 10 fS, at pH 8, but
it also varies only 3-fold over the range of pH 6 to 10. The
proton conductance at pH 8 corresponds to 6.103 s 1 at 100 mV
[129]. This is much faster than needed for ATP synthesis, but
also exceeds the expected rate of delivery of H+ by free
diffusion from the bulk solution at a concentration of 10 8 M.
With the typical second order rate constant used for H+
reactions, kH = 4 1010 M 1 s 1, the delivery rate should not
exceed 4 102 s 1.
The problem of diffusive supply was originally considered in
the context of ion channels, perhaps first by Luger [130], but
the approach is inherent in the most basic formulation of a
second order rate constant by the steady state Smoluchowski
equation:
NA
k 4p R1 R2 D1 D2 1
1000
R1, R2 and D1, D2 are the radii and diffusion coefficients for
two reactive species (particles) in solution. If one is significantly
more mobile than the other, then (D1 + D2) is well approximated
by the faster one, and this applies well to reactions between small
molecules and proteins or membranes. The sum of the radii
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
small and large molecules, or with membranes, the capture
radius is not defined by molecular dimensions but requires
specification of the reactive surface, which is often not well
known. (R1 + R2) therefore becomes an empirical parameter, the
capture radius, R0. For H+, with DH 10 4 cm2 s 1, the
commonly used empirical value of kH = 4 1010 M 1 s 1
corresponds to an encounter radius of about 5 , not
unreasonable for a soluble base or buffer.
The encounter rate described by the simple Smoluchowski
equation is also modified by any forces between the particles
[131]. As expected, attractive forces, e.g., oppositely charged
particles, increase the rate of encounter while repulsive forces
decrease it. The effect of interaction can be addressed explicitly,
if the form of the interaction is known analytically, or implicitly
as a modified encounter or capture radius. For electrostatic
interactions, the equation can be modified according to Debye
[37,126,128,132]:
NA d
k 4p R1 R2 D1 D2  d
1000 e 1
  2
jR0
exp d
1 jR0
The characteristic parameter, here, is , the signed ratio of
the Bjerrum length, RB, which is the distance at which the
electrostatic interaction between two ions becomes equal to
kBT, at zero ionic strength, and the unmodified encounter
distance, R0:
z1 z2 RB jz1 z2 je2
d d where RB 3
jz1 z2 j R0 4per e0 kB T
The third factor in Eq. (2) modifies the interaction according
to the ionic strength of the medium 1 is the Debye length.
The resulting, effective capture radius is often referred to as the
Coulomb length, and the associated volume as the Coulomb
cage.
For membrane systems especially, an important situation
arises when charged species are close enough for their Coulomb
cages to overlap and merge. Under these circumstances, a
proton or other mobile charge can diffuse a considerable
distance within the cage before escaping to the bulk phase
[126]. For protons, this may be further facilitated by dynamic
structuring of water in the presence of the charges [133].
In the case of membrane channels, Luger [130] considered
the influence of the capture radius on single channel
conductances by conceptually adding a hemisphere, of radius
R0, at each end of the pore, and computing the diffusive flux.
The convergence permeability (reciprocal of the access
resistance) then becomes:
Pa 2pR0 D cm3 s1 4 protons at generally much higher concentrations (mM) than
and the corresponding molar ionic conductance:
K 103 FPa F=RT S=M 5 phosphates, are negatively charged, which diminishes their
For protons, with a bulk phase diffusion coefficient of
10 4 cm2 s 1, we can estimate the capture radius necessary to
achieve the observed conductance Fo at pH 8, i.e., = (10 fS)/
(10 8 M) = 10 6 S/M. This requires R0 to be about 40 . This is
large and cannot be simply interpreted as a hemispherical
capture volume, as it would extend much too far into the bulk
phase to correspond with the Coulomb length at any likely ionic
strength. However, as suggested by Eigen's analysis of
dimensional effects on diffusion [134], it could be indicative
of a surface area of similar proportions, comprising the
overlapping Coulomb cages of many groups covering the
protein and the surrounding membrane.
Transposing this result to a near-surface domain assumes
that the diffusion coefficient of the proton remains similar to
the bulk phase. In general, this is not to be expected as a
retentive Coulomb cage would be mostly generated by ionized
carboxylates residues with pKa 5 and a dissociation rate
constant of about 106 s 1. This is slow enough to substantially
retard the average progress of a proton as it hops, via water,
from one site to another. On the other hand, if the acids are
close enough to transfer protons directly, or with only a single
intervening water molecule, the PT rate is much faster, i.e.,
between more or less matched pKa groups, and the process
becomes Grotthuss-like.
Also, after much controversy, there seems to be some
consensus on the lateral diffusion coefficient for protons, along
the lipid/water interface [135,136]. A recent determination on a
phosphatidylcholine (zwitterionic) lipid bilayer yielded
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
in the bulk phase. It is considered compatible with a Grotthuss-
mechanism involving surface water.
At the present time, the information on Fo is of insufficient
resolution to say whether the requisite surface charges are
present to make the large capture radius a possibility. In terms of
protein alone, it is probably unlikely, but with lipid headgroups
included, especially considering the apparent propensity for
negatively charged lipids, like cardiolipin, to associate with
bioenergetic proteins, it is possible. However, there is now
growing belief in the existence of water structure at membrane
interfaces that can restrain ion movement in the absence of net
surface charge. This is possibly related to the layering effects at
hydrophobic surfaces seen in the long molecular dynamics
simulations of Sansom and coworkers [94,95]. The evidence for
an interfacial barrier is discussed at length by Mulkidjanian et al,
in this volume [137].
A number of other mechanisms exist that can relieve the
limitation of proton availability. These have been widely
discussed [126,128,138] and will only be briefly summarized
here. They include non-specific effects like transport by soluble
buffers and the related phenomenon of water hydrolysis
[17,124,125,139]. Soluble buffers represent sources of labile
free protons (< M), and their diffusion rates are usually within
an order of magnitude of the proton. A limiting feature may be
that the most prevalent physiological buffers, including
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
local concentration at and accessibility to negative surfaces,
like natural membranes.
The action of water as a proton donor (hydrolysis) is a
special case of this buffering activity. It was first brought to
prominence in biological systems by Kasianowicz et al.
[139] who were forced to invoke it to account for the high
rates of proton transport effected by S-13, a very potent
uncoupling agent. Although water is an extremely poor
donor, with a pKa of 15.74 on a molar scale, it is of course
present at very high concentration (55 M) and is therefore
effective in the last resort. The effective rate constant
depends on the pKa of the acceptor, and is given approx-
imately by 3.1010 55 10pK 15.74 = 3 1010 + pK 14 s 1 where
3.1010 M 1 s 1 is the second order rate constant for reaction of
OH with the neutral acid:
H2 O A OH AH
Water was also suggested to be the dominant proton donor to
the secondary quinone, QB, in reaction centers in native
membranes [140], although this did not appear to be the case
for isolated RCs [141]. However, the new thinking concerning a
dielectric barrier at membrane surfaces may bring these two
experimental results in to concordance [137].
A more specific, structural solution is local proton reservoirs
or buffers. These are amino acid residues, notably histidine, that
are significantly protonated at physiological pH and can act as
immediate proton sources for donation to the protein [142]. A
role for histidines has often been correlated with sensitivity to
Zn2+ ions and other divalent transition metals, and has
implicated histidines at the input sites of pathways in several
proton transfer proteins, including bioenergetic proteins and
proton channels [128,143146]. The diagnostic value of
divalent metal ion sensitivity has been given substantial
credence by the recent identification and cloning of the long-
sought voltage-gated proton channel [147,148], which is
inhibited by Zn2+ [146]. Site specific mutagenesis identified
two histidines as the source of the Zn-sensitivity [148].
A related motif is the combination of histidines and acidic
residues, which is found in cytochrome c oxidase and in
bacterial photosynthetic reaction centers (see Sections 7.1, 7.4
and 7.5). This arrangement may simultaneously attract protons
from the bulk phase, through the negatively charged carbo-
xylates, and provide immediate proton injection from the
protonated histidines [149].
6.2. Proton selectivity
In discussing proton transfer in aquaporin and gramicidin,
there is some tendency to equate the issue of the Grotthuss
mechanism with whether structural reorientation is kinetically
determining, e.g., [87]. Warshel's view is that electrostatic and
desolvation energies exert an over-riding influence in con-
trolling the rate of proton transfer and, indeed, most processes.
It is important, however, not to let this obscure the crucial role
that the Grotthuss mechanism plays in both the mechanism and
the remarkable specificity of many proton transport processes.
895
The relative selectivity of the gramicidin channel for H+, for
example, lies in the lower diffusive mobility of alkali metal
cations compared to the Grotthuss-conducted protons. Even
greater selectivity is seen in the (LSLLLSL)3 peptide helical
bundle of Lear et al. [106], which exhibits a proton
conductance of 120 pS in 0.5 M HCl, but no detectable
(5 pS) conductance for alkali metal cations at much higher
concentrations 10 M in the case of Li+.
Quite remarkable selectivity for protons is manifested by
some transport devices, such as the Fo component of H+-
translocating F-type ATPases. In addition to the very high
proton conductance in the face of very low proton concentra-
tions, the conductance to any other ion is negligible. The
selectivity (H+/Na+ permeability ratio) of Fo is in excess of 107
[129]. This likely reflects chemically specific steps in the
transfer, involving protonation and deprotonation of at least one
carboxylic acid residue near the center of the membrane
[150,151]. Additional buffer groups are needed to account for
the remarkably small pH dependence of the proton conductance
[129]. It should be noted that the powerful selectivity of Fo is
not required for its activity, per se, as the closely related Na+-
translocating F-type ATPases, which have a very similar Fo
sequence and structure, function well with Na+ and a much
lower selectivity against H+ [150].
Such a high degree of selectivity may well require one or
more chemical steps of this sort, i.e., if a simple water filled
channel cannot be so discriminating. This is evidently true for
gramicidin A. In contrast, a modified gramicidin, with a
CH2OH group protruding into the lumen, is almost perfectly
H+-selective, while maintaining H+ conductance that is within
a factor of 3 of the unmodified channel (S. Cukierman,
personal communication). This latter result also suggests a role
for a serine sidechain in the H+ selectivity of the synthetic
(LSLLLSL)3 peptide channel [106], although experimentally
the selectivity of this peptide channel is only known as a lower
limit (> 40). The simulations of Wu and Voth [105] suggested
that the water column was continuous in this channel, but
these were carried out on a model structure since it is not
known experimentally. At the present time, therefore, these
and other molecular dynamics studies of hydrophobic channels
that imply the possibility of high proton conduction with
negligible water diffusion [102,103] should be viewed with
caution.
7. Proton coupled electron transfer and conformationally
coupled proton transfer
7.1. Cytochrome oxidasethe D-pathway
A water-filled channel, the D-pathway, is responsible for
delivery of at least 6 and possibly 7 of the 8 protons consumed or
transferred in the full turnover of cytochrome oxidase [152154]:
4e 8H in O2 2H2 O 4H out
The D-pathway is marked by an eponymous aspartic acid
(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
H5O2+) ready for the next reductive step of the binuclear center.
The kinetic basis of active pumping does provide motivation
for fast proton transfer on demand, in order to maximize the
yield of pumping and minimize side reactions or unproductive
back reactions (slip). In this context, an interesting suggestion
is that the water column of the D channel could collapse
transiently after PT to the terminal Glu, breaking contact at
this point and retarding back transfer or proton slip ([157],
and Martyn Sharpe (Michigan State University), personal
communication).
7.2. Transient vs. static PT paths
The D channel in cytochrome oxidase and gramicidin seem
to provide examples of well defined and highly connected PT
pathways, but functional connectivity can also be achieved
transiently with highly mobile water molecules. This could also
provide a mechanistic component of gating or catalysis. The
idea that a PT pathway of water molecules can be transiently
organized at a specific place and point in time is inherent to
proposed gating mechanisms of proton pumping in the central
cavity of cytochrome oxidase [162165], and in bacteriorho-
dopsin (see below). It is also of possible significance in acid
base catalysisfor example, the transient assembly or or-
ganization of a proton conducting water chain has been
proposed as a prerequisite for rate limiting PT in the active
site turnover of carbonic anhydrase [168171]. However, this is
controversial [172,173].
7.3. Bacteriorhodopsin
Transient modifications of water chains and networks are
essential to the H+ pumping mechanism of the bacteriorho-
dopsin photocycle (Fig. 4), and are well documented by an
exceptional collection of X-ray structures of intermediates
[174176], by FTIR [177], and by electrostatics calculations
[178,179] and molecular dynamics simulations [180182]. The
action is partitioned between two domainsthe extracellular
and cytoplasmic domains, separated by the photoactive retinal-
lysine Schiff base. Following light absorption, the all-trans
retinal isomerizes to a 13-cis, 15-anti configuration, which is
held in a highly twisted form by constraints of the protein.
Subsequent relaxation of the 13-cis form drives protein
conformational changes and coupled pKa shifts that lead to
proton release from the extracellular space and proton uptake
from the cytoplasmic side.
In the extracellular domain, a proton is transferred from the
protonated Schiff base of retinal-Lys216 to Asp85 within about
500 s. Coupled to this, Arg82 moves down and away from
Asp85, and a proton is transferred to the protein surface from
the proton release group, formed by Glu194, Glu204 and about
4 water molecules. This proton escapes to the bulk phase on a
much slower time scale, which roughly corresponds to
reprotonation of the Schiff base from the cytoplasmic side.
The reprotonation of the Schiff base occurs via proton transfer
from Asp96 over a distance of about 11 , and involves the
structuring of water molecules to act as a bridge between donor
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
release group, Arg82 recovers its original position, and the
resting state is restored.
7.3.1. Proton release from bacteriorhodopsin
The nature of the proton release group (PRG) was initially
problematical, as FTIR studies could not identify appropriate
changes in the protonation state of any relevant amino acid
side chain. This has now been resolved by the recognition of a
delocalized proton, in the form of H5O2+, associated with
Glu194 and Glu204, both of which are ionized, Arg82, and at
least two additional water molecules [178,183]. Delocalized
protons, of which H5O2+ is the simplest, are characterized by
broad IR absorption bands [43], and time resolved changes in
the 18001900 cm 1 region have been identified and shown to
fit the expected behavior of the PRG [45,183]. With bulk
phase pKa values of 4.4 for glutamic acid and approximately
1.7 for H5O2+ , this would appear to be an unusual
arrangement, perhaps requiring a delicate balance of electro-
static forces. Indeed, it is abolished when either Glu194 or
Glu204 are replaced by glutamine, but it is partly retained
when aspartatic acid substitutes for Glu204 and in many other
mutants in the general vicinity [45]. It is, therefore, at least
minimally robust to small perturbations of local structure and
electric potential.
In fact, the dominant electrostatic contributions are likely to
be quite local and the stabilization of the ionized species by
mutual interaction could be a general phenomenon. Spassov et
al. [178] modeled the electrostatic interactions in this region and
found that, in the absence of a protonatable model for water,
Glu194 and Glu204 interacted so strongly that one was always
ionized (pKa < 0) and the other was always protonated
(pKa > 15). When H5O2+ was permitted in the calculations, it
became the preferred protonation site, with both Glu194 and
Glu204 ionized. The net interaction energy among these three
groups (Glu194, Glu204 and H5O2+) is sufficiently favorable
to oppose the intrinsic propensity for H5O2+ to transfer its proton
to Glu.
This balance is upset when the retinal isomerizes and
disrupts the interaction between the protonated Schiff base and
ionized Asp85, which is mediated by a water molecule.11 The
Schiff base proton is transferred to Asp85, causing Arg82 to
move away and down towards the PRG, and this drives the
proton from the PRG to the surface, where it is delayed by
surface forces for up to a millisecond [185188]. Molecular
dynamics simulations suggest that as the Arg82 moves down,
the water cluster near the Schiff base can merge with the water
cluster of the PRG [181,182]. It was suggested that this could
allow rapid, Grotthuss-type, proton transfer, but for what reason
is not clear.
The movement of Arg82 and the coupled pKa change for
Asp85, effectively inhibit back transfer of a proton from the
extracellular side to the Schiff base, at least until the 13-cis
11
Interestingly, the correct placement of this water (wat402) in computational
analyses is one of the few examples where quantum mechanical refinements, or
an unusual parametrization of the polarizability, are required [184].
configuration has relaxed and the nitrogen lone pair is
comfortably facing the cytoplasmic domain. When the Schiff
base is reprotonated from the cytoplasmic side and begins to
reisomerize back to the all-trans form, Asp85 protonates the
PRG and Arg 82 returns to its up position.
7.3.2. Proton uptake by bacteriorhodopsin
At the opposite extreme from the well defined and connected
water chains of gramicidin, the D channel of cytochrome
oxidase, and the networks of the proton release complex of
bacteriorhodopsin, is the proton uptake pathway at the
cytoplasmic side of bacteriorhodopsin. Here the proton
conducting structure linking the cytoplasm to the Schiff base
is assembled from scratch by structural changes that draw in
water from the aqueous phase. This allows transfer of a proton
from Asp96, which is protonated in the ground state; it is
subsequently reprotonated from the cytoplasmic medium.
These events are initiated by relaxation of the 13-cis
retinylidene, which forces movement of helices F and G that
opens a channel and draws water in from the bulk phase. A PT
pathway is established between Asp96 and the deprotonated
Schiff base and, at the same time, structural changes around
Asp96, including water movements, lower the pKa of Asp96
thereby driving the proton transfer to the Schiff base. The water
chain from Asp96 to the protein surface, which allows
reprotonation of the acid, is completed as the inner water
chain to the Schiff base is collapsing.
This sequence of events in the cytoplasmic domain rapidly
builds and destroys two short water chains, employing one or
two waters that are already sequestered within the protein in the
ground state, but importing at least 2 or 3 others. This is an
expensive business, as the creation of water-sized cavities costs
on the order of 5 kcal/mol [189]. Bacteriorhodopsin can afford to
do this as it has a great deal of excess free energy derived from
the photochemical isomerization of the retinal (12 kcal/mol),
but it is unlikely to be a generally applicable design strategy. In a
recent description of events on the extracellular side, Gerwert
[190] has proposed that the remodeling of the hydrogen-bonded
network in the PRG constitutes a significant fraction of the
energy stored in the protein, transferring it from the photo-
chemical product as the distorted 13-cis retinal relaxes.
Similarly, the cavity formation on the cytoplasmic side could
readily represent a similar magnitude of free energy storage, and
would reflect the transfer of the strains from the proton release to
the proton uptake theater of operations.
7.3.3. Is the presence of a Zundel ion (H5O2+) significant?
It is clear that water plays crucial dynamic and structural
roles in the proton transfers leading to proton release in
bacteriorhodopsin. Not only does it provide much of the
pathway for transfer but it is also the repository of the labile
proton, H5O2+, in the ground state. This is the first unequivocal
identification of a delocalized proton in a functional biological
role, but it may turn out to be a widespread phenomenon, e.g., in
RCs (see below), and recently proposed for the D channel of
cytochrome oxidase (Martyn Sharpe, personal communication).
The question arises whether there is any particular advantage to
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
it or is it just one of many possible structural solutions to a given
functional requirement?
Strong electrostatic interactions, leading to marked pKa
shifts, are widely encountered in proteins and underlie many of
their unique attributes. The operational pKa of an acidbase
group is determined by the summed interactions with its charge
environment and by its intrinsic pKa, where the usual suspects
for protein groups range from about 4.5 (for Asp and Glu) to
12.5 (for Arg). Water adds to this a significantly stronger acidic
pKa of 1.7, identified with H3O+. The Zundel cation is not
expected to have a significantly different intrinsic pKa, but it
does represent a more delocalized charge, which will help in
sequestering it in a lower dielectric environment.
An assortment of amino acid side chains could very readily
provide the same charge balance as the native PRG (net 0 or
1), from among the protonated Schiff base (SBH+), Asp85,
Arg82+, Glu194, Glu204, and possibly Asp212. In fact,
several mutants do just this, but they tend to be functionally
impaired, like E204Q and E194Q, and the continuum band is
lost. The inclusion of protonated water, with low intrinsic
pKa, in the native PRG may allow more rapid proton release
when the charge balance is upset. When H5O2+ snaps back to
H3O+, the low pKa is manifested with a proton dissociation
rate of > 1011 s 1, and a correspondingly fast pairwise PT
rate to the nearest acceptor. This may be important in
directing the proton transfer to the out-going path, before
structural changes allow back transfer to the Schiff base. This
could be an effective device in any situation where rapid,
intermittent proton delivery must compete with other pro-
cesses, i.e., pumps.
7.4. Bacterial photosynthetic reaction centers
At the present time, there appears to be no evidence for
specialized, ultrafast PT in proton pumping bioenergetic
systems, beyond the incoherent hopping of Grotthuss-type
mechanisms. Some might consider this to be specialized, but it
is a natural manifestation of acidbase features common to
many chemical functionalities. If this is so, then non-vectorial
(in the sense of non-transmembrane) PT is likely to arise with
the same natural designs as encountered in proton pumps, and
the two classes can be considered together in terms of
identifying underlying principles. Where pumping mechanisms
might have additional kinetic requirements is in the gating
mechanisms required to prevent slippage.
The bacterial photosynthetic reaction center (RC) provides
a good and experimentally amenable system for studying
long distance PT, although it is, for the most part, non-
vectorial. In reaction centers, the transfer of electrons in the
primary photochemical events generates most of the electrical
component, while electron-coupled proton uptake and release
accompanying the redox reactions of secondary acceptors
and donors is largely responsible for the proton concentration
gradient (pH). The reduction of quinone (Q) to quinol
(QH2) represents the initial step of a chemiosmotic proton
pumping redox loop [191,192]. However, this process is
only minimally electrogenic, and the charge moved into the
899
membrane dielectric represents only about 15% of the
average transmembrane charge transfer per electron turnover
[140,193195].
The reactions involving PT are summarized in the acceptor
quinone cycle shown in Fig. 5 (left). Reducing equivalents are
stored in the double reduction of the secondary quinone, QB
(ubiquinone in Rhodobacter (Rb.) sphaeroides), via the primary
quinone, QA, and quinol is released into the membrane after two
light-activated turnovers of the RC [196,197]. The oxidized
primary donor, P+, is reduced by a secondary donor (cytochrome
c2 in vivo) after each photoactivation, and only the quinone
events are shown in the cycle [149,198].
Sub-stoichiometric (H+/e < 1) proton uptake occurs on the
first flash, due to the electrostatic influence of the semiquinone
anions, QA and QB , on the pKas of nearby ionizable amino
acid residues [149,199202]. Super-stoichiometric proton
uptake occurs on the second flash, as QB is reduced to quinol
and released into the membrane. The average stoichiometry is
H+/e = 1, as stipulated by the overall chemical reaction.
The RC quinones are well buried in the protein, and proton
transfer to QB must extend over a distance of about 15 . Site
directed mutagenesis studies have implicated several members
of a large cluster of ionizable residues (predominantly acidic)
as being involved in the proton delivery pathway (reviewed in
[149,198,203]). However, it is barely possible, from simple
kinetic studies, to make the distinction between actual proton
carriers and those having influence, for example, by electro-
static interaction. Direct spectroscopic evidence of a proton
carrying role has been presented only for Asp-L210 and Glu-
L212, and possibly a surface histidine, on the basis of static
and time resolved FTIR studies of the first turnover [204
209]. However, compelling evidence comes from radical
changes in the mechanism of the second electron transfer upon
mutation of AspL213, SerL223, HisH126 and HisH128,
and AspM17 (together with AspL210) [210220].
The pathway is outlined in Fig. 5 (right). The path from the
entry point near HisH126 and HisH128 at least up to Asp
L210 is shared by both the first and second H+ taken up from
the aqueous medium. The H+ taken up on the first flash settles
predominantly on GluL212, at neutral pH, although it is
probably distributed over other residues, too. The H+ taken up
on the second flash is delivered directly to the C1O group of
QB , via AspL213 and SerL223. This is followed by
electron transfer from QA to form QBH. The quinol is fully
protonated by transfer from GluL212, possibly as it unbinds
[221224].
In wild type RCs, the second reduction of QB to QBH is rate
limited by electron transfer, i.e., proton transfer from the
aqueous phase is very fast [225]:
fast PT slow ET
Q 
A QB X Q
A QB H Y QA QB H

The net rate of ET is given by kobs = kET[QBH] [226]. The ET
rate constant, kET, has been estimated at 106 s 1, which places
the PT equilibration rate at > 107 s 1 [149,227]. The rate of
equilibration is the sum of forward and back transfer rates,
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,
a deprotonation rate to water of about kH10pK. Using the bulk
solution value of kH 4.1010 M 1 s 1, the computed off-rate
would be inadequate: kH = 4.1010 10 4.5 106 s 1. However,
measurements of the rate of H+ uptake to RCs in the absence of
ET, i.e., uptake to the QA state, show a substantially larger
second order rate constant [141], which increases with pH
above the isoelectric point of the protein (pI 6.1). This
probably reflects the influence of surface potential on the local,
surface pH. In spite of the different binding site locations of QA
and QB, proton binding in response to uptake by the QA state of
the protein involves most of the same residues [202,228] and
occurs via the same pathway as for the QB state [218].
Related to this, the pKa of QB is pH dependent because it is
influenced by the local potential in the QB binding pocket, and
this is determined by the ionization state of amino acid residues
around it [149,202,228]. The pKa 4.5 was estimated for pH 7.5
[227]. At lower pH, the pKa decreases, indicating a faster off rate,
and the on rate, kH[H+], also increases. At higher pH, the pKa
increases, but the apparent rate constant, kH, also increases, so
the deprotonation rate constant changes little. Thus, the rate of
proton equilibration to QB /QBH remains fast compared to the ET
rate over most or all of the accessible pH range (pH 3.511.5).
Several amino acid residues have been strongly implicated
as proton carriers because mutation to non-ionizable forms
causes a dramatic change in the mechanism of the reduction of
QB to QBH, from ET-limited to PT-limited. These include
AspL213 Asn (mutant L213DN), SerL223 Ala
(L223SA), the double mutant AspL210 Asn + Asp
M17 Asn (L210DN/M17DN), and the double mutant His
H126 Ala + HisH128 Ala (2xHis) (reviewed in
[149,198,203,229]). In the case of mutant L213DN, the second
electron transfer rate was inhibited by at least 104 fold at pH > 7
[211]. However, because PT is now rate limiting [220], the
proton transfer rate was much more strongly inhibited
estimated as inhibited by 107-fold in the mutant. Interes-
tingly, this mutant could be partially reactivated (rescued) by
small weak acid anions, such as azide (N3) and nitrite [212].
In RCs with the mutation GluH173 Gln (mutant
H173EQ), the 2nd electron transfer was also strongly inhibited,
by more than 102 fold, and could be fully rescued by azide
[230]. However, in this case, the reaction remained ET-limited
[220] and the effect on the PT rate is indeterminate, although it
could be substantially impaired. The effect of this, and other
impaired mutations in which ET is still rate limiting, is
consistent with an alteration in the pKa of QB a decrease in
pKa will decrease the equilibrium population of QBH, and
therefore decrease the net rate of ET. In such circumstances, full
restoration of the 2nd electron transfer rate by azide must
include recovery of the native pKa value for QB . This could be
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
achieved by binding of the azide anion, N3, restoring the
negative electrostatic potential lost upon mutation of ionized
GluH173 to Gln [230]. Activity of the protonated species,
N3H, as a proton carrier could also supplement the proton
delivery function of the protein [212].
Several primary mutations of acidic residues are partially
recovered by second site revertants, some at considerable
distance, which can be understood as restoring negativity to the
QB domaineither by adding a new acidic residue or removing
a wild type basic residue [231235]. This is an attractive
general explanation but the molecular mechanism in some
cases, at least, involves significant structural changes, including
relocation or introduction of water molecules [236238].
7.4.1. Proton entry to bacterial reaction centers
The proton entrance site was first indicated by the striking
inhibition of the 2nd electron transfer by divalent transition
metal cations, especially Zn2+ and Cd2+ [145,213,239241],
and a lesser effect on the 1st electron transfer [145,242].
Both effects follow from the inhibition of H+ uptake, and
identify the proton entry site as the same for both protons
[213,219].
The metal binding site was shown by X-ray crystallography
[243] and by EPR [244,245] to be a surface patch, comprising
histidines H126 and H128, and AspM17, with AspL210 just
below the surface. Mutation of the two histidines to alanine
(mutant 2xHis) inhibited both the 1st and the 2nd electron
transfer at pH > 8, and the 2nd electron transfer switched from
being ET to PT limited [219].
This strongly supports the notion that the histidine residues
function as a surface buffer or reservoir of protons for local
injection into the proton pathway [128,142]. This design is
adequate to keep proton delivery non-rate limiting in the wild
type at pH values well above the functional pKas of the
histidines (pKa = 6.36.8 [241]), while the 2xHis mutant is
proton limited [219,246], because even a small degree of
protonation of the histidines will provide much greater, effective
proton availability than the nanomolar concentrations of free H+
at alkaline pH.
Both 1st and 2nd electron transfer reactions were restored in
the 2xHis by addition of imidazole [219], and Paddock et al.
[246] found that the 1st electron transfer in 2xHis mutant RCs
could be rescued by a variety of cationic buffers (BH+) in a
manner that was proportional to the buffer pKa over a wide
range of values (Brnsted plot). Analysis in terms of rate
limiting proton transfer from the protein surface revealed the
involvement of a low pKa intermediate (pKa 4) in the transfer
pathway, on the way to the final destination, GluL212, which
has a higher pKa. This would be consistent with the presence of
several carboxylates in the QB domain.
7.4.2. Proton carriers in bacterial reaction centers
In the double mutant, AspL210 Asn + AspM17 Asn
(L210DN/M17DN), the 2nd electron transfer is severely PT
limited over a wide pH range, but can also be rescued by added
buffers [247]. Thus, one or both of these aspartic acids is a
functional proton carrier, and not just an electrostatic contributor
901
to setting the pKas of other proton transfer groups. Function can
be restored to mutant RCs by small buffers but, unlike the 2xHis
mutant, proton transfer is only substantially restored by weak,
neutral acids, (AH) [247]. Rescue activity seems to be mainly
limited by steric and electrostatic factors restricting access
through a narrow channel. The acid forms were active at low
concentrations, but, because of their generally low pKa values,
the total concentration of salt required near neutral pH was high.
A Brnsted plot of activity vs. pKa of the rescuing acids was
linear, with a slope of 1, and extrapolated to diffusion-limited
behavior at pKaapp 1. However, the maximum rate achieved
under saturating concentrations of acid (salt) did not correlate
with pKa. This behavior suggested a simple model in which the
acid and anion species compete for binding, both with weak
affinity. The model predicts that the intercept at the diffusion
limit corresponds to a true pKa = 45, again consistent with a
carboxylic acid or even QB , itself.
The binding affinities for both acid and anion forms was very
weak (1 M) for all active species, and the efficacy and
properties of the rescue relied on very rapid on and off rates
the on-rates were near the diffusion limit. However, the
stringent size limitation indicates a need to access a small
internal volume, which we consider to be the proton channel to
the QB domain. Such rapid, diffusive access to a buried site may
seem a tall order, but there are several clear precedents.
Dramatic examples include acetylcholinesterase [248,249] and
catalase [250,251], both of which support diffusion limited
entry to active sites at the end of long (2030 ), narrow
channels. Perhaps this should not be too much of a surprise after
our consideration of transmembrane ion channels, except that
the RC pathway to QB was not designed to accept small weak
acids and, as discussed below, may well be designed to exclude
small, potentially redox-active species.
7.4.3. The proton path in bacterial reaction centers
The entry site and path for proton delivery to QB in wild type,
and the probable access route for weak acids in the L210DN/
M17DN mutant RCs is shown in Fig. 6. Water molecules are
largely absent from this view at the present resolution of RC X-
ray structures (< 2 ), and modeling adds none with clear
consensus among different structures. Water molecules are
present elsewhere, in the QB domain, however, and void
volumes are available in the X-ray structures to accommodate
others in the putative pathway. It seems likely therefore that
water molecules are present but highly mobile.
A functional role for water in the protonation reactions of the
quinone states is supported by infrared spectroscopy, through
the characteristic IR spectra (Zundel bands) of polarizable
protons [43]. Breton and Nabedryk observed a broad and
featureless component in the QA /QA FTIR difference spectrum
of Rps. viridis and Rb, sphaeroides, which they assigned to a
Zundel band [206]. This is an indicator of water in a hydrogen
bond network containing an excess charge.
Remy and Gerwert have made time resolved FTIR
measurements of the 1st electron transfer, by the step-scan
method, and see direct evidence for transient proton transfer
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,
and deprotonation in 150 s.
7.4.4. Proton delivery in bacterial reaction centers
Mutation of Asp-L213 to a non-ionizable residue, e.g.,
mutant L213DN, causes a dramatic, 104-fold inhibition of the
2nd electron transfer and a switch to PT limitation [220,252].
The extent of PT inhibition is on the order of 107 fold. This
mutant is partially rescued by some of the same small weak
acids as are active in L210DN/M17DN [212], but the
recovery is very weak and shows no sign of saturating at
the highest concentrations available (E. Takahashi and C. A
Wraight, unpublished observations). Examination of the X-ray
structures indicates that there is little volume available on the
other side of Asp-L213, where it transfers a proton to Ser-
L223 and thence to QB , and no water molecules have shown
up there. This would clearly inhibit the action of a weak acid,
which would have to surmount the steric hindrance of the
mutation site (Asn-L213, for example) to access Ser-L223, at
least momentarily.
Interestingly, the 2nd electron transfer in AspL213 Glu
mutant RCs (L213DE) is also substantially inhibited (approxi-
mately 102-fold) but it is not PT-limited [252]. A likely
explanation is that changes in the network of electrostatic
interactions, due to the longer side chain or slightly higher
intrinsic pKa of glutamic acid, lead to GluL213 being neutral,
whereas AspL213 is ionized. The local change in electrostatic
potential (less negative) would downshift the pKa of QB , as in
many other mutations of acidic sites. The L213DE mutant is
similar to the GluH173 Gln mutation (H173EQ) in the extent
of inhibition, while maintaining PT faster than ET [220,230].
Finally, SerL223 is implicated both as a hydrogen bond
donor to the semiquinone and as the immediate proton donor,
preceding the 2nd electron transfer [209,228,253,254]. Muta-
tion of SerL223 to threonine or aspartic acid retains activity,
but asparagine and alanine do not [215,216]. Remarkably,
replacement with glycine does yield activity, suggesting that
the absence of a side chain in Gly allows room for a water
molecule, which functions in the same way as the serine
hydroxyl [254].
Even without taking into account local electrostatics and
pKa perturbations, the relevant donor species to QB-(apparent
pKa 4.5) must almost certainly be SerOH2+ (solution
pKa 2), rather than SerOH (solution pKa 16), because
the pKas in the latter case are much more unfavorable.
SerOH2+ could be formed at very low occupancy by proton
transfer from AspL213COOH (pKa 4), which may also
be at low occupancy depending on its functional pKa.
(Functional pKa values in the QB domain acid cluster are
very hard to determine and to specify because of the large
number of ionizable groups and the strength of interactions
[202,228,255257]). However, the transfer from SerOH2+ to
QB will be very favorable.
After the 2nd electron transfer and protonation of the C1O
group, the C4O group is protonated and the quinol unbinds
and leaves the QB site. The proton source is GluL212, which
appears to have complex titration behavior, and is partially
ionized over a wide pH range [202,204,208,228,258261]. It
becomes fully protonated after the first flash and functions with
a high effective pKa 9. It is unlikely that GluL212 donates
directly to the quinol, but perhaps via one or more water
molecules that could move into position as the quinol anion
attempts to detach from a hydrogen bond with HisL190. In
mutants where the second proton transfer is inhibited, quinol
unbinding is greatly slowed down and follows the slow proton
uptake kinetics [221].
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
7.5. Selectivity revisited
The ability for small weak acids to penetrate the proton
transfer pathway of reaction centers, apparently with ease,
raises the question of selectivity again. For the reaction center,
this may not be a critical issue, since the proton path dead-
ends at QB and is not part of a transmembrane, pumping
activity. Nevertheless, there is no indication that the wild-type
structure permits entry of small ions, i.e., proton delivery is
not inhibited by high concentrations of salts, and is only
affected at all by virtue of well-understood ionic strength
effects [141,247].
It is possible that the path is sufficiently flexible that it could
accommodate some occupancy by other ions without impairing
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
mutant could also restore low levels of activity in L213DN
mutant RCs, but were more than two orders of magnitude less
effective. This suggests that circumventing AsnL213 is
extremely difficultor, possibly, that the native aspartates at
positions L210 and M17 act with the surface histidines as an
effective proton-specific filter.
For proton pumps, the issue of selectivity is potentially more
serious. The D channel of cytochrome oxidase, for example,
might accommodate an alkali metal cation, but this would
almost certainly have a highly deleterious effect on proton
translocation. No such inhibition has been observed. Inhibition
by Zn2+ ions does occur, but this is associated with binding to
surface histidine residues, impairing their function as local
proton sources [144,262,263]. It is also worth noting that
movement of a cation in the D channel would require flux of
water molecules, as in gramicidin. But this would be in the
opposite direction from any likely water movement and would
deposit unwanted waters in the catalytic center. Thus, it seems
that the entry point of the D channel is well served by the
structure, including the key aspartic acid and surface histidines,
to be proton specific. Also, the water continuity is broken at the
ring of asparagines just inside the channel, which would
presumably necessitate the complete desolvation of the cation in
an energetically prohibitive step.
For bacteriorhodopsin, proton specificity is not due to the
Schiff base alone, as indicated by the remarkable similarity
between bacteriorhodopsin and halorhodopsin [264]. However,
the local electrostatics and proton acceptor activity provided by
Asp85 (which is threonine in halorhodopsin) ensure that
bacteriorhodopsin acts as a proton pump. The covalent pro-
tonation of the Schiff base ensures that no other cation is
translocated. Whether other ions freely enter the extracellular
cavity seems very unlikely, given the balance of electrostatic
interactions necessary to stabilize the protonated water ion in the
proton release group. Furthermore, none are evident in the high-
resolution X-ray structures, so they would have to be highly
mobile. The excluding factor is presumably electrostatics
including the ubiquitous desolvation costs.
The limits on selectivity for these systems have not be tested
in the same systematic way as for the H+-selective Fo, but
903
common salts in the 100 mM range do not detectably alter the
proton transferring activity of any of these proteins, with H+ at
submicromolar levels. It would seem, therefore, that achieving
considerable selectivity is not a difficult engineering challenge,
and that ionizable amino acids can perform this function well.
Although the resolution of the available X-ray crystal
structures for most of these examples is not definitive, it
seems likely that the ionizable sidechains interrupt any water
column present, and impart chemical specificity by necessita-
ting protonationdeprotonation of the acidic group. For such
a mechanism, rapid proton throughput requires fast deproto-
nation rates. This implies low pKa functional groups, and the
common occurrence of carboxylic acids in these roles is
entirely consistent with this.
8. Why are there long proton paths for non-vectorial
reactions?
For transmembrane-vectorial proton transfer, as in proton
pumping systems, the ultimate goal is to move protons across
the significant dimensions of the membrane and this may
motivate evolutionary design to maximize the distances covered
by any one process. However, it is not obvious why essentially
non-vectorial proton uptake and release reactions should be
long range.
In the L210DN/M17DN double mutant, we observed that
H3O+ was not as active in rescuing function as its pKa would
imply, and that NH4+ was also weaker than predicted from a
linear Brnsted plot [247]. This suggests that cationic donors
are less effective than neutral acids in this interior environment,
contrary to their action at the surface as seen in the rescue of
the 2xHis mutant. Of course, the mutations themselves
introduce some change in net and surface charge, and
L210DN/M17DN mutant RCs might be expected to have
diminished affinity for cationic species compared to wild type
RCs, and the 2xHis mutant RCs relatively more. Nevertheless,
the native QB-site engineering appears to be primarily directed
at stabilizing the anionic semiquinone, achieved by specific
hydrogen bonding interactions and by a positive electrostatic
potential [149,228,257]. As pointed out by Rongey et al. [232],
a positive potential may favor electron transfer reactions at QB
but it is incompatible with the equally important function of
delivering H+ ions to QB for full reduction. Thus, to the extent
that the QB site is net positive, simple accessibility to H3O+ is
not a good solution for proton delivery, in spite of its strong
proton donor potential. This, of course, is in addition to its very
low concentration at physiological pH. Consequently, a
designed proton conduction path, mostly via neutral acid
groups, is necessary for kinetic competence.
However, since the proton carrying activity of the aspartic
acids L210 and M17 can be restored by soluble neutral buffers,
albeit small ones, and the proton injector function of the surface
histidines is fully rescued by soluble cationic buffers, perhaps
we should askwhy does the QB site need a specially
constructed proton delivery path? Why is it not just accessible
to the numerous physiological buffers?
904 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
8.1. Long-lived states
The proton pathway of the QB domain clearly has some
attributes in common with those of proton pumps like
bacteriorhodopsin and cytochrome oxidase, but the RC does
not pump. Furthermore, the thermodynamic properties of the
one-electron redox couples of QB appear to derive from quite
simple principles that need not require elaborately obscured
binding sites [149,257]. However, QB acts to accumulate two
electrons from sequential photochemical events, and it must
be able to store one electron in a metastable formQB is
stable for many minutes. This makes it susceptible to non-
specific reactions, which would partially short circuit the
energy storing reactions of light driven electron transfer and
proton uptake. We have previously shown the propensity for
QA to be reoxidized even by the highly charged ion,
ferricyanide [265]. However, electron exchanges between
one-electron agents may be more readily reversible than
charge accumulating reactions. The long lifetime of QB
requires it to be sufficiently buried to protect it from even the
most pedestrian scavenging reactions with cytoplasmic
electron acceptors and donors, some of which are quite
small and might be able to approach QB if it were not well
sequestered.
Similar considerations may apply to other examples where
one-electron and two-(or more) electron redox chemistries
meet. For example, Complex II (succinate dehydrogenase),
which is also a non-proton-pumping bioenergetic membrane
complex, has a long and highly connected chain of water
molecules that links the aqueous-membrane interface to the
deeply buried quinone site [266]. Like the D-channel in
cytochrome oxidase, it is terminated at each end by ionizable
side chains. The Qo site of the cytochrome bc1 complex has a
particularly tightly coordinated two-electron chemistry to
perform, and just the assembly of reactive components
cytochrome bL, quinol, and the Rieske ironsulfur protein
creates a fully encased quinone site that has no direct access to
solvent. It, too, has a chain of water molecules that link the
active site to the periplasmic or intermembrane space, which is
considered a likely conduit for proton release [267]. Like
reaction center proton uptake, the bc1 proton release is
essentially a non-vectorial reaction and is generally considered
non-electrogenic (but see [268]).
Hydrogenases are also noteworthy for their deeply buried
active sites. Hydrogen gas is able to move through the protein
by hopping between small, transiently formed, non-polar
cavities[269]. Protons, however, are thought to access the
active site via water-filled channels [270,271].
It may therefore be generalizable that redox cofactors with
metastable reduction states will be deeply buried, and, if they
are also protonation sites, they will require proton transfer
paths.
8.2. How buried is that?
This is quite readily answered with the principles that govern
non-adiabatic electron transfer in natural systems. For this, the
simplest forms of electron transfer theory are completely
adequate [272]:
k k0 expbRexpDG*=kB T 7
As Dutton and coworkers have effectively shown, for
purposes of understanding evolution and natural design of
intramolecular electron transfer proteins, the electronic cou-
pling parameter, , is essentially constant with a value of
1.4 1 [1,2,273]. Thus, ET rates are determined by the
distance, R, and the free energy of reaction, which modifies the
activation free energy G* according to the usual Marcus
formula [274]:
 
k DG- 2
DG* 1 8
4 k
The reorganization energy, , is a measure of how much
the molecular framework and surroundings change when the
electron is transferred. Since electronic and electrostatic
energies are large, is usually large, too. If both reactants
are in protein and therefore somewhat shielded from the
solvent, is on the order of 1 eV, or slightly less. On the
other hand, free energies of biological reactions are
commonly small. With these guidelines, it is apparent that
physiologically meaningful reactions, generally faster than
1 ms, are limited to distances less than 15 (edge-to-edge)
[1]. This is a sufficiently strong statement that reactions
purported to occur over distances greater than this should be
examined for possible alternative mechanisms, and even
errors.
For intermolecular electron transfer, binding of reaction
partners must be taken into account. Otherwise the same
principles apply, although has not been so well sampled and
could be somewhat larger. For a physiological reaction,
molecular recognition and binding produce a productive
association in which the electron transfer distance is minimized
and binding is of sufficient duration to allow transfer. However,
the affinity should be no stronger than necessary, since
disassociation is an equally important part of turnover.
Functional proteinprotein dissociation constants are com-
monly on the order of Kd 1 M. For a diffusion limited
reaction between medium sized, soluble proteins (50 kDa, 50
diameter), the bimolecular rate constant is about 5 108 M 1
s 1 even in the absence of any attractive forces. Thus, the
dissociation rate constant, koff, is about 5 102 s 1 and the
lifetime of a complex is about 1 ms. This is long enough for a
reaction with G 0 to occur over a distance of about 15 .
Shorter complex lifetimes could service reactions at shorter
distances (determined by the location of the redox cofactors in
each protein).
Conversely, an undesirable reaction might involve a small
redox-active molecule binding non-specifically to the protein
with a low affinity. If non-specific binding typically has
Kd 10100 mM, this corresponds to koff 107 s 1, or a
lifetime of about 0.1 s. To limit the chance of the redox
reaction occurring in the lifetime of the complex, also with
 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
G 0, the distance to the protein cofactor should be
10 .12 Clearly a compromise has to be reached for a
protein that is involved in physiological bimolecular asso-
ciations because the cofactors cannot be buried that deep in
both partners without violating the productive reaction distance
limit of 15 . In fact, exchange with one-electron centers may
not be especially at risk in this way, as the reactions are more
freely reversible. However, for charge accumulating reactions,
in which one-electron transfers communicate with n-electron
centers, e.g., quinone (n = 2), with metastable intermediate
states, such parasitic reactions are potentially highly dele-
terious. The intermediate states can also be very long-lived,
making them vulnerable to even very rare events and very
feeble reagents.
For a redox center not involved in intermolecular ET, there
is no such constraint, except modesty of cost. Thus, to find
the QB site sequestered 16 inside the protein is not
unreasonable. The semiquinone lifetime is tens of minutes in the
dark, although the relevant lifetime should be taken from low
light conditionsthe lowest at which the organism can grow
phototrophically.
The result of such a defensive design for electron transfer is
that physiological proton transfer must be facilitated over
similar distances and in such a way that it does not open up the
redox center to small redox active species, i.e., a proton specific
pathway is required.
9. Implications for intraprotein proton transfer pathway
design
Unlike electron transfer, the necessary pathways for proton
transfer are highly dependent on structural details, and yet
robustness remains an important and inevitable criterion for
natural, evolutionarily selected design.
It is evident that water is the major constituent in many of
the long distance PT paths actually encountered in proteins. In
contrast, Onsager's original concept of hydrogen bonded
chains in protein function emphasized a role for amino acid
side chains [39,40]. In their further development of this idea,
Nagle and Morowitz certainly recognized that water was a
candidate [41,49], but they initially focussed on protein
functional groups. As the idea matured, however, Nagle
recognized that water was likely to be more than a candidate
and was an important, major component of proton conducting
hydrogen bonded chains [275]. His motivation was the relative
dearth of hydrogen-bonding residues in the membrane-
spanning regions of bacteriorhodopsin that were being revealed
by electron diffraction studies at that time [276]. This was
confirmed by high resolution X-ray crystallography, and has
subsequently been shown for some other proteins, too, notably
cytochrome oxidase. Nagle speculated briefly, but presciently,
on the possible properties of water-wires as proton conducting
pathways [275].
12
For a small molecule bound at the surface and therefore essentially in
water is likely to be larger than 1 eV, which will slow down the rate of
reaction and shorten its range.
905
9.1. Why water?
An obvious difference between water and, say, serine is the
mobility that water has in situ. A serine hydroxyl is restrained to
rotate about the CC bond, which means that the geometric
constraints for productive proton transfer are much greater. If
the OH is also hydrogen bonded, then the group is essentially
fixed in space. In contrast, Hummer et al. noted that, despite the
quasi-one-dimensional order of the single-file waters in
nanotubes, the water chain retained considerable entropy due
to their free rotation about their aligned hydrogen bonds,
resulting in a degenerate energetic ground state [92]. Similarly,
Tian and Cross noted that cations inside gramicidin maintained
a relatively large positional entropy in the channel sites, and
suggested that this was important for their high transport
mobility [80].
The local mobility of water provides a much greater
dielectric response than would otherwise be seen, and this
will contribute significantly to the energetics of ion/proton
entry. For protons, the potential for charge delocalization could
also be a significant factor in the energetics. These considera-
tions also underlie the computational truth that unless water is
handled with full dynamics, rather than discrete sampling of
conformations, it is usually better to throw them out and use
= 80 in the voids.
A unique advantage of water as a proton carrier component is
that it is resistant to mutationcavities capable of accommodat-
ing water are more robust to mutations than specific amino acids,
i.e., mutational substitutions of nearby residues, including those
that define the cavity, will often have similar molecular volumes
and will not change the volume of the cavity or may only
displace it slightly. There are some suggestions, from computa-
tional studies, that a water-filled channel could be highly proton
specific, but this would require fine engineering to hold the water
rather rigidly in a limited range of coordinations, and it would
then be susceptible to mutational breakage. A simpler solution is
to utilize as much mobile water as possible and add selectivity
only where needed, in the form of ionizable residues, ideally at
the entrance. This is the basic design of the D channel of
cytochrome oxidase. Far greater sophistication is present
elsewhere, at the top of the channel and in the central cavity,
to handle the gating requirements of the pump.
In reaction centers, the requirements are less stringent, and
the path functions very effectively with a mixed bag of acids and
waters. It is clear from this example that specific pKa values are
not very important. Provided the main players are intact, the
local electrostatics, and hence the functional pKas, are not critical
[229]the PT rate stays fast compared to ET. The pKa of QB, of
course, is an exception, because that alters the population of the
active form (QBH) in the rate determining reaction.
10. Conclusions
Unlike electron transfer, proton transfer has a great deal of
structural requirement, but still less than might be expected. Fast
proton transfer depends on close proximity, to the point of
hydrogen bond formation. The constraints that this could
906 C.A. Wraight / Biochimica et Biophysica Acta 1757 (2006) 886912
impose are considerably eased by exploiting mobile water as a
major constituent. Given sufficient connectivity in this way, the
susceptibility to mutational failure can be restricted to a few
residues that provide the necessary chemical specificity of the
path, and these can even be provided redundantly. There are few
restrictions on pKa values, even for the chemical gateways, so
long as they are ionizable, i.e., the path is either fast or it is
non-functional. Although some key residues may be irre-
placeable, peripheral mutations do not substantially alter the
rapid functionality of the path. This is a practical definition of
evolutionary robustness.
Acknowledgments
I would like to thank Sam Cukierman for suffering
interminable questions about gramicidin, with good humor,
and O. S. Andersen, B. Corry, W. Junge, R. Poms, E.
Tajkhorshid and A. Warshel for answering some others. I am
also indebted to three anonymous reviewers for many insightful
comments. The mental effort was supported by NIH (RO1
GM53508) and NSF (MCB 03-44449).
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