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Volume 272, Number 50, Issue of December 12, 1997 pp. 31209-31212
From the Department of Physiology and Biophysics, University of California, Irvine, California 92697
INTRODUCTION
Structure and Photochemical Reaction Cycle
Partial Reactions of the Photocycle Observed without
Illumination
Mechanism of Transport
Role for Bound Water in Proton Transfers
Nature of the Active Site
Conclusions
FOOTNOTES
REFERENCES
INTRODUCTION The high resolution structures now available for
mitochondrial ATPase (1) and mammalian and bacterial cytochrome
c oxidases (2, 3) have raised hopes that the long sought
description of the proton translocation mechanism in transmembrane
pumps might be near. According to the simplest version of the
alternating access hypothesis (reviewed in Refs. 4-6), such transport
is based on the energy-dependent cycling through two
protein conformations in which the access of a single ion binding site
changes from one membrane side to the other. It has been difficult to
put this idea to a test because most proton pumps are large or
multisubunit proteins in which the site of ion translocation is at some
distance from the chemical reactions that drive it, and the chemical
reactions are themselves complex. If there is a general mechanism in
proton pumps, clues to it are more likely to come from a simpler
system. Recent progress with bacteriorhodopsin, a small retinal protein in which the thermal reisomerization of photoisomerized retinal drives
the proton transport, has yielded a step-by-step mechanism for the
translocation cycle. The principles and perhaps some of the details in
this mechanism, described briefly below, may prove to apply to other
ion pumps. They seem relevant also to signal receptors (7).
Structure and Photochemical Reaction Cycle Bacteriorhodopsin is a seven-helix transmembrane protein, with an
all-trans-retinal lying at a small angle to the membrane surface and linked via a protonated Schiff base to Lys216
near the middle of helix G (Fig. 1). Most
of the structure has been known at 3.5-Å resolution from
two-dimensional crystals (8), and more recently the entire structure
was described at 3.0 Å (9). Higher resolution from three-dimensional
crystals, at 2.5 Å is now available (10). The interhelical cavity is
divided by the Schiff base into extracellular and cytoplasmic
"half-channels" that together describe the trajectory of the
transported proton. The extracellular half-channel contains numerous
charged or hydrogen-bonding residues, whereas the cytoplasmic region is
simpler and mostly hydrophobic. In the first transport event after
absorption of a photon, the Schiff base proton is mobilized by
photoisomerization of the retinal to
13-cis,15-anti, and transferred to
Asp85 in the extracellular region, causing the release of a
proton to the surface. The Schiff base is then reprotonated from
Asp96 from the cytoplasmic side. Asp96 in turn
is reprotonated from the surface. These proton transfers together add
up to translocation across the membrane. They and other reactions of
the retinal and the protein during the cycle have been measured by
various spectroscopic methods and consist of the interconversions of
the intermediate states designated as J, K, L, M, N, and O, and
substates of several of these. Much effort has been expended to
describe these reactions and the protein residues involved (4, 11-15).
But to understand bacteriorhodopsin as a proton pump we must know also
what determines the rates of the proton transfers and how the
pKas and the geometry of the donors and acceptors
change so as to give them a cytoplasmic-to-extracellular direction.
[View Larger Version of this Image (85K GIF file)]
Partial Reactions of the Photocycle Observed without
Illumination Although the proton transport is the consequence of a
"photocycle," mechanistic clues have been gained recently from
reactions of the unphotolyzed protein that correspond to various single photocycle steps. First, the observation of a biphasic titration curve
for Asp85 suggested that its pKa is
linked to the protonation of another residue (16, 17) that turned out
to be Glu204 (18-20) perhaps together with liganded water
but certainly other residues near the extracellular surface, such as
Glu194. The nature of the linkage is that protonation of
Asp85 will cause deprotonation of the Glu204
site and vice versa. This is the kind of coupling that would cause
proton release at the extracellular surface after proton transfer from
the Schiff base to Asp85 (21-25) even though
Asp85 itself remains protonated until the end of the
photocycle (26-28). Conversely, once the proton is released at a pH
higher than the pKa of the Glu204 site,
the pKa for Asp85 will rise. Under
physiological conditions, where the difference between the pH and the
pKa for the release is large, the proton release
will shift the protonation equilibrium between Asp85 and
the Schiff base toward virtually complete and unidirectional proton
transfer.
The second observation, made from x-ray diffraction, was that
deprotonation of the Schiff base of the D85N mutant by raising the pH
in the dark caused the protein to assume an equilibrium mixture of
conformations that exhibit structural changes seen otherwise only in
the M photointermediate (29). In the D85N/D96N double mutant the
equilibrium contained a large amount of the M-like conformation even
with the Schiff base protonated. The pKas for the
protonated Schiff base and the changes in crystallographic parameters
for D85N were the same, and the isomeric composition of the retinal was
indifferent to the shift of protein structure (30). As expected from
this, the pKa of the Schiff base in D85N was nearly
unchanged when the retinal was replaced with an analogue locked in the
all-trans configuration. These observations provided a hint
to the cause of the proton transfer switch in the photocycle that
allows reprotonation of the Schiff base from the cytoplasmic side; if
it is a result of the shift of the global protein conformation, it
depends on deprotonation of the Schiff base, i.e. loss of
interaction between the protonated Schiff base and its complex
counterion rather than directly on the isomeric state of the
retinal.
In the third study (31), mutations in the extracellular proton channel
were shown to cause parallel decreases in the rate of the final
photocycle step and the deprotonation of Asp85 in pH jump
experiments in the dark. The correlation of the rates over 3 orders of
magnitude suggested that the loss of proton from Asp85 to
the Glu204 site or directly to the medium depending on the
conditions is the rate-limiting step in the recovery of the initial
state. Other mutations, nearer the retinal, also caused the slowing of
the photocycle, but for these there was no correlation with the
deprotonation of Asp85. In these cases a perturbed
interaction between the 13-methyl or 9-methyl groups of the retinal and
the protein interfered with reisomerization to all-trans, as
suggested earlier by other kinds of evidence (32-34).
Mechanism of Transport These recent results contributed some missing pieces in the puzzle
and helped to refine the proton transport mechanism that follows. The
active site consists of the protonated Schiff base and the anionic
Asp85 and bound water (35-39) that stabilizes the buried
charges. Photoisomerization of the retinal from all-trans to
13-cis,15-anti changes this geometry (40, 41) and
redistributes the When the proton transfer converts the charged Schiff
base-Asp85 complex to a neutral pair, stabilization of the
initial structure of the protein is removed, and an alternative
conformation is assumed (30). Difference maps from neutron, x-ray, and
electron diffraction of the M state show density increases at the
cytoplasmic segments of helices B and G (48-54). Blue shift of the
absorption maximum of the chromophore with unprotonated Schiff base at
this time in the D96N mutant but not in the wild type (55) suggested that the Schiff base enters into hydrogen bonding, most likely through
bridging water molecules (8, 56) with Asp96. Thus, after
proton transfer back from Asp85 is made energetically
unfavorable by the elevated pKa of this residue, the
connection of Schiff base is switched to Asp96. The
existence of such an accessibility change, based on geometrical rearrangement at the Schiff base, is suggested also by the observation that under some conditions transport can be demonstrated even when both
Asp85 and Asp96 are replaced with neutral
residues (57, 58). The change in local geometry initiates the second
phase of proton translocation (Fig. 1, right
panel).
At this time in the photocycle the pKa of the Schiff
base is about 8 (59), i.e. much lower than its initial value of >13 (60), but the pKa of Asp96 is
presumed to be still at least as high as 11, as in the unphotolyzed protein (61). The latter pKa will decrease to about 7 so as to make Asp96 a proton donor (62). The reason
appears to be a further protein conformation change in the M
intermediate (observed when this state is stabilized in the D96N mutant
and termed MN) that consists of the outward tilt of the
cytoplasmic end of helix F (48-54). The effects of dehydration by
osmotic agents (63) and hydrostatic pressure (64) suggested that it is
the increased hydration of the cytoplasmic region that lowers the
pKa of Asp96 and makes this residue a
proton donor to the Schiff base. The observed consequences of chemical
modifications of engineered cysteines and cross-linking at the
cytoplasmic end of helix F, and not elsewhere, attributed the increased
hydration to the cleft that is formed upon tilt of this helix (65).
Reversal of the tilt of helix F restores the initial high
pKa of Asp96 (66), and this residue is
then reprotonated from the cytoplasmic surface. The increase of the
lateral cross-section of the protein in the bilayer during this
process (50) may be the cause of the cooperativity in the
two-dimensional lattice of the purple membrane upon
photoexcitation (67).
Reisomerization of the retinal to a twisted all-trans
configuration (not shown in Fig. 1) is followed by proton transfer from Asp85 to the Glu204 site and relaxation of the
twisted retinal chain (31), completing the photocycle. Thus, the
recovery of the initial proton affinities of Asp85
(pKa about 2.5) and the Glu204 site
(pKa about 9) at the end of the photocycle is the reason for the irreversibility of this reaction. It is because the
pKa of Asp85 returns at this time to its
initial very low value that proton transport can proceed against a high
transmembrane proton potential, and sufficient protonmotive force is
created to drive the light-driven synthesis of ATP (68, 69).
Role for Bound Water in Proton Transfers Much indirect evidence indicates that there is specifically bound
water near the Schiff base and Asp85, and its hydrogen
bonding undergoes changes throughout the photocycle. Water near the
Schiff base and Asp85 was suggested to mediate the initial
proton transfer (41, 70). Hydrogen-bonded water molecules that connect
Asp85 to Glu204 in structures calculated from
molecular dynamics (71, 72) may play the major role (19) in the linked
pKas of Asp85 and the Glu204
site. The changed access of the Schiff base to Asp96 has
been suggested to be through reorganized water molecules at the active
site (73). Water molecules between the Schiff base and
Asp96 were postulated to mediate proton transfer across the
12-Å distance in the interior of the protein (8, 9). The effects of
osmotic agents on this reaction (63) suggested that first an increased, then a decreased, amount of bound water near Asp96
associated with the formation and the closing of a cleft at the cytoplasmic surface is what regulates the proton affinity of this residue as a proton donor to the Schiff base and then an acceptor from
the cytoplasmic surface. Finally, in the last step of the sequence
proton is lost from Asp85 to Glu204 most likely
through the mediation of the hydrogen-bonded water chain that connects
them. Indeed, the large D2O isotope effect (5-10-fold)
associated with both proton release and the deprotonation of
Asp85 that takes place later (31) strongly suggest that
bound water plays an important role in these processes.
Nature of the Active Site The Schiff base and its acceptor appear to constitute the single
active site in this proton pump. The function of the rest of the
protein may be simply to alternately deliver to and remove a proton
from this site. If this is true, we should distinguish between an
active site specific for the initial and critical ion transfer and a
nonspecific matrix that responds to it passively by redirecting its
access to either membrane surface.
There is support for this view from an observed change in the ion
specificity of bacteriorhodopsin. Upon replacing Asp85 with
a threonine, the equivalent residue in the light-driven chloride pump
halorhodopsin (74), bacteriorhodopsin acquired the spectroscopic
properties and the chloride transport activity (that proceeds in the
opposite direction from the proton transport) of halorhodopsin (75).
Although the chloride transport mechanism in this recombinant
bacteriorhodopsin and in halorhodopsin (76-79) is not yet well
understood, it must involve binding of chloride at the active site and
its translocation to the cytoplasmic domain. Evidently, the alternating
pathways of access in this protein allow the movement of not only
protons but also chloride.
Halorhodopsin, in turn, can be made to transport protons. This will
occur in a two-photon reaction in which the deprotonated 13-cis and protonated all-trans-retinal Schiff
bases are forcibly interconverted, with an accompanying protonation and
reprotonation that results in net translocation of charge (80).
Analogously to the mutational change of specificity of
bacteriorhodopsin, halorhodopsin also becomes a proton pump when an
artificial proton acceptor, the azide anion, occupies a site near the
Schiff base where otherwise the transported chloride is bound (81).
Thus, when provisions are made for a proton acceptor to the Schiff
base, the access change and the conduction pathways for chloride will also accommodate protons.
The hypothesis that the functioning of bacteriorhodopsin depends less
on proton conduction pathways to and from the surface than on the
initial proton transfer at the active site is supported by the
remarkable fact that of the many hundreds of site-specific mutations
studied (12, 14, 15) none inactivate transport entirely. Those that
have the greatest effect prevent proton transfer from the Schiff base
to Asp85, e.g. by replacing the aspartate with a
neutral residue or by keeping it permanently protonated. However, even
in the former case where transport of the kind in the wild type is
inactivated, proton transport is observed under some conditions (57,
58). Two other mutations that are less disruptive are also worthy of note. In the first, E204Q, proton release to the extracellular surface
upon protonation of Asp85 is abolished. Although proton
release is delayed until the end of the photocycle when the low
pKa of Asp85 is reestablished, the
direction of the proton transfers is preserved (18, 82). In the second,
D96N, reprotonation of the Schiff base and the subsequent proton uptake
from the cytoplasmic surface are hindered. Protonation of the Schiff
base is directly from the surface but again with the correct
directionality (62, 83, 84). Thus, the proton conduction pathways to
both membrane surfaces can be seriously perturbed without loss of
transport activity. On the other hand, interference with these pathways
does affect the turnover rate of the pump, and the rate of proton
transport at high light intensities will be decreased. At physiological pH the slowing of the photocycle in D96N amounts to 2-3 orders of
magnitude, as the rate-limiting step becomes the inefficient capture of
a proton at the cytoplasmic surface (63). Replacement of residues at
the cytoplasmic surface, such as Asp38, that might also
have roles in proton conduction causes a severalfold slowing of the
photocycle (45). Similarly, at the extracellular surface, the E204Q
mutation causes decrease of the turnover by 1-2 orders of magnitude,
as the loss of the proton of Asp85 becomes rate-limiting
(18, 31). Avoiding such problems must be the rationale for the
evolution of the proton transfer pathways described above. Indeed, the
arrangement of acidic residues in the structure suggests that multiple
pathways may exist for both release and uptake of protons at the two
surfaces (9).
Conclusions Thus, the transport in bacteriorhodopsin is explained by an
alternating access mechanism. The change of the access of the occluded
proton binding site occurs in sequential steps. In the first and last
steps the direction of the transfer of proton between the Schiff base
and the two membrane surfaces is determined by the magnitude and sign
of a changing
Fig. 1.
Proton transfers in the two phases of the
bacteriorhodopsin photocycle. The structure of the unphotolyzed
protein is shown with all-trans-retinal at 3-Å resolution
(9). The proton moves from the cytoplasmic membrane surface to the
extracellular in several segments (proton transfers are shown with
curved green arrows). A detailed description of these
events, some supported by more evidence than others, is given in the
text. Briefly, in the first phase (left panel) the
protonated Schiff base of the photoisomerized
13-cis,15-anti-retinal protonates the anionic Asp85. By electrostatic interaction (blue
arrow), this causes proton transfer from Glu204, or
water liganded to it, to Glu194 and from there proton
release to the surface. In the second phase a large scale conformation
change (shown in the right panel as tilt of helix F) occurs
and causes increased hydration of the cytoplasmic region and thus a
decrease of the pKa of Asp96. This
causes reprotonation of the Schiff base. Recovery of the initial
protein conformation results in reprotonation of Asp96 from
the bulk and reisomerization of the retinal to all-trans. The final proton transfer from Asp85 to Glu204
completes the pathway of the proton across the protein. The access of
the retinal Schiff base to its proton acceptor and donor is therefore
determined by directed shifts of pKas and probably also by a geometrical reorientation that connects it with first the
acceptor and then the donor. (Coordinates were generously provided by
Y. Kimura.)
-electron system along the retinal chain (42, 43),
causing the pKa difference between the Schiff base
and Asp85 to narrow from about 5 to <1 (44). Fig. 1 shows
schematically the proton transfer steps in the early and late phases of
the transport cycle that ensues. As Asp85 becomes
protonated, linkage of the pKas of Asp85
and the Glu204 site (Fig. 1, blue arrow in
left panel) causes the pKa of the
Glu204 site to drop from 9 to about 5. As a result,
Glu204 protonates Glu194, and the latter
releases the proton to the
surface.1 At neutral
(physiological) pH release of the proton to the extracellular surface
is strongly favored. This in turn causes the pKa of
Asp85 to further rise (17, 19, 46), and its protonation
equilibrium with the Schiff base shifts toward nearly full proton
transfer (24). This transition, identified as the M1 
M2 reaction that was deduced from the kinetics of
absorbance changes (47), completes the first phase of the transport
cycle (Fig. 1, left panel). Reprotonation of the Schiff base
from Asp85 is now no longer possible.
pKa relative to proton acceptor and
donor groups. Although this would ensure, by itself, the direction of
the transport, there may be in addition a change of the local geometry
to switch the connectivity of the Schiff base. The lesson for other
pumps may be that the local ion transfer reactions, which deliver the
transported ion to the active site and remove it, are more crucial and
more stringently regulated than the conduction pathways between the
binding site and the two membrane surfaces.
FOOTNOTES
REFERENCES
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