580
Bacteriorhodopsin, Molecular Biology of
maximum, near 410 nm. The kinetics of
this conversion, on a timescale of tens of
microseconds as measured in the visible
and the infrared, is multiphasic. Analysis
of the
kinetics suggests
that
L
is in
equilibrium with an early M state, M
1
,and
the mixture of L and M
1
decays together
to form M
2
and then the late M state,
M
0
2
(Fig. 2). At pH
>
6, the latter occurs
in a unidirectional reaction, and thus L
d
isappea
rsasM
0
2
is formed. The reason
for this appears to be that at the higher
pH, a proton dissociates from a site in
the extracellular region that interacts with
Asp85. The proton is then released to the
extracellular surface. The site is likely to
consist of bound water, and its function
depends on Arg82 and the carboxyl groups
of Glu194 and Glu204.
The anomalous titration properties of
Asp85 reveal how proton release is coupled
to protonation of Asp85. The appearance
of two apparent p
K
a
s for Asp85 could be
modeled by interaction between the proton
af±nities of the aspartate and the proton
release site. They interact in such a way that
either may be protonated but not both. This
seemstooccuralsoduringthephotocycle.
When Asp85 becomes protonated by the
Schiff base, the p
K
a
of the proton release
group is lowered and it dissociates. When
the proton is released to the bulk at a
pH higher than the p
K
a
of the proton
release site, the p
K
a
of Asp85 is in turn
driven higher, and the deprotonation of
th
eS
ch
i
f
fb
a
s
eb
e
c
om
e
sc
omp
l
e
t
e
.Th
e
identity
of
the
proton
release
is
still
uncertain. Although Arg82, Glu194, and
Glu204 are all involved, the released proton
is most probably a proton delocalized in
the water network rather than one that
originates from these amino acid residues.
Solid-state NMR spectra show that at
this time a guanidinium group becomes
strongly asymmetrical, suggesting reloca-
tion of the side chain of an arginine, very
probably Arg82. In the crystallographic
structures of the M
2
and M
0
2
states, the
side chain of Arg82 moves away from the
region of the now-protonated Asp85 and
approaches the region of Glu194/Glu204.
This shuttling of Arg82 is therefore the
means by which Asp85 interacts with the
proton release site and the direct cause of
the release of the proton to the surface in
M
0
2
.InM
1
, the Schiff base remains initially
more or less in the same position as before
its reprotonation, but in the M
1
to M
2
reac-
tion it rotates to face the cytoplasmic side.
Thus, after deprotonation of the Schiff base
there is a drastic change in the geometry of
the retinal. Before deprotonation, it faces
in the extracellular direction, toward wa-
ter 402 and Asp85, its proton acceptor,
but after its deprotonation it turns to the
cytoplasmic direction, toward its eventual
proton donor, Asp96. This ‘‘reorientation
switch’’ as well as the modulation of the
p
K
a
of Asp85 prevent reprotonation of the
Schiff base by reversal of its deprotonation
and confer directionality on the pump.
In the X-ray diffraction structures of the
intermediate states, the retinal continues
to relax as it passes through the M states,
and in M
0
2
it reaches the con±guration
bent at C
13
, as expected for the 13-cis
isomer. This thrusts the 13-methyl group
against the indole ring of Trp182, which
moves toward the cytoplasmic side. The
movement of Trp182 and rearrangements
of the side chains between helices F
and G initiate the outward tilt of helix
F and an ‘‘opening’’ of the cytoplasmic
region. As a result, in M
2
,ac
lu
s
t
e
ro
f
three water molecules accumulates near
Asp96. One of these, water 504, separates
the otherwise hydrogen-bonded Asp96 and
Thr46. Another, water 503, connects water
504 with water 502, located near, although
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