Bacteriorhodopsin, Molecular Biology of
581
not within hydrogen-bonding distance, the
Schiff base. The water cluster in M
2
may be expected to (1) lower the p
K
a
of
A
sp96soa
stom
ak
ei
tap
ro
tondono
r
and (2) constitute the beginnings of a
hydrogen-bonded chain that would, in the
next photocycle step, conduct the proton
to the Schiff base.
3.4
The N State and Proton Uptake from the
Cytoplasmic Side
Reprotonation of the Schiff base by Asp96
produces the N intermediate, which ab-
sorbs near 560 nm but with a lower extinc-
tion than the initial BR state, as expected
for its 13-
cis
retinal. A large-scale protein
conformation change in N is evident from
electron diffraction, measured either at
various times after flash illumination and
freezing or at an ambient temperature in a
photostationary state of a mutant with long
N lifetime. The extensive displacement of
the cytoplasmic end of helices F and G in
N is con±rmed by distance measurements
with pairs of spin labels, and with maps
of Hg attached to engineered cysteines.
The changed accessibilities of spin labels
to aqueous quenching agents, and engi-
neered cysteine residues to hydrophilic
reagents, further de±ne the conforma-
tional changes of the helices and the
interhelical loops.
The effects of osmotic agents, hydro-
static pressure, and humidity on the M
0
2
→
N reaction and on the protein conforma-
tion change, and kinetic analysis of the
in-plane cooperativity in the purple mem-
brane lattice, suggest that the rationale of
the helical tilt is to increase the hydration
of the cytoplasmic region and thereby de-
crease the p
K
a
of Asp96. Although widely
assumed, the hydrogen-bonded chain of
water molecules between Asp96 and the
Schiff base has not been directly observed.
The conformation with such a chain of
water might be a transient state that leads
to N, one that does not accumulate in mea-
surable quantities. Recovery of the tilt of
helix F is during decay of the N state to
the O intermediate, as it is absent in the
crystal structure of a mutant that should
have an O-like structure. Recovery of the
initial state of the cytoplasmic region will
reestablish the initial high p
K
a
of Asp96,
and it will have been reprotonated from
the cytoplasmic surface.
3.5
The O State
Reisomerization of the retinal to all-trans
is made possible by the lowered barrier
to bond rotations in the polyene chain
upon protonation of the Schiff base. It
occurs in the N to O transition, coupled
to the reprotonation of Asp96. Residues
that contact the chain near the 9-methyl
and 13-methyl groups, such as Trp182
and Leu93, facilitate the reisomerization,
through steric interaction, that transmits
displacements of the protein to the retinal
and
vice versa
. The O state has a strongly
red-shifted maximum in the visible, at least
partly because Asp85 is still protonated,
and thus the main component of the
counterion to the protonated Schiff base
is lacking. Large-amplitude hydrogen out-
of-plane vibrations indicate that, as in
the K state, the retinal chain is twisted.
These
features
disappear
in
the
±nal
O
→
BR reaction, which appears to be
limited by the rate of proton transfer
from
Asp85
to
the
still
unprotonated
proton release site. As expected from the
recovery of the low initial p
K
a
of Asp85,
this reaction is unidirectional under all
conditions and ensures not only the full
repopulation of the initial state but also the
