Bacteriorhodopsin, Molecular Biology of
579
Fig. 2
Photocycle scheme for
bacteriorhodopsin. The intermediate
states, described in the text, are shown
with the isomeric state of the retinal
indicated. In all states other than BR and
O, the retinal is 13-cis. Proton release is
to the extracellular side of the
membrane; uptake is from the
cytoplasmic side.
O
all-trans
BR
all-trans
K
13-cis
h
n
L
13-cis
M
1
13-cis
M
2
13-cis
N
13-cis
(cytoplasm)
H
+
H
+
(extracell.)
protein in the infrared. They correspond
to stages of the progress of the photocycle.
3.1
The K State
In the K intermediate that arises on the
picosecond timescale, the retinal assumes
a twisted 13-cis,15-anti conFguration, as
indicated
by
the
observation
of
high-
amplitude hydrogen out-of-plane bands,
an
dc
onF
rm
e
db
yt
h
eX
-
r
a
yd
i
f
f
r
a
c
t
i
on
structure of the retinal. The latter had
shown that the bond angle at C
13
is anoma-
lously wide, attributable to the fact that the
retinal binding site resists deformation on
the timescale in K. This keeps the retinal
linearly extended in spite of rotation of
the C
13
=
C
14
bond that would otherwise
result in a bent polyene chain. Counterro-
tations of the C
14
C
15
and C
15
=
Nbonds
ensure that the direction of the N
Hbond
remains roughly in the extracellular direc-
tion. However, the N
H
O angle that the
Schiff base forms with water 402 is now
more sharply angled, making its hydro-
gen bond weaker. This is in accordance
with an anomalously low Schiff base band
frequency and loss of its deuterium isotope
effect, as well as a shift of the O
Hstretch
bands of a water molecule (assigned to be
water 402) to higher frequencies.
3.2
The L State
Kconve
r
tsinabou
t1
µ
s into the L state
that absorbs at about 540 nm. ±ourier-
transform infrared (±TIR) spectra indicate
that in L, changes begin to appear in
the protein also, in both extracellular and
cytoplasmic regions. The Schiff base band
increases in frequency and regains its
deuterium isotope effect, indicating that
it forms a stronger hydrogen bond than
in K. The X-ray diffraction structure of L
indicates that this is because rotations of
the C
13
=
C
14
and C
15
=
N bonds return the
N
H
O geometry toward its initial angle.
This means that further relaxation of the
retinal by proton transfer from the Schiff
base to water 402 and thence to Asp85 is
made possible.
3.3
The M States and Proton Release to the
Extracellular Side
The M states, with deprotonated Schiff
base, have strongly blue-shifted absorption
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