(Received for publication, July 7, 1995; and in revised form, September 18, 1995)
From the
Bacteriorhodopsin is a light-driven proton pump, which undergoes
a photocycle consisting of several distinct intermediates. Previous
studies have established that the M N step of this photocycle
involves a major conformational change of membrane embedded
-helices. In order to further investigate this conformational
change, we have studied the photocycle of the high pH form of the
mutant Asp-85
Asn (D85N
). In contrast to wild type
bacteriorhodopsin, D85N
has a deprotonated Schiff base
and a blue-shifted absorption near 410 nm, yet it still transports
protons in the same direction as wild type bacteriorhodopsin (Tittor,
J., Schweiger, U., Oesterhelt, D. and Bamberg, E.(1994) Biophys.
J., 67, 1682-1690). Resonance Raman spectroscopy of
D85N
and D85N
regenerated with retinal
labeled at the C-15 position with deuterium reveals the existence of an
all-trans configuration of the chromophore. Fourier transform
infrared difference spectroscopy shows that the photocycle of this
light-adapted form involves similar events as the wild type
bacteriorhodopsin photocycle including the M
N protein
conformational change. These results help to explain the ability of
D85N
to transport protons and demonstrate that the M
N conformational change can occur even in the photocycle of an
unprotonated Schiff base form of bacteriorhodopsin.
Bacteriorhodopsin (bR) ()is a M
26,000 protein found in the purple membrane of Halobacterium
salinarium(1, 2) . Although detailed information
exists about the bR structure (3) and the protonation changes
which occur at specific amino acid
residues(4, 5, 6, 7, 8, 9) ,
key questions still remain about the detailed mechanism of light-driven
proton transport from the cytoplasmic to the extracellular medium.
Perhaps the most fundamental question concerns understanding how the
light-induced isomerization of the retinal chromophore couples to
protein conformational changes. A closely related question applies to
sensory rhodopsin I (10) and rhodopsin(11) , whose
signaling mechanisms also depend on the triggering of protein
conformational changes by retinal isomerization.
Several recent FTIR
studies indicate that a major conformational change involving membrane
embedded -helices occurs in the bR structure during the M
N
transition(12, 13, 14, 15, 16, 17) .
For example, an intense negative band near 1669 cm
appears in the amide I region of the time-resolved FTIR
difference spectrum of bR during formation of the N intermediate (13, 14, 15) and disappears during N
decay(15, 17) . Similar results are also obtained from
low temperature steady-state measurements, where the N intermediate is
stabilized by either high pH or low
temperature(16, 18) . In a recent study combining
attenuated total reflection (ATR) FTIR difference spectroscopy and
site-directed isotope labeling (SDIL), it was found that part of the M
N transition involves the Tyr-185/Pro-186 region of the F-helix (19) . This region may function as a hinge which allows
reorientation of the F-helix. An interesting possibility is that this
structural change is responsible for a switch in the Schiff base
accessibility from an extracellular to a cytoplasmic proton conducting
pathway, termed the E and C conformations(20, 21) . In
general, such a switch has been postulated to be present in a variety
of active transport pumps (21, 22, 23, 24, 25) .
In this report, we have used FTIR difference and resonance Raman
spectroscopy to study the photoreactions of the high pH form of the
Asp-85 Asn mutant (D85N
). Several earlier studies
have established that the Schiff base of D85N deprotonates at high pH
forming a new state with a
near 410
nm(20, 26, 27, 28, 29) .
Surprisingly, this deprotonated Schiff base species is still able to
pump protons under blue light illumination in the same direction as
wild type bR(30, 31, 32) . Our results show
that the photocycle of the light-adapted form of D85N
involves a conformational change of the protein, which is very similar
to that occurring in wild type bR, despite the fact that the original
all-trans state of the retinylidene chromophore is
deprotonated. This result, as well as studies on other bR mutants,
suggests that this conformational change may constitute a key step in
the proton pumping mechanism of bacteriorhodopsin. It also indicates
that the protonation state of the Schiff base is not directly coupled
to this conformational change.
The 15D-all-trans-retinal
was obtained as described in (41) by LiAlD (Aldrich), reduction of all-trans-retinoic acid to give
15D
-all-trans-retinol. The latter was then
oxidized with activated manganese dioxide to give
15D-all-trans-retinal. Crude product was purified on a silica
gel column using step gradient of ethyl acetate in hexane and finally
recrystallized from petroleum ether. The sample obtained was
characterized by
H NMR, by analytical high performance
liquid chromatography (>96% trans) and by the CI-MS spectrum
(M
= 285.03; isotopic purity >97%). D85N
containing the 15D-all-trans-retinylidene chromophore
(15D-D85N) was produced by photobleaching D85N in the presence of 1 M hydroxylamine, washed two times with bovine serum albumin
solution to remove the retinal oxime, and then regenerated with the
15D-all-trans-retinal.
Figure 1:
FTIR difference spectra of dark-adapted
D85N. The blue light excitation (
<440 nm) FTIR difference spectra of dark-adapted D85N at pH
9.6 recorded using ATR (a) and transmission (b)
methods. Both D85N
spectra were recorded at 2
cm
resolution and represent the average of at least
50 pairs of dark-light cycles (5 min for a and 15 min for b). The ATR spectrum was measured at 7 °C and the
transmission spectrum at 22 °C. Further details are given under
``Experimental Procedures.'' Increasing the dark
portion of the cycle did not alter the individual difference spectrum
significantly, indicating that all the photoproducts decayed within the
dark period. Absorbance scale shown (0.00025 absorbance units) is for trace b. c, the wild type M
N spectrum was
recorded under conditions previously
reported(12) .
Fig. 1also shows that steady-state illumination
of dark-adapted D85N with blue light produces a mixture
of photoproducts that are very similar to the N and O intermediates of
the light adapted bR photocycle. For example, the positive 1510
cm
band and the pair of positive bands at 1200 and
1168 cm
correspond closely to the frequencies of
major chromophore bands found in the O
intermediate(15, 46) . Furthermore, the positive bands
at 1530/1554 cm
along with the strong peaks at 1186
cm
are similar to bands assigned to the N
intermediate(14, 47) . This can be seen by comparing
the D85N
difference spectra with the M
N
difference spectrum of wild type bR (Fig. 1), where positive
bands also appear at 1554, 1530, and 1186 cm
. As
discussed later, we believe these photoproducts occur because blue
light first light-adapts D85N
, thereby producing an
M-like form with an all-trans chromophore
(M
), which then undergoes a blue
light-driven photocycle that includes N- and O-like intermediates.
A
remarkable feature of the D85N difference spectra is the
appearance of a pair of bands near 1669/1661 cm
(-/+) in the amide I region, which have been
previously associated with the major conformational change occurring
during the M
N transition of bR(12, 13, 14, 15, 16, 17) (see Fig. 1). The appearance of these bands is a strong indication
that a similar conformational change occurs during blue light
excitation of D85N
. (
)There is also a negative
band at 1742 cm
in the D85N
difference
spectra (Fig. 1). In the case of wild type bR, this is assigned
to the carboxyl stretch of Asp-96 (13, 14, 15) and is associated with the
deprotonation of this residue and the reprotonation of the Schiff base
during the M
N transition(48) . Thus, we conclude that
changes in both the conformation and protonation of the protein,
similar to those which occur during the M
N step of the wild
type bR photocycle, also occur in the blue light-driven photocycle of
D85N
.
Figure 2:
Low temperature FTIR difference spectra of
D85N. All spectra shown were recorded at -3 °C
at 2 cm
resolution by the transmission method (see
Refs. 4, 35, and 36 for further details concerning sample preparation
for FTIR). The excitation scheme used to record each difference
spectrum is indicated. For example, (dark
blue) denotes a
sequence whereby the sample is first measured in the dark and then with
blue light. a, the difference spectrum obtained by subtracting
a spectrum of dark-adapted D85N
from the first
illumination with blue light (440-nm narrow band filter). b,
subsequent difference spectrum obtained by subtracting the measurement
made during blue light illumination (see a) and the following
dark measurement made after 1 h in darkness. c, average of
subsequent difference spectra obtained by subtracting a spectrum of the
sample recorded under illumination with green light (500-nm narrow band
filter) from one recorded under illumination with red light (650 nm),
which acts to photoreverse the effects of the 500-nm illumination. d, O
N difference spectrum of the bR mutant Y185F
(adapted from (15) ). Absorbance scale shown (0.0001 absorbance
units) is for trace c.
The photochemistry of the N- and O-like blue light
photoproducts of D85N trapped at low temperature were
studied by recording the FTIR difference spectra produced by using
different wavelengths of illumination. Illumination with green light
(500 nm) followed by photoreversal with red light (650 nm) produces a
difference spectrum (Fig. 2, trace c) that compares
well with the O
N difference spectrum measured for wild type bR (17) and the mutant Y185F (15, 49) (Fig. 2, trace d). For
example, the difference bands in the 1150-1250
cm
fingerprint region are highly characteristic of
an all-trans to 13-cis isomerization of the
chromophore(44, 50) . This effect can be explained by
the selective photoreaction of the low temperature trapped O-like
photoproduct with red light to form an N-like species. A similar effect
has been previously observed for the O intermediate formed by the bR
mutant Y185F(39) . At room temperature, the O photocycle
appears to involve a K-like intermediate and a long-lived N
intermediate(49, 51) . The N species formed in D85N
can also be photoreversed back to O using green light. Notice that
photoreversal between O and N produces a characteristic switch in the
protein conformation as indicated by the bands at 1669 and 1649
cm
. This is again similar to the photocycle of wild
type bR, where this conformational change occurs between M
N and
is reversed during the N
O step(15) .
Interestingly,
we also observed a difference spectrum similar to the O N
difference spectrum (green
red) shown in Fig. 2when blue
light was used for excitation. A possible explanation for this effect
is that the O-like species, which contains an all-trans protonated chromophore, exists in equilibrium with an M-like
species, which contains an all-trans chromophore and
deprotonated Schiff base (M
).
Photoconversion by blue light of M
to N
would then be expected to cause a depletion of the O-like species and
the appearance of the negative O-like chromophore bands.
Figure 3:
Panel A, resonance Raman spectra of
D85N and M intermediate of wild type bR (WT).
15D-D85N
denotes D85N
regenerated with a
retinylidene chromophore labeled with deuterium at the C-15 position,
whereas a D85N
sample regenerated with nonlabeled retinal
was used to obtain the 15H-D85N
spectrum. No additional
light was used to maintain light adaptation other than the probe beam. Inset, subtraction between the 15H- and 15D-D85N
spectra in the 1100-1300 cm
region. Both
the spectra were scaled to the same ethylenic intensity at 1563
cm
before subtraction. Panel B, resonance
Raman spectra of the N-like photoproduct of D85N
and the
N intermediate of wild type bR. See ``Experimental
Procedures'' for additional details. An asterisk is used
to label the 1048 cm
band from KNO
used
for frequency calibration.
In order to determine the
isomeric composition of D85N, we examined the 15D induced
changes in the structurally sensitive fingerprint region
(1100-1400 cm
). In the case of model compounds
containing an all-trans unprotonated retinal Schiff base, a
band appears at 1178 cm
and downshifts to 1158
cm
for the 15D isotope substitution(50) .
However, no such isotope shift is observed for the 13-cis unprotonated Schiff base. In addition, 15D substitution causes a
characteristic 1225 cm
band in the spectrum of
13-cis unprotonated Schiff base to become more intense. In
contrast, only a small band that is isotope-insensitive is observed at
this frequency for all-trans unprotonated Schiff base
compounds.
In the case of 15D-D85N, we observe isotope
effects that are typical of the all-trans unprotonated retinal
Schiff base but which are not observed for 13-cis unprotonated
retinal Schiff bases. First, a decrease in the intensity of the 1175
cm
band occurs along with the appearance of a
distinct band at 1157 cm
characteristic of the
all-trans configuration. This effect is clearly observable in
the scaled subtracted spectrum of 15H- and 15D-D85N
(see inset to Fig. 3, panel A). Second, unlike the
M-intermediate, no isotope effects are seen at 1225 cm
in 15D-D85N
compared to the 15D
analog(50) . These results strongly indicate that the
D85N
chromophore exists in a predominantly all-trans configuration with an unprotonated Schiff base. However, since we
do not observe a complete disappearance of the 1175 cm
band in 15D-D85N
and a drop also occurs in the band
near 1200 cm
as seen in 13-cis model
compounds(50) , a species with a 13-cis chromophore
configuration may also be present(28, 30) . This may
be due to the presence of a fraction of D85N
, which is
not light-adapted by the 413.1-nm exciting light.
FTIR difference spectroscopy indicates that a major
conformational change involving membrane buried -helical structure
occurs during the M
N transition and is reversed during the N
O transition of
bacteriorhodopsin(12, 13, 14, 15, 16, 17) .
These events are correlated with the transfer of a proton from Asp-96
to the Schiff base (M
N) and the reprotonation of Asp-96 along
with the reisomerization of the chromophore from a 13-cis to
an all-trans configuration (N
O)(15, 17, 46, 48, 53) .
Structural changes in the C-D loop near Asp-96 are also detected by
site-directed spin labeling, which are correlated with the decay of the
M intermediate(54) .
Although the exact nature of the
structural changes occurring upon N formation are not yet known, most
available data are consistent with the reorientation of
membrane-embedded -helices(12) . For example, electron
diffraction detects structural changes in the bR photocycle that
involve a tilt of a portion of the F and C helices on the cytoplasmic
side of the protein(55) . Furthermore, the 1669 cm
band observed in the M
N FTIR difference spectrum (Fig. 1) exhibits out-of-plane dichroism which is consistent
with the tilt of
-helices away from the membrane normal. (
)Recent studies based on SDIL have established that a
portion of this band contains contributions from the amide carbonyl
group of Tyr-185 and may reflect structural rearrangements in the
Tyr-185/Pro-186 amide bond(19) . Changes in this bond are
detected as early as the bR
K phototransition(56) ,
which is most likely directly triggered by the all-trans
13-cis isomerization of the retinylidene
chromophore. Thus, one possibility is that the initial chromophore
isomerization initiates structural changes in the Tyr-185/Pro-186
region of helix F that results later in the photocycle in a
reorientation of part of this as well as other helices in the protein.
In order to explore further the nature of this conformational change
(termed here the RT-conformational change) ()and its
functional significance in the bacteriorhodopsin proton pump, we have
focused on the alkaline form of the mutant Asp-85
Asn in this
work. Earlier studies have established that blue light-driven
D85N
exhibits proton pumping in the same direction as
wild type bR(30, 32) , despite the fact that it has a
deprotonated Schiff base. It was also predicted in these works that the
proton pumping occurs due to the photocycle of an
M
form of D85N
. In analogy
with light-adapted bR (bR
), it might then be expected
that the photocycle of D85N
would include a step similar
to the RT-conformational change.
The results of our FTIR and
resonance Raman measurements establish the following. (i) Light-adapted
D85N contains a retinylidene chromophore with an
all-trans configuration (M
).
(ii) The photocycle of light-adapted D85N
involves
formation of N- and O-like intermediates. (iii) The photocycle of
light-adapted D85N
involves an RT-conformational change
and proton transfer from Asp-96 to the Schiff base. (iv) The N- and
O-like photoproducts of the light-adapted D85N
photocycle
can be trapped at low temperatures and exhibit a photoreversible N
O conversion, which also involves reversal of the
RT-conformational change.
A simplified scheme similar to one
proposed earlier (30) which accounts for these findings and the
ability of light-adapted D85N to pump protons in the same
direction as wild type bR is shown in Fig. 4. Blue light
adaptation of dark-adapted D85N
(species not shown)
produces an equilibrium mixture of two light-adapted species containing
all-trans retinal chromophores, M
and O. Both of these species exist in the R conformation and
differ mainly by the Schiff base protonation state. Blue light
absorption by the M
species triggers a
photocycle that consists of a number of early intermediates (not shown)
including an M-like intermediate with a deprotonated Schiff base and
13-cis retinal configuration (20, 31) and
later, an N-like species containing a
13-cis/C=NH
chromophore. Formation of
the N-like species involves a net change in protein conformation from
the R to T state and the internal movement of a proton from Asp-96 to
the Schiff base. The thermal decay of the N-like to O-like species
involves the reisomerization of the chromophore, uptake of a proton by
Asp-96 from the cytoplasmic medium, and a switch of the protein
conformation from T to R. The Schiff base of the O-like species then
deprotonates releasing a proton into the external medium, thereby
reestablishing its thermal equilibrium with the
M
form.
Figure 4:
Simplified model of the photocycle and
proton pumping of wild type bR (left) and D85N (right). Only the light-adapted states (bR
(dark gray) and M
(light gray)) and the corresponding N (gradient
gray) and O (sandpaper gray) states of the WT and
D85N
photocycles, respectively, are shown. Additional
intermediates are also likely to be present in the D85N
including an M-like intermediate in analogy with wild type bR.
The chromophore configuration (all-trans/C=N anti,
13-cis/C=N anti), Schiff base protonation state
(N-H
, N), protein states (R, T), and Asp-96
protonation state (COOH, COO
) are indicated along
with the movement of protons during each step of the photocycle.
Cytoplasmic and extracellular sides of the protein are top and bottom, respectively (the protein is represented in the figure
by an oval shape). The dotted arrow indicates steps
not shown in the D85N
photocycle and the dashed arrow the equilibrium between the M
and O-like intermediate. See ``Discussion'' for
additional details.
An important feature of the
proposed D85N photocycle is its similarity to the bR
photocycle. In both photocycles, an RT-conformational change is
observed, which is accompanied by proton transport from Asp-96 to the
Schiff base. In the case of bR, this involves deprotonation of the
Schiff base and ejection of a proton to the external medium. However,
in the case of D85N
, proton ejection does not occur at
this stage since M
already has a
deprotonated Schiff base. The late photocycles in both cases involve
the reprotonation of Asp-96 via uptake of a proton from the cytoplasmic
medium, a reversal in protein conformation, and reisomerization of the
chromophore to an O-like species. Finally, both photocycles involve the
decay of the O-like species. However, unlike native bR where
light-adapted bR (bR
) has an all-trans chromophore with a protonated Schiff base, the
M
form of D85N
contains a
deprotonated Schiff base.
It can also be seen from Fig. 4that the major differences between native bR and
D85N arise as a natural consequence of the Asp
Asn
substitution, which acts to lower the Schiff base pK
from about 13 in wild type bR down to 8.7 in
D85N(26, 27, 28, 30) . This drop in
pK
causes the all-trans retinal Schiff
base to deprotonate and produces the observed equilibrium between the
all-trans protonated (O-like) and deprotonated
(M
) forms of the chromophore. Note that
spectroscopic and stop-flow measurements show the transition between
these two forms is not rapid(20, 32) . This may be due
to the ability of the Asn residue in D85N to form as strong a hydrogen
bond as Asp in wild type bR(38) .
An additional conclusion
from our work is that light-adapted wild type bR and D85N both exist in the R conformation despite the fact that the
protonation state of the Schiff base is different. In particular, we
observe the RT-conformational change in the photocycle of both
M
and bR
. This also
implies that the switch from the R to T conformation is not dependent
on the disruption of the Schiff base-counterion interaction. (
)This contrasts with a recent study of D85N
based on x-ray scattering measurements of D85N at high pH (
11)
along with the effects of azide on the photocycle of D85N and
D85N/D96N(20) . This study concluded that D85N
exists in the ``C'' conformation in contrast to D85N at
lower pH and light-adapted bR, which exists in the ``E''
conformation. (
)It was also deduced from these experiments
that the switch between the E and C conformations is due mainly to the
disruption of ionic interactions of the positively charged Schiff base
inside the retinal binding pocket (20, 21) . However,
recent FTIR-ATR studies show that a global conformational change occurs
in the wild type bR (57) above pH 11, which is concomitant with
ionization of Asp-96. Thus, the C conformation of D85N measured at pH
11 by x-ray might be more related to the high pH disruption of
interactions near Asp-96 rather than at the Schiff base. Recent, pH
induced ATR-FTIR difference measurements on D85N support this
possibility. (
)
In conclusion, our results establish that
the photocycle of D85N includes as a key step a
conformational change, which has previously been observed to occur in
wild type bR between the M and N intermediates. While the equivalence
of this RT- conformational change and the required EC switch for
bacteriorhodopsin has not yet been established, such an equivalence
would help to account for the observed proton pumping of D85N
in the same direction as wild type bR(30) . Our results
also indicate that electrostatic interactions in the Schiff base region
are not essential for stabilization of the two major conformations of
bR observed by FTIR spectroscopy. In contrast, the configuration of the
chromophore appears to play an essential role, since only the
isomerization of retinal from an all-trans to 13-cis configuration has been associated with triggering the
RT-conformational change. This can be simply understood if the
all-trans/C=N anti and 13-cis/C=N syn
forms of retinal act to stabilize the light-adapted conformation of bR,
whereas the 13-cis/C=N anti configuration causes strong
steric interactions in the retinal binding pocket eventually causing
the protein to change conformations. Future studies which combine FTIR,
site-directed mutagenesis, and site-directed isotope labeling (58) are likely to shed further light on the nature of this
conformational change and the chromophore-protein interactions that
trigger it.