(Received for publication, July 13, 1995; and in revised form, August 29, 1995)
From the
We have measured proton release into the medium after proton
transfer from the retinal Schiff base to Asp in the
photocycle and the C=O stretch bands of carboxylic acids in wild
type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but
not in E204D, the normal proton release is absent. Consistent with
this, a negative band in the Fourier transform infrared difference
spectra at 1700 cm
in the wild type, which we now
attribute to depletion of the protonated E204, is also absent in E204Q.
In E204D, this band is shifted to 1714 cm
, as
expected from the higher frequency for a protonated aspartic than for a
glutamic acid. Consistent with their origin from protonated carboxyls,
the depletion bands in the wild type and E204D shift in D
O
to 1690 and 1703 cm
, respectively. In the protein
structure, Glu
seems to be connected to the Schiff base
region by a chain of hydrogen-bonded water. As with other residues
closer to the Schiff base, replacement of Glu
with
glutamine changes the O-H stretch frequency of the bound water
molecule near Asp
that undergoes hydrogen-bonding change
in the photocycle. The results therefore identify Glu
as
XH, the earlier postulated residue that is the source of the released
proton during the transport, and suggest that its deprotonation is
triggered by the protonation of Asp
through a network that
contains water dipoles.
Bacteriorhodopsin is the light-driven proton pump in the cell
membranes of halobacteria. The transport cycle
(``photocycle'') is initiated by photoisomerization of
retinal from all-trans to 13-cis and the ensuing
transfer of a proton from the retinal Schiff base to Asp,
an anionic residue that lies between the Schiff base and the
extracellular surface of the protein (Mathies et al., 1991;
Tittor, 1991; Ebrey, 1993; Krebs and Khorana, 1993; Lanyi, 1993). After
protonation of Asp
, a proton is released to the aqueous
phase. A protein conformational shift observed while the Schiff base is
unprotonated (Subramaniam et al., 1993) has been associated
with the kinetically detected M
to M
reaction
(Váró and Lanyi, 1995). It
was suggested to change the access of the Schiff base away from
Asp
and toward the initially protonated Asp
that lies near the cytoplasmic surface (Kataoka et al.,
1994) and to lower the pK
of Asp
(Cao et al., 1993b; Brown et al., 1995a).
Asp
thus becomes a proton donor and reprotonates the
Schiff base. This is followed by reprotonation of Asp
from
the cytoplasmic surface, reisomerization of the retinal to
all-trans, and deprotonation of Asp
in the
extracellular domain, thereby recovering the initial state. The
intermediate states that arise and decay in this cycle have been named
J, K, L, M, N, and O (Lozier et al., 1975).
The proton
transfers inside the protein to and from the Schiff base trigger the
proton transfers between groups near the surfaces and the aqueous
phase. The pK of the release group in the
extracellular domain is lowered after protonation of Asp
(Zimányi et al., 1992), and the
pK
of the proton uptake group in the
cytoplasmic domain is raised after deprotonation of Asp
(Zimányi et al., 1993). In this
way the extracellular and cytoplasmic peripheral domains facilitate the
unidirectional passage of protons. The details of this coupling are not
yet clear because both regions are structurally complex (Henderson et al., 1990) and contain several protonatable and
hydrogen-bonding residues, as well as functionally important bound
water (Maeda et al., 1992, 1994; Brown et al., 1994;
Fischer et al., 1994; Kandori et al., 1995; Yamazaki et al., 1995).
The relevant buried protonatable residues to
the extracellular side of the Schiff base are Asp,
Asp
, and Arg
and possibly Tyr
and Tyr
. The first three and coordinated water
constitute a diffuse counter-ion to the Schiff base (De Groot et
al., 1989, 1990; Dér et al., 1991).
Interaction of the anionic Asp
with Arg
is
indicated by the fact that the pK
of
Asp
is increased by about 5 pH units in the R82Q and R82A
mutants (Subramaniam et al., 1990; Thorgeirsson et
al., 1991; Balashov et al., 1992; Brown et al.,
1993), and interaction of Asp
with the Schiff base is
inferred from the fact that the pK
of the
Schiff base is decreased by about 4 pH units in the D85N mutant (Otto et al., 1990; Marti et al., 1992; Brown et
al., 1993; Tittor et al., 1994; Kataoka et al.,
1994). The participation of Asp
in these interactions is
indicated by the observations that in D212N the Schiff base does not
deprotonate after photoisomerization of the retinal near neutral pH
(Otto et al., 1990; Needleman et al., 1991; Cao et al., 1993b), and in the D212N/R82Q mutant the
pK
of Asp
is not raised as
in R82Q, but the pK
of the Schiff base
appears to be several pH units higher than in the wild type (Brown et al., 1995b). Understanding the first half of the photocycle
means understanding the nature of dipole interactions and proton
transfers within this complex. There are some suggestive clues. A newly
formed hydrogen bond between water and Asp
appears to play
an essential role in destabilizing the Schiff base proton in the L
intermediate (Maeda et al., 1994). After transfer of this
proton to Asp
in the L to M reaction, a proton is released
to the extracellular surface. The released proton originates not from
Asp
, because this residue remains protonated until the
final reaction of the photocycle, but from an unknown group termed XH.
The pK
of XH must be high in the
unphotolyzed protein (Kono et al., 1993; Balashov et
al., 1993) but will have decreased at this time to about 6
(Zimányi et al., 1992). Above pH 6 XH
releases its proton, but at more acid pH it remains protonated
throughout, and a proton is released at the extracellular surface only
at the end of the photocycle, presumably directly from Asp
as its pK
returns to its initial
low value (about 2.5). Arg
and Tyr
have been
proposed as candidates for XH, but the former would have to be in a
very unusual environment for its pK
to
become as low as 6 (Brown et al., 1993), and as for the
latter, neither UV Raman (Ames et al., 1990) nor NMR (Herzfeld et al., 1990; McDermott et al., 1991) indicate the
formation of a tyrosinate in the photocycle. In view of these problems,
until now a water liganded to Arg
(Braiman et
al., 1988) seemed to be the most likely source of the released
proton.
In this report we identify Glu, a residue
located nearly at the surface end of the extracellular proton channel
and about 15 Å from the Schiff base (Henderson et al.,
1990), as part of the proton release mechanism. The evidence indicates
that Glu
is protonated in BR but unprotonated in the M
state and strongly suggests therefore that Glu
is the
proton release group XH. This role for Glu
was recently
predicted on the basis of molecular dynamics calculations (Scharnagl et al., 1995).
The site-specific residue replacements E204Q and E204D were introduced into the bop gene and expressed in Halobacterium salinarium with a vector based on a halobacterial plasmid to be described elsewhere. The mutant R82Q was described before (Brown et al., 1993). The purple membranes containing these proteins were purified by a standard method (Oesterhelt and Stoeckenius, 1974).
Time-resolved spectroscopy was as described previously (Cao et al., 1993a, 1993b). The experiments that measured proton release were done in 2 M NaCl at the pH indicated, without buffer, and with or without 50 µM pyranine. When it occurred several milliseconds after photoexcitation or later, the absorption change of pyranine was assumed to approximate the kinetics of the interaction of the protons with the protein, because protons at the surface and in the bulk equilibrate in about 1 ms (Heberle and Dencher, 1992). All spectroscopy in the visible was at a regulated 22 °C.
For the Fourier transform infrared
(FTIR) ()measurements, 40-µl aliquots of the samples in
2.5 mM phosphate buffer, pH 7, were dried on a BaF
window, humidified with H
O or D
O before
mounting into an Oxford cryostat, and illuminated at 274 °K for 2
min with >500 nm light for light adaptation. The M minus BR
spectra were measured at 230 K after 1 min of illumination with >500
nm light in a Bio-Rad FTIR spectrometer FTS60A/896 with 2
cm
resolution. The spectra shown are differences of
the averages of four to six recordings, each calculated from 128
interferograms before and after illumination. All the spectra are
scaled by normalizing to the amplitude of the 1169 cm
band.
Figure 1: Time-resolved difference spectra in the photocycle of E204Q bacteriorhodopsin. The spectra were measured at increasing delay times after photoexcitation. The directions of the changes are indicated by upward and downward arrows. Delay times: A, 100, 250, and 600 ns and 1.5, 4, 10, 25, and 60 µs; B, 100 and 600 µs and 1.5, 4, 10, 15, 25, and 40 ms; C, 100, 150, 250, 400, and 600 ms and 1 s. Conditions: 15 µM bacteriorhodopsin and 2 M NaCl, pH 6.4.
Fig. 2A shows the time-courses of absorbance changes
at selected wavelengths after photoexcitation of wild type
bacteriorhodopsin. At 410 nm the M intermediate is measured, at 660 nm
the O intermediate is measured, and at 570 nm mostly the depletion of
the BR state is measured. From the latter, the presence or the absence
of the N intermediate during and after decay of the M state can be
inferred also (Zimányi et al., 1993). In
addition to these traces, the net absorption of the pH indicator dye
pyranine is included (Fig. 2, dashed line) to show the
release of protons (from the extracellular surface) and their uptake
(at the cytoplasmic surface) that occurs roughly during the decay of N.
Although pyranine detects the protons only once they are in the bulk,
the pH-dependent kinetics had argued (Zimányi et al., 1992) that when the protons are released to the
membrane surface during the formation of M, it occurs during the
M to M
reaction.
Figure 2: Absorbance change at selected wavelengths and proton kinetics as measured with pyranine. A, wild type bacteriorhodopsin. B, R82Q bacteriorhodopsin. The pyranine traces are shown as dashed lines. Conditions for both A and B: 15 µM bacteriorhodopsin, pH 7.3.
As described earlier (Otto et al., 1990; Thorgeirsson et al., 1991; Cao et
al., 1993a; Balashov et al., 1993), replacement of
Arg with glutamine or alanine causes distinct changes in
the chromophore and proton kinetics. Fig. 2B shows that
in R82Q (a) the formation of M is more rapid than in wild
type, (b) proton release is delayed until after proton uptake at the other membrane surface, and (c) very
little of the O intermediate accumulates. Proton transport occurs in
spite of the late release of the proton to the extracellular surface,
apparently because the M
to M
reaction can
proceed without deprotonation of the XH group, as in the wild type at
pH < 6 (Zimányi et al., 1992). The
effects of the R82Q mutation on the deprotonation of the Schiff base
and the proton release have suggested that Arg
has a role
in proton conduction between the Schiff base and the aqueous phase. The
effect of Arg
on the accumulation of O was suggested to
reflect a requirement for its positive charge for the early
reisomerization of the retinal from 13-cis to all-trans in the N to O reaction (Otto et al., 1990; Cao et
al., 1993a; Balashov et al., 1993).
Fig. 3A shows single wavelength kinetics for the chromophore and for
protons in E204Q at pH 7.3. This mutant resembles R82Q in that (a) the formation of M is more rapid than in wild type (by
about 3), but more importantly in that (b) proton
release is delayed until after proton uptake. Thus, as in
R82Q, the proton release mechanism that utilizes deprotonation of XH is
made inactive upon replacing Glu
. However, unlike in
R82Q, the O intermediate accumulates in amounts even larger than in the
wild type. The decay of M contains a second phase, larger than in wild
type, with a time constant that roughly corresponds to the rise of the
O state. The proton uptake coincides with the rise of the O state.
Figure 3: Absorbance change for E204Q (A) and E204D (B) bacteriorhodopsins at selected wavelengths and proton kinetics as measured with pyranine. The pyranine traces are shown as dashed lines. Conditions as described in the legend to Fig. 1, except the pH was 7.3.
Fig. 4shows M minus BR spectra in the C=O
stretch region for the wild type protein, E204Q, and E204D. The
spectrum of the wild type (Fig. 4A) contains the
prominent positive band of the protonated Asp at 1761
cm
, characteristic of the M state (Braiman et
al., 1988; Fahmy et al., 1992). As in many earlier
reported spectra under these conditions, there is no obvious negative
band that would indicate the deprotonation of Glu
.
However, the appearance of a negative band at 1714 cm
in E204D (Fig. 4C) raises the strong possibility
that when residue 204 is an aspartic acid, it becomes deprotonated. If
this is indeed the depletion band of the protonated Asp
,
its frequency will be downshifted in D
O, similarly to the
positive band of the protonated Asp
. The appearance of a
negative band at 1703 cm
in D
O (Fig. 4C, dotted line) confirms this. The
downshift of 11 cm
in D
O is similar to
the 12 cm
downshift for Asp
in the same
spectra. Fig. 4shows also the differences between the spectra
measured in H
O and D
O for E204Q and E204D.
Recordings of two independent experiments are shown to illustrate the
reproducibility of these difference spectra. In the E204D the
H
O/D
O difference spectrum has the positive and
negative features expected from the spectra in Fig. 4C.
They are absent in E204Q (Fig. 4B). Importantly, the
results with the wild type (Fig. 4A) show that there is
a similar H
O/D
O difference feature at lower
frequencies, where the original bands are obscured by the amide bands.
The negative band is at 1700 cm
, and the positive
band is at 1690 cm
. We suggest that the 1700
cm
band is the depletion band of the protonated
Glu
. The small bands in the 1720-1740
cm
region are ascribed to perturbation of Asp
and Asp
(Sasaki et al., 1994). The broad
bands in the 1680-1705 cm
region in E204Q and
E204D are likely due to perturbation of peptide C=O and arginine
C-N (Braiman et al., 1994). These perturbations could be
different in the different mutants. In E204Q Gln
might
contribute also to these bands.
Figure 4:
M minus BR difference spectra in
the C=O stretch region for wild type bacteriorhodopsin and E204Q
and E204D measured with FTIR. For each sample a spectrum in
HO (solid line), a spectrum in D
O (dotted line), and H
O minus D
O spectra from two independent experiments (solid
lines below the other two) are shown.
The amplitude of the C=O band
ascribed to Glu is smaller than that of Asp
.
Increasing the pH from 7 to 10, which would result in more complete
deprotonation of Glu
in the photocycle but in less
observed amplitudes if this pH is higher than the pK
of Glu
in the unphotolyzed state, did not change
the amplitudes (not shown). Tilting the samples, which would reveal any
orientational effects of the C=O group in question, did not
increase the amplitude relative to that of the Asp
band
(not shown). Measuring the M minus BR spectrum in
photostationary states at room temperature, which might result in more
efficient proton release, did not increase the amplitude either (not
shown). Thus, the low intensities might originate from some degree of
delocalization of the proton of residue 204 within the extracellular
hydrogen-bonded network. The greater intensity of the band in
Asp
than that in Glu
might originate,
likewise, from better localization of the proton on the aspartate than
on the glutamate. However, we cannot exclude the possibility that the
proton is localized fully on Glu
, but the intensity of
the C=O stretch band is lower than expected for some other
(unknown) reason.
In the region of the O-H stretch of water,
the negative band of the wild type at 3643 cm is
replaced in E204Q with a band shifted to a lower frequency, at 3638
cm
(Fig. 5). The water that changes its
hydrogen bonding is therefore more strongly bound in this mutant, as in
D212N (Kandori et al., 1995). Replacing Glu
with
aspartic acid preserved the higher frequency, although a second weaker
depletion band of unknown origin at a lower frequency appeared also (Fig. 5C).
Figure 5: M minus BR difference spectra in the water O-H stretch region for wild type bacteriorhodopsin and E204Q and E204D measured with FTIR.
We report here that proton release at the extracellular
surface of bacteriorhodopsin does not occur at its normal time in the
photocycle when Glu is replaced with a glutamine. The
influence of this mutation is through removing the carboxyl group,
because proton release is normal in E204D. Glu
is located
near the extracellular surface (Henderson et al., 1990), and
in a structural model from molecular dynamics calculations (Humphrey et al., 1994), it is connected by a chain of three
hydrogen-bonded water molecules to Arg
in the cluster of
residues that directly participate in the counter-ion to the Schiff
base. In considering the possible role of Glu
in the
proton release, it is instructive therefore to recall what is known
about the hydrogen bonding of water in this region during the
photocycle.
The properties of the water near Asp,
detected in the L and M states by FTIR, can be summarized as follows.
In the wild type unphotolyzed protein it is weakly
hydrogen-bonded (O-H stretch at 3643 cm
) but
becomes somewhat more strongly bonded (shift to 3636-3638
cm
) when either Asp
(Kandori et
al., 1995) or Glu
(Fig. 5) is replaced. It
becomes much more strongly hydrogen-bonded in the L intermediate of the
wild type protein (shift to a diffuse region between 3460 and 3560
cm
). These bands of the O-H stretch are
eliminated when Asp
is replaced (Maeda et al.,
1994), suggesting that either Asp
participates in the
increased hydrogen bonding or possibly this water is absent altogether
in the D85N mutant. The extent of the shift is greatly reduced when
Asp
is replaced with asparagine (Kandori et al.,
1995), suggesting that Asp
is an important participant in
the stronger hydrogen bonding. Therefore, it seems likely that water is
held in a hydrogen-bonded network by Arg
,
Asp
, Glu
, and Asp
, and this
network in the unphotolyzed state is characterized by a balance of
opposing hydrogen bonds. After photoexcitation the water becomes more
strongly bound to Asp
and creates the conditions in the L
state for the role of Asp
as proton acceptor to the Schiff
base in the L to M reaction and for the subsequent release of a proton
to the surface.
Arg and Glu
have
different functions from Asp
and Asp
in this
network. When Asp
or Asp
is replaced, the
complex in L becomes nonfunctional for proton transfer and the Schiff
base remains protonated. Replacing Arg
or
Glu
redistributes the hydrogen bonds of bound water to
favor the remaining residues and accelerates rather than abolishes the
proton transfer from Schiff base to Asp
. However, the
release of a proton to the surface cannot occur at this time in the
photocycle when Arg
or Glu
is replaced. This
does not necessarily identify either of these residues as the group XH,
the source of the proton, however. There has never been any direct
evidence that the origin of the released proton is Arg
itself, and we had estimated (Brown et al., 1993) its
pK
in the photocycle as much higher than that of
XH. A normal proton release in the photocycle of D212N/R82Q (Brown et al., 1995b) argues that Arg
is not XH. On the
other hand, we find not only that proton release depends on Glu
but that Glu
is protonated in BR and deprotonated
in the M state. It appears therefore that Glu
is XH, and
the proton to be released in the M state is either localized entirely
on this carboxyl group or to some degree delocalized in a
hydrogen-bonded network that contains also water.
Although much
closer to the aqueous interface than the residues near the Schiff base
(Henderson et al., 1990), Glu can probably
fulfill the requirements we had set earlier for XH
(Zimányi et al., 1992). Because it is
the source of the released proton, its pK
should
be high in the unphotolyzed protein but decrease to about 6 in the
photocycle. Molecular dynamics and electrostatic calculations have
estimated the pK
of Glu
as 13 before
photoexcitation but 6-8 in the M state (Scharnagl et
al., 1995). We therefore suggest the following sequence of events
after photoisomerization of the retinal. First, the Schiff base proton
is transferred to the anionic D85. Second, the hydrogen bond between
Arg
and the now neutral Asp
is broken, and
the water dipoles in the network redistribute so as to transfer some of
the positive charge of Arg
toward Glu
.
Although the location of Arg
in the model from electron
diffraction is ambiguous (Henderson et al., 1990), in several
structural models based on molecular dynamics (Humphrey et
al., 1994; Scharnagl et al., 1995), Arg
does
assume the position between Asp
and Glu
required for this. Alternatively, the side chain of
Arg
could move from Asp
toward
Glu
. Third, Glu
loses its proton to the
surface and the aqueous phase. This sequence would account for the so
far poorly understood events that result in proton release in the
photocycle.