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Biophys J, February 2000, p. 967-976, Vol. 78, No. 2
and
*Max-Planck-Institut für Molekulare Physiologie,
D-44227 Dortmund, Germany; and Max-Planck-Institut
für Biophysik, D-60596 Frankfurt/Main, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The phototaxis of halophilic archaea is mediated
by two receptors, the sensory rhodopsins I and II (SRI and SRII), which
are responsible for positive and negative responses of the bacteria toward light (for a recent review see, e.g., Spudich, 1998
). SRI and
SRII are membrane proteins with seven transmembrane spanning helices
and all-trans retinal bound to the opsin. On light
excitation the archaeal sensory rhodopsins undergo a photocycle during
which the physiological reaction is triggered. Sensory rhodopsins from Halobacterium salinarum have been sequenced (Blanck et al.,
1989; Zhang et al., 1996) and in the case of the photophobic receptor also from the phylogenetically distinct Natronobacterium
pharaonis (pSRII) (Seidel et al., 1995). The biochemical and
physiological properties of pSRII are quite similar to those of SRII
from H. salinarum as demonstrated in measurements of the
photocycle (Scharf et al., 1992) and in their physiological response
toward blue-green light (Scharf and Wolff, 1994). The signal
transduction (reviewed in Marwan and Oesterhelt, 1999) is homologous to
the eubacterial chemotactic so-called two-component system (reviewed
in, e.g., Falke et al., 1997) in which the incoming signal triggers the activation of a cytoplasmic His kinase (CheA) from which the
information is transferred to response regulators (e.g., CheY, CheB).
Whereas the chemotactic receptors transfer an incoming signal directly to their cytoplasmic domains, SRI and SRII form complexes with their
corresponding transducers (halobacterial transducer of rhodopsin, Htr)
(Ferrando-May et al., 1993; Lüttenberg et al., 1998; Sasaki and
Spudich, 1998; Yao et al., 1994).
The amino acid sequence of the photophobic receptor pSRII is quite
homologous to that of bacteriorhodopsin (BR) (Seidel et al.,
1995). Residues involved in the proton release, such as Asp-85-BR or
Arg-82-BR, are also present in the pSRII sequence. However, potentially charged amino acids such as Asp-96-BR, located in the
cytoplasmic channel of the molecule, are not found. Furthermore, Asp
residues on the cytoplasmic surface of BR are replaced by neutral amino
acids in pSRII. Generally, the extracellular channels are quite
homologous (with the exception of E194, which is replaced by P183 in
pSRII). However, the cytoplasmic channel of pSRII has lost most of the
amino acids that are known to facilitate the reprotonation of the
Schiff base. Consequently, one can predict functional differences
between the two pigments that are related to the second part of the
reaction cycle. Indeed, a decreased rate of reprotonation of the Schiff
base has been verified in recent work on the photocycle of pSRII
(Chizhov et al., 1998).
Numerous publications on the mechanism of the proton pump in BR
revealed the decisive participation of BR-Asp-85 and BR-Asp-96 in the
proton transfer. These residues fulfill steering tasks, thereby
increasing the photocycle turnover and the proton pump efficiency.
Currently there is not a single mutant known that blocks the proton
pump of BR entirely. Therefore, it is probable that the sensory
rhodopsins can also pump protons. This has already been verified for
SRI, although attached to its transducer the efficiency is abolished
(Bogomolni et al., 1994; Spudich, 1995) or at least reduced (Haupts et
al., 1996). The capability to pump protons has not yet been rigorously
proven for SRII or pSRII. According to Sasaki and Spudich (1999) SRII
takes up a proton from the extracellular side during the M 
O
transition and releases it concomitantly with the O-decay to the same
side. This timing and location of proton transfer steps leads to a
futile proton cycle. These data have to be reconciled to the
observation that in pSRII Asp-75 is protonated probably by the proton
of the Schiff base during the L M transition (Engelhard et al.,
1996).
In the present work we describe the light-activated proton transfer
reactions of pSRII and its channel-mutants D75N-pSRII and F86D-pSRII,
which had been attached to black lipid membranes (BLM) (Bamberg et al.,
1979). It can be shown that pSRII and F86D-pSRII can function as proton
pumps and that the addition of external proton donors such as azide or
imidazole increases the stationary photocurrent considerably.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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All reagents used were of analytical grade.
Expression in H. salinarum
For the expression of pSRII and its mutants in H. salinarum a strain (Pho81/w) was used whose genes for BR, HR, SRI,
SRII, and the transducers HtrI and HtrII are
defective (Olson and Spudich, 1993; Spudich and Spudich, 1993). Based
on the shuttle vector pUSNovo, an expression vector pBL7Novo was
designed in which the coding sequence of pSRII started downstream of
the precursor sequence of bop, including the first two amino
acids of the mature BR. H. salinarum Pho81/w was transformed
essentially using the method of Cline and Doolittle (1987). The cells
were spread on Novobiocin plates and positive clones were selected and
subsequently genetically characterized by Southern blot analysis. A
transformant containing a double insertion (probably by a double
crossover event) of the psop gene into the bop
gene locus (Pho81/wpSRII) was selected for the isolation of pSRII.
The psopII mutants (psopII-D75N;
psopII-F86D) were prepared by PCR using the
overlap-extension method (Higuchi et al., 1988; Ho et al., 1989).
Escherichia coli cells were transformed by electroporation (Dower et al., 1988).
Expression in E. coli
For DNA manipulation E. coli strain XL1 was used.
Gene expression was carried out in E. coli BL21 (DE3). The
plasmid pET27bmod, derived from pET27b (Novagen, Madison, WI) was used
to construct the expression vectors for the C-terminal 6x His-tagged
pSOPII and F86DpSOPII genes. A detailed description of the cloning
strategy for His-tagged pSOPII inserted between the 5'-NcoI
and 3'-HindIII restriction sites is given in Shimono et al.,
1998 and Hohenfeld et al., 1999. For the site-specific introduction of
the F86D mutation extension-overlap PCR was used as described above (Ho
et al., 1989). The His-tagged pSOPII gene was cloned into the pUCBM20 vector (Boehringer Mannheim, Germany) via
NcoI/HindIII restriction. As a final PCR product
the His-tagged pSOPII gene carrying the F86D mutation was ligated after
restriction back into pET27bmod; pSRII and F86D-pSRII were expressed
according to Shimono et al., 1998 and purified using the method of
Hohenfeld et al., 1999.
Reconstitution into PM lipids
For reconstitution into native purple membrane lipids the solubilized proteins were shaken 16 h in a buffer (20 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH = 8.0) containing a 15-fold molar excess of lipids and detergent-adsorbing biobeads (Biorad Müchen, Germany, SM2, 100 mg/mg protein). After filtration the reconstituted proteins were pelleted by centrifugation at 100,000 × g (1 h, 4°C) and resuspended in 20 mM NaCl, NaH2PO4/Na2HPO4, pH = 7.5).
Laser flash photolysis
The laser flash photolysis setup and the data evaluation were
essentially identical to those described by Chizhov et al., 1996. Laser
flash-induced transient absorption was recorded at 20°C in the
spectral range from 360 to 660 nm in steps of 10 nm. The experimental
data were fitted using a multiexponential least-square fit procedure
(Müller et al., 1991; Müller and Plesser, 1991).
Photocurrent measurements
Black lipid membranes with an area of
10
2 cm2 were formed in a
Teflon cell filled with the appropriate electrolyte solution (1.5 ml in
each compartment). The membrane forming solution contained 1.5% (w/v)
diphytanoyllecithin (Avanti Biochemicals, Birmingham, AL) and 0.025%
(w/v) octadecylamine (Riedel-de Haen, Hannover, Germany) in n-decane to
obtain a positively charged membrane surface (Dancshazy and Karvaly,
1976). Membrane formation was controlled by eye and the capacitance of
each individual membrane was determined.
Membrane fractions containing pSRII or its mutants were suspended in
distilled water and sonicated for 1 min in a sonication bath. Aliquots
of 30 µl were added under stirring to the rear compartment of the
cell containing the appropriate buffer. Photosensitivity (photocurrents) developed in time and reached a maximal and constant value after ~40 min. As light sources, a xenon lamp (100 W) or mercury lamp (100 W) were used. The intensity of the lamps in the plane
of the membrane were 2 W/cm2 and 4 W/cm2, respectively. Light reached the membrane
after passing a heat protection filter. For "white" or "yellow"
light cutoff filters 
> 360 nm or > 495 nm (Schott,
Mainz, Germany) respectively, were used. A K40 broadband interference
filter served for "blue" light excitation (maximal intensity: 2 W/cm2). Light intensity was measured as described
previously (Fendler et al., 1987). To obtain stationary currents the
black lipid membrane was doped with the light-insensitive protonophores
1799 in combination with the Na+,
K+/H+ exchanger monensin.
Further details of the system were described earlier (Bamberg et al.,
1979). It should be noted that the BLM experiments, which were done
with both E. coli- and H. salinarum-expressed proteins, led to the same results.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Photocycle kinetics of pSRII, D75N-pSRII, and F86D-pSRII
Photocycle kinetics
In Fig. 1 the light-activated transient absorption changes of pSRII, F86D-pSRII, and D75N-pSRII are depicted at representative wavelengths. At 500 nm the depletion and reformation of the initial state of the pigments is monitored. The traces at 400 nm and 550 nm are indicative for the rise and decay of the M400 and O550 intermediates, respectively. The turnover rates of the photocycles of pSRII and F86D-pSRII are quite similar and the reaction cycles are completed in the range of seconds. Compared with BR, this slow cycling rate reflects the physiological function of pSRII as a photoreceptor (for a detailed analysis of the pSRII photocycle see Chizhov et al., 1998). Whereas the deprotonation (M400 formation) is accomplished similarly to BR in ~200 µs, the second part, which depicts the reprotonation of the Schiff base, is slowed down by about
two orders of magnitude.
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).
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; Zhu et al., 1997). Another effect of
this mutation concerns the photocycle (Fig. 1 C). Faster
than the dead time of the instrumental setup (200 ns), an intermediate
appears which is stable over a time interval of ~10 ms. It decays
back to the ground state with 
2 = 28 ms. No
M-like intermediate could be detected. The global fit analysis and a
modeling of the data to a scheme of irreversible first-order reactions
reveals only two spectrally defined species (see Chizhov et al., 1996,
1998) for a detailed description of the data evaluation procedure). The
first intermediate has an absorption maximum at 565 nm (Fig.
2 A) and decays with a
half-life of 1 = 7 ms. The product of this
reaction absorbs maximally at 520 nm (Fig. 2 B). A shoulder
is clearly visible on the long wavelength side of the absorption
maximum. A Gaussian fit of the spectrum provides two species absorbing
maximally at ~565 nm (X565) and 520 nm
(Y520). The two peaks can be explained by a
mixture of two rapidly equilibrating species. The rates of the forward
and back reactions of this equilibrium must be fast compared with the
rate of reformation (2 = 28 ms) of the
original resting state. The photocycle of D75N-pSRII may then be
described by the following scheme:
![<UP>pSRII</UP><SUP><UP>D75N</UP></SUP><SUB>520</SUB> <LIM><OP><ARROW>→</ARROW></OP><LL><SUB><200 ns</SUB></LL><UL><SUB>hv</SUB></UL></LIM> <UP>K</UP><SUB>565</SUB> <LIM><OP><ARROW>→</ARROW></OP><UL><SUB>7 ms</SUB></UL></LIM>[<UP>X</UP><SUB>565</SUB> <LIM><OP><ARROW>↔</ARROW></OP><UL><SUB>fast</SUB></UL></LIM> <UP>Y</UP><SUB>520</SUB>]<LIM><OP><ARROW>→</ARROW></OP><UL><SUB>28 ms</SUB></UL></LIM> <UP>pSRII</UP><SUP><UP>D75N</UP></SUP><SUB>520</SUB>](/content/vol78/issue2/fulltext/967/img001.gif)
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L or an N O equilibrium. Which of these
assignments or other possibilities turns out to be correct has to be
seen. It should be noted that the thermal relaxations of the
corresponding BR mutant (D85N-BR) proceed from K to L and N before
reforming the initial state (Braiman et al., 1988; Needleman et al.,
1991).
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Effect of external proton donors on the photocycle kinetics of pSRII and F86D-pSRII
The transient absorption changes of pSRII at different concentrations of azide are illustrated in Fig. 3. As reported previously (Miyazaki et al., 1992; Takao et al., 1998) the experiments show that azide only
accelerates the reprotonation of the Schiff base without having an
influence on the M-formation or the overall turnover rate.
Consequently, the decay of O550 denotes the
rate-limiting step of the photocycle. The fast M-decay and the
unaltered other rate constants are the reason for the accumulation of
the O-intermediate at higher azide concentrations, as can be depicted
from the traces at 550 nm. Similar results were obtained for the mutant
F86D (data not shown). However, the BR mutant D96N-BR responds
differently to azide. Not only the M-decay is significantly
accelerated, but also the turnover rate (Tittor et al., 1989). It
should be noted that the azide effect is pH-dependent (Takao et al.,
1998). At a low pH ~ 4.7, azide enhances the reprotonation of
the Schiff base, indicating that HN3 rather than
N3 is the active species.
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reported a similar observation
for SRI from H. salinarum. Imidazole also had no influence on the decay of the SRI373 (M)-intermediate, but
the M-decay of the H166A mutant is 10-fold, accelerated upon the
addition of 10 mM imidazole. The authors conclude that His-166 plays a
critical role in the protonation pathways of SRI and therefore
imidazole might mimic His-66. However, in pSRII the corresponding
residue (Leu) is not conserved, which points to a more direct
participation of imidazole in the proton transfer process.
Photocurrent measurements of pSRII, D75N-pSRII, and F86D-pSRII
Photocurrent measurements of pSRII and D75N-pSRII
The photocurrents (excitation wavelength > 495 nm,
green) of pSRII are shown in Fig. 4 at
different conditions. At neutral pH (Fig. 4 A) only a
transient downward deflection can be detected, which corresponds to a
movement of positive charges toward the protein-free side of the BLM.
It is the same direction as that observed for BR (Fahr et al., 1981) or
D96N-BR (Tittor et al., 1989), which indicates (assuming a preferential
attachment of the extracellular side of pSRII to the BLM, as was found
for BR) that the charge displacement has the same vectoriality in BR
and pSRII, namely from the cytoplasm to the external medium. The sign of the transient current is in agreement with the
deprotonation of the Schiff base (formation of M) and the
protonation of Asp-75. At this pH no photostationary current is
observed.
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; see Table
1 for the photocycle data at pH 6.8). Also, the kinetic data at pH 5.5 are almost identical, with the exception of 7
(O-decay), which decreases by a factor of 2. Unfortunately, the
evaluation of the photocycle data at lower pH values is impaired
because Asp-75 becomes protonated (pKa = 5.6), which leads to a photocycle (data not shown) similar to that of
the mutant D75N-pSRII (see above). Taking the decrease of
7 between pH 6 and pH 5.5 into account, it
seems likely that a further increase in the turnover rate at lower pH
4.7 is responsible for the observed small stationary photocurrent,
though the concentration of active species is reduced.
The above considerations imply that at pH 4.7 two species are
photoactivated and may contribute to the transient and/or stationary photocurrent. This is substantiated by a closer inspection of the
transient photocurrent. As can be seen from Fig. 4 B the
onset of the transient photocurrent is a superposition of two signals of opposite sign, which is not seen for pSRII at pH 6.8 (Fig. 4
A), indicating the presence of at least two species. The
upward deflection corresponds to the transient current obtained with the mutant D75N (Fig. 4 D).
The stationary photocurrent of the pSRII wild type can be considerably
enhanced by the addition of azide (30 mM) and reaches values of ~40
nA/cm2 (Fig. 4 C). A 10-fold increase
of the steady-state signal can already be observed at a concentration
of 1 mM azide, and saturates at 50-100 mM azide with an 
70-fold
larger amplitude. From this saturation curve an apparent binding
constant can be estimated (Km = 7 mM).
A similar increase of the steady-state photocurrent is also measured at
higher pH values, though for the same enlargement the azide
concentration has to be increased.
Replacement of Asp-75 as the acceptor for the Schiff base proton by Asn
inverts the direction of the transient photocurrent (Fig. 4
D), whereas a photostationary current was not observed. The
corresponding BR mutant D85N (Ganea et al., 1998; Tittor et al., 1995)
and SRI (Haupts et al., 1996), when illuminated with red light (>630
nm), similarly display transient photocurrents that are directed toward
the cytoplasmic side. However, in contrast to the D85N-BR mutant,
D75N-pSRII responses quite different to blue light in the presence of
azide (100 mM azide at pH 4.7). Under these conditions a very small
inwardly directed photostationary current develops and not, as observed
for the BR-mutant, an outwardly directed charge movement. This former
observation has also been described for SRI590,
which can be selectively excited by red light (Haupts et al., 1996).
In pSRII typical blue-light quenching of the photocurrent is observed
(Fig. 5 A) which is much
better resolved in the presence of 10 mM azide (Fig. 5 B).
This inhibition of the proton transfer has originally been described in
BR and has been explained by the photoexcitation of the M-intermediate
with a second photon that short-circuits the proton pumping cycle
(Ormos et al., 1978). Control experiments at pH 6.8 with the addition
of azide gave similar results (data not shown). These experiments
clearly demonstrate that pSRII, like BR, functions as an
outward-directed proton pump. As in BR, the long-lived M-intermediate
can be photochemically reverted back to the initial ground state,
resulting in an inhibition of the photostationary current.
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= 380-420 nm) becomes more and more inefficient for quenching. The absorption spectrum of pSRII has considerable extinction in this region
(Chizhov et al., 1998) so that its photocycle can also be triggered by
blue light. Since the flux of photons is constant throughout the
experiments, these two effects could result in an enhancement of the
photostationary current by blue light.
Photocurrent of the mutant F86D-pSRII
The photocurrent measurements of F86D-pSRII are shown in Fig. 6. At neutral pH illumination of the sample with green light generates an outwardly directed transient current that is followed by a stationary current in the same direction (Fig. 6 A). The signal unequivocally proves that the incorporation of a carboxyl group into the cytoplasmic channel (Asp in position 86) is sufficient to generate proton pump activity.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The amino acid sequence of pSRII is quite similar to that of BR,
however notable exceptions are especially found in the cytoplasmic proton channel. Taking recent x-ray diffraction data of BR (Belrhali et
al., 1999; Essen et al., 1998; Luecke et al., 1999; Pebay-Peyroula et
al., 1997) into account, it is obvious that residues of the cytoplasmic
proton pathway are not found in pSRII. From five polar residues
possibly involved in proton transfer only three are altered to neutral
amino acids, namely D38, T46, and D96. However, residues of the
extracellular channel are almost all (with the notable exception of
E194) conserved. The photocycle data of wild-type pSRII clearly reflect
this pattern (see Table 1; Chizhov et al., 1998). A similar behavior is
found in BR mutants in which Asp-96 is replaced by a neutral amino acid
(e.g., D96N-BR; Tittor et al., 1989). Although the time courses are
quite similar, the reformations of the ground states are following
different pathways. Whereas in D96N-BR the M-decay is greatly
simplified without any transient accumulation of N and O species (Nagle
et al., 1995; Zimányi et al., 1999), these latter intermediates
can readily be detected in pSRII (Chizhov et al., 1998).
Comparing the response of pSRII and D96N-BR toward azide, another
striking difference becomes apparent. Although azide catalyzes the
reprotonation of the Schiff base in D96N-BR as well as in pSRII, the
reformation of the original ground state is not accelerated in the
latter pigment. Moreover, as is apparent at increasing azide
concentrations, the reprotonation of the Schiff base is considerably
accelerated. However, reactions within the protein that finally lead
back to the resting state are not at all influenced. Obviously, the
positive charge of the Schiff base seems to influence neither the
kinetics of the cis-trans isomerization of retinal or the
deprotonation of Asp-75, which is still protonated in the O-intermediate (F. Siebert, personal communication) nor the timing of
conformational changes that restore the original ground state of pSRII.
Another trigger has to reestablish the initial state. Such a crucial
step could be, e.g., the deprotonation of Asp-75. It should be noted
that the analogous SRII mutant from H. salinarum is
constitutively active (Spudich et al., 1997). Taking these data into
account, one could speculate that the neutralization of Asp-75 is an
important factor to produce the signaling state of the photophobic
receptor. It will be interesting to analyze the charge transfer
properties of this SRII mutant and to study the physiological
properties of D75N-pSRII.
At pH 6.8 a stationary photocurrent, as was demonstrated for BR
under the same conditions, cannot be detected for pSRII. Taking the
amplitude of the transient signal into account, it can be excluded that
the absence of a steady-state photocurrent is due to a low number of
active cycling proteins. Generally, the size of a photostationary
current is dependent on the turnover number of the ion pump (Fahr et
al., 1981). In BR a photostationary current of ~100
nA/cm2 has been measured. Taking the turnover
numbers of BR and pSRII into account one could expect a photocurrent of
~1 nA/cm2 for pSRII, which is close to the
detection limit of the system. Similarly, for D96N-BR, whose monophasic
M-decay is ~500 ms, almost no photostationary current has been found
(Tittor et al., 1989).
Lowering the pH to 4.7 establishes a small but distinct photostationary current. It has already been mentioned that at lower pH the absorption maximum of pSRII shifts from 500 to 520 nm, forming a "pink" membrane, which gives rise to the inwardly directed transient photocurrent. It is therefore unlikely that the "pink" membrane can also transfer protons from the inside to the outside of the cell. It follows that at pH 4.7 only few active species (<25%) of pSRII could contribute to the photostationary current. These active species must have a high turnover number to generate the stationary current. As was shown above, a decrease of the pH from pH 6 to pH 5.5 accelerates the reformation of the initial state by at least a factor of two. Under the assumption that a further increase of the proton concentration (pH 4.7) would additionally shorten the photocycle turnover, the observed stationary photocurrent can be explained. In conclusion, the data of the photostationary and transient currents indicate that pSRII, like D96N-BR, is an outward-directed proton pump.
The kinetic data (Figs. 1 and 3) demonstrated that external proton donors such as azide or imidazole increase the rate of the reprotonation of the Schiff base although the overall duration of the photocycle has not changed. Because the rate-limiting steps in the catalytic cycle are not considerably affected, a photostationary current should not be observed. The discrepancy between photocycle data and the presence of a photostationary current in pSRII (in the presence of azide) and F86D-pSRII poses a problem because the proton pump activity is obviously not dependent on the slow photocycle turnover.
A conceivable mechanism for the proton pump activity of pSRII in the
presence of azide and that of F86D-pSRII might be that the
reprotonation of the Schiff base can occur from both the extracellular and cytoplasmic channel. This model would imply that although the
photocycle turnover is quite slow, the Schiff base in the wild type is
reprotonated from the extracellular side. The mutation from Phe to Asp
in F86D-pSRII, azide, or lowering the pH (assuming that increasing
proton concentrations only affect the reprotonation rate of the Schiff
base from the cytoplasmic, but not the extracellular channel) would
facilitate protonation of the Schiff base from the cytoplasmic channel
(fast equilibrium between two M intermediates; local access model?
(Brown et al., 1998)), thereby increasing the photostationary current.
The photostationary currents measured in the presence of azide would
represent maximal output governed by the photocycle turnover. However,
at neutral pH the reprotonation of the Schiff base would occur in pSRII
from the extracellular channel, thus being unable to generate a
photostationary current. Recent experiments that support this model
indicate that the uptake of protons from the bulk medium (during the M
O transition) precedes the release of protons (Sasaki and Spudich,
1999; N. Kamo, personal communication). Furthermore, Sasaki and Spudich (1999) provide evidence that the proton uptake and release both occur
from the same extracellular side. This observation could explain why a
photostationary current could not be observed in wild-type pSRII at pH
6.8.
The photocurrent results could also be explained by a two-photon
process. Whereas the measurements of the photocycle are single-turnover experiments, the photocurrent is determined under constant illumination (excitation wavelength > 495 nm) of the protein. The (photo)
steady-state mixture of pSRII consists mainly of the M intermediate
(Engelhard et al., 1996), which absorbs maximally at 390 nm (Chizhov et
al., 1998) and is therefore unable to absorb the second photon.
However, if the addition of azide or the mutation F86D would shift the photo-steady-state mixture toward the N- and O-intermediates that appear later in the photocycle and have considerable absorption at
wavelengths above 495 nm, a two-photon process would become possible.
Excitation of the N- and/or O-intermediate would accelerate the
photocycle considerably and the turnover could reach the millisecond range. A similar two-photon process in pSRII at pH 6.8 cannot be
excluded. However, under these conditions the steady-state concentration of N and O is apparently too low (Engelhard et al., 1996)
to contribute significantly to the photostationary current.
These assumptions are corroborated in preliminary flash photolysis experiments on background-illuminated (white light) samples of pSRII and F86D-pSRII at neutral pH. Under these conditions the steady-state concentration of at least the O-intermediate is significantly increased. Additional measurements of FTIR-spectra of pSRII in the presence of azide and of F86D-pSRII under continuous illumination clearly indicate that the steady-state mixtures of both samples contain species with a protonated Schiff base (F. Siebert, personal communication). These data are in line with two-photon processes to explain the proton pump activities of pSRII and its F86D mutant (Table 2). It is interesting to note that the switch separating the cytoplasmic from the extracellular accessibility of the Schiff base must have occurred before the second photon was absorbed. Otherwise, an inhibition of the photocurrent should have been observed.
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The two explanations for the photocurrent behavior of pSRII are not exclusive. It could be possible that mechanism I (proton uptake and release from the same side) holds for wild-type pSRII at neutral pH, whereas mechanism II (two-photon process) is valid in cases where external and/or internal proton donors are present.
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ACKNOWLEDGMENTS |
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We thank M. Kolleck for excellent technical assistance.
The work was supported by Deutsche Forschungsgemeinschaft Grants En87 10-2 (to M.E.) and SFB 479 (to E.B.). G.S gratefully acknowledges a fellowship by the Boeringer Ingelheim Fonds.
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FOOTNOTES |
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Received for publication 6 July 1999 and in final form 4 November 1999.
Address reprint requests to Dr. Martin Engelhard, Max-Planck-Institut für Molekulare Physiologie, Otto Hahn Strasse 11, D-44227 Dortmund, Germany. Tel.: 49-231-1332302; Fax: 49-231-1332399; E-mail: martin.engelhard{at}mpi-dortmund.mpg.de.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.
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8:3477-3482[Abstract]. Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling.
Proc. Natl. Acad. Sci. USA.
96:4414-4419[Abstract/Full Text].
Biophys J, February 2000, p. 967-976, Vol. 78, No. 2
© 2000 by the Biophysical Society 0006-3495/00/02/967/10 $2.00
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