Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.

The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.     

Fourier transform infrared spectra of a late intermediate of the bacteriorhodopsin photocycle suggest transient protonation of Asp-212.

We measured time-resolved difference spectra, in the visible and the infrared, for the Glu-194 and Glu-204 mutants of bacteriorhodopsin and detected an anomalous O state, labeled O', in addition to the authentic O intermediate, before recovery of the initial state in the photocycle. The O' intermediate exhibits prominent bands at 1712 cm(-1) (positive) and 1387 cm(-1) (negative). These bands arise with the same time constant as the deprotonation of Asp-85. Both bands are shifted to lower frequency upon labeling of the protein with [4-(13)C]aspartic acid. The former band, but not the latter, is shifted in D2O. These shifts identify the two bands as the carboxyl stretch of a protonated aspartic acid and the symmetric carbonyl stretch of an unprotonated aspartate, respectively, and suggest that in O' an initially anionic aspartate enters into protonation equilibrium with Asp-85. Elimination of the few other candidates, on various grounds, identifies Asp-212 as the unknown residue. It is possible, therefore, that in the last step of the photocycle of the mutants studied the proton released from Asp-85 is conducted to the extracellular surface via Asp-212. An earlier report of a weak band at 1712 cm(-1) late in the wild-type photocycle [Zscherp and Heberle (1997) J. Phys. Chem. B 101, 10542-10547] suggests that Asp-212 might play this role in the wild-type protein also.     

pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy.

Previous C13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pKa of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pKa = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.     

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.     

Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation

We have individually replaced all 7 of the arginine residues in bacteriorhodopsin by glutamine. The mutants with substitutions at positions 7, 164, 175, and 225 showed essentially the wild-type phenotype in regard to chromophore regeneration, chromophore lambda max, and proton pumping, although the mutant Arg-175----Gln showed decreased rate of chromophore regeneration. Glutamine substitutions of Arg-82, -134, and -227 affected proton pumping ability, and caused specific alterations in the bacteriorhodopsin photocycle. Finally, electrostatic interactions are proposed between Arg-82 and -227, and specific carboxylic acid residues in helices C and G, which regulate the purple to blue transition and proton transfers during the photocycle.     

Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M.

Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.     

Structural changes in bacteriorhodopsin during the photocycle measured by time-resolved polarized Fourier transform infrared spectroscopy.

The structural changes in bacteriorhodopsin during the photocycle are investigated. Time resolved polarized infrared spectroscopy in combination with photoselection is used to determine the orientation and motion of certain structural units of the molecule: Asp-85, Asp-96, Asp-115, the Schiff base, and several amide I vibrations. The results are compared with recently published x-ray diffraction data with atomic resolution about conformational motions during the photocycle. The orientation of the measured vibrations are also calculated from the structure data, and based on the comparison of the values from the two techniques new information is obtained: several amide I bands in the infrared spectrum are assigned, and we can also identify the position of the proton in the protonated Asp residues.     

High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4-13C]Asp resonances.

Solid state 13C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with [4-13C]Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues. (2) Relaxation studies and T1 filtering allow one to discriminate between resonances with different mobility. (3) A comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of the chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp96 and Asp115). All other detected resonances, including Asp85 and Asp212, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected.     

Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212

Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.     

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point-mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild-type BR, the M formation in BR Asp85---Glu is accwelerated approximately 10-fold, whereas the M decay in BR Asp96---Asn is slowed down approximately 50-fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85---Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96---Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.     

The retinylidene Schiff base counterion in bacteriorhodopsin

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M----N transition

The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96----Ala and Asp-96----Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96----Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L----M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N----O or O----bR transition.     

Structural investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.     

Understanding structure and function in the light-driven proton pump bacteriorhodopsin

The atomic structure of bacteriorhodopsin and the outlines of its proton transport mechanism are now available. Photoisomerization of the retinal in the chromophore creates a steric and electrostatic conflict at the retinal binding site. The free energy gain sets off a sequence of reactions in which directed proton transfers take place between the protonated retinal Schiff base, Asp-85, and Asp-96. These internal steps, and other proton transfers at and near the two aqueous interfaces, add up to the translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Bound water plays a crucial role in proton conduction in both extracellular and cytoplasmic regions, but the means by which the protons move from site to site differ. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. The directionality of the translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96.     

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.     

Anion binding to the Schiff base of the bacteriorhodopsin mutants Asp-85----Asn/Asp-212----Asn and Arg-82----Gln/Asp-85----Asn/Asp-212----Asn.

Studies of bacteriorhodopsin have indicated that the charge environment of the protonated Schiff base consists of residues Asp-85, Asp-212, and Arg-82. As shown recently (Marti, T., R?sselet, S. J., Otto, H., Heyn, M. P., and Khorana, H. G. (1991) J. Biol. Chem. 266, 18674-18683), in the double mutant Asp-85----Asn/Asp-212----Asn chromophore formation is restored in the presence of salts, suggesting that exogenous anions function as counterions to the protonated Schiff base. To investigate the role of Arg-82 and of the Schiff base in anion binding, we have prepared the triple mutant Arg-82----Gln/Asp-85----Asn/Asp-212----Asn and compared its properties with those of the Asp-85----Asn/Asp-212----Asn double mutant. Regeneration of the chromophore with absorption maximum near 560 nm occurs in the triple mutant in the presence of millimolar salt, whereas in the double mutant molar salt concentrations are required. Spectrometric titrations reveal that the pKa of Schiff base deprotonation is markedly reduced from 11.3 for the wild type to 4.9 for the triple mutant in 1 mM NaCl and to 5.5 for the double mutant in 10 mM NaCl. In both mutants, increasing the chloride concentration promotes protonation of the chromophore and results in a continuous rise of the Schiff base pKa, yielding a value of 8.4 and 7.6, respectively, in 4 M NaCl. The absorption maximum of the two mutants shows a progressive red shift, as the ionic radius of the halide increases in the sequence fluoride, chloride, bromide, and iodide. An identical spectral correlation in the presence of halides is observed for the acid-purple form of bacteriorhodopsin. We conclude, therefore, that upon neutralization of the two counterions Asp-85 and Asp-212 by mutation or by protonation at low pH, exogenous anions substitute as counterions by directly binding to the protonated Schiff base. This interaction may provide the basis for the proposed anion translocation by the acid-purple form of bacteriorhodopsin as well as by the related halorhodopsin.     

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5-7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple-blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H(+). At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.     

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current-voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.     

Functional roles of aspartic acid residues at the cytoplasmic surface of bacteriorhodopsin.

The functions of the four aspartic acid residues in interhelical loops at the cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and Asp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff base and Asp-96 about 3 times more rapid, and events associated with the reisomerization of retinal to all-trans about 3 times slower. Asp-36 has the opposite effect on these processes, but to a smaller extent. Asp-102 and Asp-104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface. In the 13 bacterioopsin sequences now available, only this surface aspartate is conserved.