The proton release group of bacteriorhodopsin controls the rate of the final step of its photocycle at low pH

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.     

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.     

Time-resolved FT-IR spectroscopic investigation of the pH-dependent proton transfer reactions in the E194Q mutant of bacteriorhodopsin

The photoreaction of the E194Q mutant of bacteriorhodopsin has been investigated at various pH values by time-resolved step-scan Fourier-transform infrared difference spectroscopy employing the attenuated total reflection technique. The difference spectrum at pH 8.4 is comparable to the N-BR difference spectra of the wild type with the remarkable exception that D85 is deprotonated. Since the retinal configuration is not perturbed by the E194Q mutation, it is concluded that there is no interaction of D85 with retinal during the lifetime of the N state. At pH 6, a consecutive state to the O intermediate is detected in which D212 is transiently protonated. The comparison with wild-type bacteriorhodopsin reveals that protonation of D212 represents an intermediate step during proton transfer from D85 to the proton release group in the final stage of the reaction cycle. The described effects are more pronounced in the E194Q mutant than in the E204Q mutant demonstrating different roles of these two glutamates/glutamic acids at least in the final stages of the catalytic cycle 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.     

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.     

Photocycle in the M-form in bacteriorhodopsin mutants devoid of primary proton acceptor Asp-85

Photoinduced changes in absorption of the deprotonated M-form in the mutant bacteriorhodopsin without primary proton acceptor Asp-85 were studied and additional evidence in support of the complete transmembrane proton transfer in photocycle was obtained. Measurements of the absorption spectrum were carried out at various pH, temperature, and humidity. The direction of proton transfer was the same as in the normal photocycle of the wild-type bacteriorhodopsin: from the internal to the external side of the membrane. The effect on this process of a terminal acceptor Glu-204 was shown.     

Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).     

Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel class of amide hydrolase, which utilizes an Fe2+ ion to effect the hydrolysis of an amide bond. The ferrous ion is tetrahedrally coordinated by two histidines from a conserved HEXXH motif, a cysteine, and a water molecule. In this work, the function of the conserved glutamate (Glu-133 in Escherichia coli PDF) is evaluated by difference FTIR spectroscopic analysis of a Co(II)-substituted E. coli wild-type and E133D mutant PDF. At pH <6, the wild-type enzyme exhibited a relatively sharp C=O stretch band at 1742 cm(-1), which is assigned to the COOH group of Glu-133. The pH titration study and curve fitting to the data revealed a pK(a) of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only approximately 10-fold less active than the wild-type enzyme, a similar pH titration study of the Asp-133 C=O stretch band at 1740 cm(-1) revealed a pK(a) of 10.1. This unusually high pK(a) for a carboxyl group is likely due to its hydrophobic environment and electrostatic repulsion from the metal-bound hydroxide. These results argue that in the active form of E133D PDF, Asp-133 is protonated and therefore acts as a general acid during the decomposition of the tetrahedral intermediate by donating a proton to the leaving amide ion perhaps through a water molecule in the cavity created by the E133D mutation. In contrast, Glu-133 is deprotonated in the active form of wild-type PDF. We propose that Glu-133 acts as a proton shuttle accepting a proton from the metal-bound water and subsequently acts as a general acid during the decomposition of the tetrahedral intermediate.     

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.     

Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96]

It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.     

Structure Changes upon Deprotonation of the Proton Release Group in the Bacteriorhodopsin Photocycle

In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.     

Origins of deuterium kinetic isotope effects on the proton transfers of the bacteriorhodopsin photocycle

Deuterium kinetic isotope effects (KIE) were measured, and proton inventory plots were constructed, for the rates of reactions in the photocycles of wild-type bacteriorhodopsin and several site-specific mutants. Consistent with earlier reports from many groups, very large KIEs were observed for the third (and largest) rise component for the M state and for the decay of the O state, processes both linked to proton transfers in the extracellular region. The proton inventory plots (ratio of reaction rates in mixtures of H(2)O and D(2)O to that in H(2)O vs mole fraction of D(2)O) were approximately linear for the first and second M rise components and for M decay, as well as for O decay, indicating that the rates of these reactions are limited by simple proton transfer. Uniquely, the third rise component of M (and in the D96N mutant also a fourth rise component) exhibited a strongly curved proton inventory plot, suggesting that its rate, which largely accounts for the rate of deprotonation of the retinal Schiff base, depends on a complex multiproton process. This curvature is observed also in the E194Q, E204Q, and Y57F mutants but not in the R82A mutant. From these findings, and from the locations of bound water in the extracellular region in the crystal structure of the protein [Luecke, Schobert, Richter, Cartailler, and Lanyi (1999) J. Mol. Biol. 291, 899-911], we suspect that the effects of deuterium substitution on the formation of the M state originate from cooperative rearrangements of the extensively hydrogen-bonded water molecules 401, 402, and 406 near Asp-85 and Arg-82.     

Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.     

Photochemical reaction cycle and proton transfers in Neurospora rhodopsin.

It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterologous expression in Pichia pastoris. We report on spectroscopic properties of the Neurospora rhodopsin (NR). The photocycle was studied with flash photolysis and time-resolved Fourier-transform infrared spectroscopy in the pH range 5-8. Proton release and uptake during the photocycle were monitored with the pH-sensitive dye, pyranine. Kinetic and spectral analysis revealed six distinct states in the NR photocycle, and we describe their spectral properties and pH-dependent kinetics in the visible and infrared ranges. The phenotypes of the mutant NR proteins, D131E and E142Q, in which the homologues of the key carboxylic acids of the light-driven proton pump bacteriorhodopsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be. This implies that, if the NR photocycle is associated with proton transport, it has a low efficiency, similar to that of haloarchaeal sensory rhodopsin II. Fourier-transform Raman spectroscopy revealed unexpected differences between NR and bacteriorhodopsin in the configuration of the retinal chromophore, which may contribute to the less effective reprotonation switch of NR.     

Correlation of the O-intermediate rate with the pKa of Asp-75 in the dark, the counterion of the Schiff base of Pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR(M)) and O-intermediates (ppR(O)). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR(O) of the triple mutant was accelerated approximately 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR(O) of mutants (11 species) and those of the wild-type were well-correlated with the pK(a) value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.     

Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.     

Arginine-82 regulates the pKa of the group responsible for the light-driven proton release in bacteriorhodopsin.

In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85 microseconds in the wild type to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. 6) In the E204Q mutant only late proton release is observed at both neutral and alkaline pH, consistent with the idea that E204 is the proton release group. The proton release is concurrent with the O-->bacteriorhodopsin transition, as in R82Q at neutral pH.     

Coupling of the reisomerization of the retinal, proton uptake, and reprotonation of Asp-96 in the N photointermediate of bacteriorhodopsin

In the N to O reaction of the bacteriorhodopsin photocycle, Asp-96 is protonated from the cytoplasmic surface, and coupled to this, the retinal isomerizes from 13-cis,15-anti back to the initial all-trans configuration. To dissect the two steps, and to better understand how and why they occur, we describe the properties of two groups of site-specific mutants in which the N intermediate has greatly increased lifetime. In the first group, with the mutations near the retinal, an unusual N state is produced in which the retinal is 13-cis,15-anti but Asp-96 has a protonated carboxyl group. The apparent pK(a) for the protonation is 7.5, as in the wild-type. It is likely that here the interference with N decay is the result of steric conflict of side-chains with the retinal or with the side-chain of Lys-216 connected to the retinal, which delays the reisomerization after protonation of Asp-96. In the second group, with the mutations located near Asp-96 or between Asp-96 and the cytoplasmic surface, reprotonation of Asp-96 is strongly perturbed. The reisomerization of the retinal occurs only after recovery from a long-living protein conformation in which reprotonation of Asp-96 is either entirely blocked or blocked at low pH.     

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.