ESR - a retinal protein with unusual properties from Exiguobacterium sibiricum.

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's pKa to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.

Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum.

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.

Xanthorhodopsin: Proton pump with a carotenoid antenna.

Retinal proteins function as photoreceptors and ion pumps. Xanthorhodopsin of Salinibacter ruber is a recent addition to this diverse family. Its novel and distinctive feature is a second chromophore, a carotenoid, which serves as light-harvesting antenna. Here we discuss the properties of this carotenoid/retinal complex most relevant to its function (such as the specific binding site controlled by the retinal) and its relationship to other retinal proteins (bacteriorhodopsin, archaerhodopsin, proteorhodopsin and retinal photoreceptors of archaea and eukaryotes). Antenna addition to a retinal protein has not been observed among the archaea and emerged in bacteria apparently in response to environmental conditions where light-harvesting becomes a limiting factor in retinal protein functioning.

Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.

Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures.

Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.

Protonation reactions and their coupling in bacteriorhodopsin.

Light-induced changes of the proton affinities of amino acid side groups are the driving force for proton translocation in bacteriorhodopsin. Recent progress in obtaining structures of bacteriorhodopsin and its intermediates with an increasingly higher resolution, together with functional studies utilizing mutant pigments and spectroscopic methods, have provided important information on the molecular architecture of the proton transfer pathways and the key groups involved in proton transport. In the present paper I consider mechanisms of light-induced proton release and uptake and intramolecular proton transport and mechanisms of modulation of proton affinities of key groups in the framework of these data. Special attention is given to some important aspects that have surfaced recently. These are the coupling of protonation states of groups involved in proton transport, the complex titration of the counterion to the Schiff base and its origin, the role of the transient protonation of buried groups in catalysis of the chromophore's thermal isomerization, and the relationship between proton affinities of the groups and the pH dependencies of the rate constants of the photocycle and proton transfer reactions.

Relocation of internal bound water in bacteriorhodopsin during the photoreaction of M at low temperatures: an FTIR study.

Changes in the FTIR difference spectra upon photoconversion of the M intermediate to its photoproduct(s) M' were studied in wild-type bacteriorhodopsin and several mutants at low temperatures. The studies aimed at examining whether internally bound water molecules interact with the chromophore and the key residues Asp85 and Asp96 in M, and whether these water molecules participate in the reprotonation of the Schiff base. We have found that three water molecules are perturbed by the isomerization of the chromophore in the M --> M' transition at 80 K. The perturbation of one water molecule, detected as a bilobe at 3567(+)/3550(-) cm(-)(1), relaxed in parallel with the relaxation of an Asp85 perturbation upon increasing temperature from 80 to 100 and 133 K (before the reprotonation of the Schiff base). Two water bands of M at 3588 and 3570 cm(-)(1) shift to 3640 cm(-)(1) upon photoconversion at 173 K. These bands were attributed to water molecules which are located in the vicinity of the Schiff base and Asp85 (Wat85). In the M to M' transition at 80 and 100 K, where the Schiff base remained unprotonated, the Wat85 pair stayed in similar states to those in M. The reprotonation of the Schiff base at 133 K occurred without the restoration of the Wat85 band around 3640 cm(-)(1). This band was restored at higher temperatures. Two water molecules in the region surrounded by Thr46, Asp96, and Phe219 (Wat219) were perturbed in the M to M' transition at 80 K and relaxed in parallel with the relaxation of the perturbation of Asp96 upon increasing the temperature. Mutant studies show that upon photoisomerization of the chromophore at 80 K one of the Wat219 water molecules moves closer to Val49 (located near the lysine side chain attached to retinal, and close to the Schiff base). These data along with our previous results indicate that the water molecules in the cytoplasmic domain participate in the connection of Asp96 with the Schiff base and undergo displacement during photoconversions, presumably shuttling between the Schiff base and a site close to Asp96 in the L to M to N transitions.

The M intermediate of Pharaonis phoborhodopsin is photoactive.

The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halobacteria. Upon absorption of light, the pigment is transformed into a short-wavelength intermediate, M, that most likely is the signaling state (or its precursor) that triggers the motility response of the cell. The M intermediate thermally decays into the initial pigment, completing the cycle of transformations. In this study we attempted to determine whether M can be converted into the initial state by light. The M intermediate was trapped by the illumination of a water glycerol suspension of phoborhodopsin from Natronobacterium pharaonis called pharaonis phoborhodopsin (ppR) with yellow light (>450 nm) at -50 degrees C. The M intermediate absorbing at 390 nm is stable in the dark at this temperature. We found, however, that M is converted into the initial (or spectrally similar) state with an absorption maximum at 501 nm upon illumination with 380-nm light at -60 degrees C. The reversible transformations ppR if M are accompanied by the perturbation of tryptophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (-160 degrees C) reveals two intermediates in the photoconversion of M, which we termed M' (or M'(404)) and ppR' (or ppR'(496)). A third photoproduct, ppR'(504), is formed at -110 degrees C during thermal transformations of M'(404) and ppR'(496). The absorption spectrum of M'(404) (maximum at 404 nm) consists of distinct vibronic bands at 362, 382, 404, and 420 nm that are different from the vibronic bands of M at 348, 368, 390, and 415 nm. ppR'(496) has an absorption band that is shifted to shorter wavelengths by 5 nm compared to the initial ppR, whereas ppR'(504) is redshifted by at least 3 nm. As in bacteriorhodopsin, photoexcitation of the M intermediate of ppR and, presumably, photoisomerization of the chromophore during the M --> M' transition result in a dramatic increase in the proton affinity of the Schiff base, followed by its reprotonation during the M' --> ppR' transition. Because the latter reaction occurs at very low temperature, the proton is most likely taken from the counterion (Asp(75)) rather than from the bulk. The phototransformation of M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M'. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacteria and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II).

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant.

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.

Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp85 in the Arg82 his mutant of bacteriorhodopsin.

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Chromophore-protein-water interactions in the L intermediate of bacteriorhodopsin: FTIR study of the photoreaction of L at 80 K.

Using FTIR spectroscopy, perturbations of several residues and internal water molecules have been detected when light transforms all-trans bacteriorhodopsin (BR) to its L intermediate having a 13-cis chromophore. Illumination of L at 80 K results in an intermediate L' absorbing around 550 nm. L' thermally converts to the original BR only at >130 K. In this study, we used the light-induced transformation of L to L' at 80 K to identify some amino acid residues and water molecules that closely interact with the chromophore and distinguish them from those residues not affected by the photoreaction. The L minus L' FTIR difference spectrum shows that the chromophore in L' is in the all-trans configuration. The perturbed states of Asp96 and Val49 and of the environment along the aliphatic part of the retinal and Lys216 seen in L are not affected by the L --> L' photoreaction. On the other hand, the environments of the Schiff base of the chromophore, of Asp115, and of water molecules close to Asp85 returned in L' to their state in which they originally had existed in BR. The water molecules that are affected by the mutations of Thr46 and Asp96 also change to a different state in the L --> L' transition, as indicated by transformation of a water O-H vibrational band at 3497 cm-1 in L into an intense peak at 3549 cm-1 in L'. Notably, this change of water bands in the L --> L' transition at 80 K is entirely different from the changes observed in the BR --> K photoreaction at the same temperature, which does not show such intense bands. These results suggest that these water molecules move closer to the Schiff base as a hydrogen bonding cluster in L and L', presumably to stabilize its protonated state during the BR to L transition. They may contribute to the structural constraints that prevent L from returning to the initial BR upon illumination at 80 K.

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment.

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.

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.

Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin.

Substitution of glutamic acid-194, a residue on the extracellular surface of bacteriorhodopsin, with a cysteine inhibits the fast light-induced proton release that normally is coupled with the deprotonation of the Schiff base during the L to M transition. Proton release in this mutant occurs at the very end of the photocycle and coincides with deprotonation of the primary proton acceptor, Asp-85, during the O to bR transition. the E194C mutation also results in a slowing down of the photocycle by about 1 order of magnitude as compared to the wild type and produces a strong effect on the pH dependence of dark adaptation that is interpreted as a drastic reduction or elimination of the coupling between the primary proton acceptor Asp-85 and the proton release group. These data indicate that Glu-194 is a critical component of the proton release complex in bacteriorhodopsin.

Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; guanidine hydrochloride restores it.

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKa's of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKa's of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKa's of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKa's of E204 and D85.

Evidence that aspartate-85 has a higher pK(a) in all-trans than in 13-cisbacteriorhodopsin.

Three experimental observations indicate that the pK(a) of the purple-to-blue transition (the pK(a) of Asp-85) is higher for all-trans-bR(1) than for 13-cis-bR. First, light adaptation of bacteriorhodopsin (bR) at pHs near the pK(a) of Asp-85 causes an increase in the fraction of the blue membrane present. This transformation is reversible in the dark. Second, the pK(a) of the purple-to-blue transition in the dark is lower than that in the light-adapted bR (pK(a)(DA) = 3.5, pK(a)(LA) = 3.8 in 10 microM K(2)SO(4)). Third, the equilibrium fractions of 13-cis and all-trans isomers are pH dependent; the fraction of all-trans-bR increases upon formation of the blue membrane. Based on the conclusion that thermal all-trans <=> 13-cis isomerization occurs in the blue membrane rather than in the purple, we have developed a simple model that accounts for all three observations. From the fit of experimental data we estimate that the pK(a) of Asp-85 in 13-cis-bR is 0.5 +/- 0.1 pK(a) unit less than the pK(a) of all-trans-bR. Thus in 10 microM K(2)SO(4), pK(a)(c) = 3.3, whereas pK(a)(t) = 3.8.

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.

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.

The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin.

To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of substitution of tyrosine 57 with asparagine and phenylalanine on the properties of bacteriorhodopsin.

Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85. In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT). The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1. In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT. Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.