Structural changes of sensory rhodopsin I and its transducer protein are dependent on the protonated state of Asp76

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.     

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.     

FTIR spectroscopy of the O photointermediate in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the retinal chromophore is thermally isomerized from the 13-cis to all-trans form. We employed FTIR spectroscopy of ppR at 260 K and pH 5 to reveal that this isomerization occurs upon formation of the O intermediate (ppR(O)) by using ppR samples reconstituted with 12,14-D(2)-labeled retinal. In ppR(O), C=O stretching vibrations of protonated carboxylates newly appear at 1757 (+)/1722 (-) cm(-1) in H(2)O and at 1747 (+)/1718 (-) cm(-1) in D(2)O in addition to the 1765 (+) cm(-1) band of Asp75. Amide I vibrations are basically similar between ppR(M) and ppR(O), whereas unique bands of ppR(O) are also observed such as the negative 1656 cm(-1) band in D(2)O and intense bands at 1686 (-)/1674 (+) cm(-1). In addition, O-D stretching vibrations of water molecules in the entire mid-infrared region are assigned for ppR(M) and ppR(O), the latter being unique for ppR, since it can be detected at low temperature (260 K). The ppR(M) minus ppR difference spectra lack the lowest frequency water band (2215 cm(-1)) observed in the ppR(K) minus ppR spectra, which is probably associated with water that interacts with the negative charges in the Schiff base region. It is likely that the proton transfer from the Schiff base to Asp75 in ppR(M) can be explained by a hydration switch of a water from Asp75 to Asp201, as was proposed for the light-driven proton-pump bacteriorhodopsin (hydration switch model) [Tanimoto, T., Furutani, Y., and Kandori, H. (2003) Biochemistry 42, 2300-2306]. In the transition from ppR(M) to ppR(O), a hydrogen-bonding alteration takes place for another water molecule that forms a strong hydrogen bond.     

FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) reduced; heme a and Cu(A) oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a(3) oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a(3) decreases from 1965.3 cm(-1) at pH* 相似文献   

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a₃, including Asp399 and the Cu_B ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.     

Proton transfer reactions in the F86D and F86E mutants of pharaonis phoborhodopsin (sensory rhodopsin II)

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII), a negative phototaxis receptor of Natronobacterium pharaonis, can use light to pump a proton in the absence of its transducer protein. However, the pump activity is much lower than that of the light-driven proton-pump bacteriorhodopsin (BR). ppR's pump activity is known to be increased in a mutant protein, in which Phe86 is replaced with Asp (F86D). Phe86 is the amino acid residue corresponding to Asp96 in BR, and we expect that Asp86 plays an important role in the proton transfer at the highly hydrophobic cytoplasmic domain of the F86D mutant ppR. In this article, we studied protein structural changes and proton transfer reactions during the photocycles of the F86D and F86E mutants in ppR by means of Fourier transform infrared (FTIR) spectroscopy and photoelectrochemical measurements using a tin oxide (SnO2) electrode. FTIR spectra of the unphotolyzed state and the K and M intermediates are very similar among F86D, F86E, and the wild type. Asp86 or Glu86 is protonated in F86D or F86E, respectively, and the pK(a) > 9. During the photocycle, the pK(a) is lowered and deprotonation of Asp86 or Glu86 is observed. Detection of both deprotonation of Asp86 or Glu86 and concomitant reprotonation of the 13-cis chromophore implies the presence of a proton channel between position 86 and the Schiff base. However, the photoelectrochemical measurements revealed proton release presumably from Asp86 or Glu86 to the cytoplasmic aqueous phase in the M state. This indicates that the ppR mutants do not have the BR-like mechanism that conducts a proton uniquely from Asp86 or Glu86 (Asp96 in BR) to the Schiff base, which is possible in BR by stepwise protein structural changes at the cytoplasmic side. In ppR, there is a single open structure at the cytoplasmic side (the M-like structure), which is shown by the lack of the N-like protein structure even in F86D and F86E at alkaline pH. Therefore, it is likely that a proton can be conducted in either direction, the Schiff base or the bulk, in the open M-like structure of F86D and F86E.     

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).     

FTIR difference spectra of Wolinella succinogenes quinol:fumarate reductase support a key role of Glu C180 within the "E-pathway hypothesis" of coupled transmembrane electron and proton transfer

Electrochemically induced static FTIR difference spectroscopy has been employed to investigate redox-driven protonation changes of individual amino acid residues in the quinol:fumarate reductase (QFR) from Wolinella succinogenes. The difference spectra presented were taken in the mid-infrared region from 1800 to 1000 cm(-1), and the signals obtained represent transitions between the reduced and oxidized states of the enzyme. Analysis of the difference spectra shows evidence for structural reorganizations of the polypeptide backbone upon the induced redox reaction. Furthermore, spectral contributions were found above 1710 cm(-1) where stretching vibrations of protonated carboxyl groups from aspartic or glutamic acid side chains absorb. With the help of site-directed mutagenesis and hydrogen/deuterium isotope exchange, it was possible to identify amino acid residue Glu C180, which is located in the membrane-spanning, diheme-containing subunit C of QFR, as being partially responsible for the derivative-shaped spectral feature with a peak/trough at 1741/1733 cm(-1) in the reduced-minus-oxidized difference spectrum. This signal pattern is interpreted as a superposition of a protonation/deprotonation and a change of the hydrogen-bonding environment of Glu C180. This residue is the principal constituent of the recently proposed "E-pathway hypothesis" of coupled transmembrane proton and electron transfer in QFR [Lancaster, C. R. D. (2002) Biochim. Biophys. Acta 1565, 215-231]. Thus, the study presented yields experimental evidence which supports a key role of Glu C180 within the framework of the E-pathway hypothesis.     

Structural characterization of the L-to-M transition of the bacteriorhodopsin photocycle.

Structural intermediates occurring in the photocycle of wild-type bacteriorhodopsin are trapped by illuminating hydrated, glucose-embedded purple membrane at 170 K, 220 K, 230 K, and 240 K. We characterize light-induced changes in protein conformation by electron diffraction difference Fourier maps, and relate these to previous work on photocycle intermediates by infrared (FTIR) spectroscopy. Samples illuminated at 170 K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm resolution limit of our data. Difference maps obtained with samples illuminated at 220 K, 230 K, and 240 K, respectively, reveal a progressively larger structural response in helix F when the protein is still in the M state, as judged by the FTIR spectra. Consistent with previous structural studies, an adjustment in the position or in the degree of ordering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin experiences minimal change in protein structure until a proton is transferred from the Schiff base to Asp85. The M intermediate then undergoes a conformational evolution that opens a hydrated "half-channel," allowing the subsequent reprotonation of the Schiff base by Asp96.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.     

Characterization of the photoreduction of the secondary quinone QB in the photosynthetic reaction center from rhodobacter capsulatus with FTIR spectroscopy

The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter (Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H2O and 2H2O. The Q-B/QB FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone-protein interactions. As expected from the conservation of most of the amino acids near QB in the RCs from Rb. capsulatus and Rb. sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm-1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb. sphaeroides RCs. However, noticeable changes are observed at 1709 cm-1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm-1 (side chain and/or backbone), around 1540 cm-1 (amide II), and in the semiquinone absorption range. This FTIR study demonstrates that the environment of the secondary quinone in Rb. capsulatus is close but not identical to that in Rb. sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near QB.     

Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppR(K) minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm(-1)) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppR(K) minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm(-1)) and amide-A (3350-3250 cm(-1)) vibrations. The identical spectra for the complex of the unlabeled ppR and (13)C- or (15)N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppR(K). In addition, we observed D(2)O-insensitive bands at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.     

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.     

Active internal waters in the bacteriorhodopsin photocycle. A comparative study of the L and M intermediates at room and cryogenic temperatures by infrared spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.