Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant

We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 --> Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 --> Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 --> Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.     

The last phase of the reprotonation switch in bacteriorhodopsin: the transition between the M-type and the N-type protein conformation depends on hydration

In order to elucidate the mechanism of the reprotonation switch of bacteriorhodopsin, the protein conformation of the M intermediate of the D96N mutant was examined at various hydration conditions by X-ray diffraction and FTIR spectroscopy. We observed two distinct protein conformations at different levels of hydration. One is like in the N photointermediate, although in this case with an unprotonated Schiff base. It is stabilized in highly hydrated samples. The other is a protein conformation identical to that in the normal M intermediate of wild-type bacteriorhodopsin, which is stabilized in partially dehydrated samples. The hydration dependence of the structural transition between the M-type and the N-type conformations suggests that there is a change in the binding of water at the cytoplasmic surface. Thus, more water molecules bind in the N-type structure than in the M-type. This is consistent with the idea that the conformational change from the M-type to the N-type corresponds to the opening of the proton channel to the cytoplasmic surface by tilt of the cytoplasmic end of helix F, and that this is required for proton transfer from Asp-96 to the retinal Schiff base.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

Hydration of the counterion of the Schiff base in the chloride-transporting mutant of bacteriorhodopsin: FTIR and FT-raman studies of the effects of anion binding when Asp85 is replaced with a neutral residue

The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.     

Annexin I is a local mediator in neural-endocrine feedback control of inflammation

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.     

Transmembrane signaling mediated by water in bovine rhodopsin

Unhydrated air-dried films of rhodopsin from bovine rod outer segment membranes do not produce its active state, metarhodopsin II. In order to reveal requirements for its formation, we studied changes in H-bonding of water, peptide carbonyl and carboxylic acid in the photochemical reactions by means of difference Fourier transform infrared spectroscopy, under both hydrated and unhydrated conditions. A water molecule near Glu113, which undergoes H-bonding change in bathorhodopsin, remained in the unhydrated film, but with a weaker H-bonding state than in the hydrated film. The other water molecules, which shfit in lumirhodopsin and metarhodopsin I as well as in bathorhodopsin of the hydrated film, were not observed in the unhydrated film. Effects of the dehydration were detected in all the C=O stretching vibrations of the peptide backbone and of Asp83 in the formation of bathorhodopsin. The C=O stretching band of Asp83 of lumirhodopsin and metarhodopsin I is intensified in the unhydrated film. We propose that structural changes at the intradiscal site in the interaction between the Schiff base and Glu113 affect water molecules, the peptide backbone, Asp83 and Glu122 in helices B and C through consecutive photochemical processes to metarhodopsin II.     

Conformational changes in the core structure of bacteriorhodopsin

Bacteriorhodopsin (bR) is the light-driven proton pump found in the purple membrane of Halobacterium salinarium. In this work, structural changes occurring during the bR photocycle in the core structure of bR, which is normally inaccessible to hydrogen/deuterium (H/D) exchange, have been probed. FTIR difference bands due to vibrations of peptide groups in the core region of bR have been assigned by reconstituting and regenerating delipidated bR in the presence of D2O. Exposure of bR to D2O even after long periods causes only a partial shift of the amide II band due to peptide NH --> ND exchange only of peripheral peptide structure. However, the amide II band completely downshifts when reconstitution/regeneration of bR is performed in the presence of D2O, indicating that almost the entire core backbone structure of bR undergoes H/D exchange. Peripheral regions can then be reexchanged in H2O, leaving the core backbone region deuterated. Low-temperature FTIR difference spectra on these core-deuterated samples reveal that peptide groups in the core region respond to retinal isomerization as early as the K intermediate. By formation of the M intermediate, infrared differences in the amide I region are dominated by much larger structural changes occurring in the core structure. In the amide II region, difference bands appear upon K formation and increase upon M formation which are similar to those observed upon the cooling of bacteriorhodopsin. This work shows that retinal isomerization induces conformational changes in the bacteriorhodopsin core structure during the early photocycle which may involve an increase in the strength of intramolecular alpha-helical hydrogen bonds.     

Proton transfer from Asp-96 to the bacteriorhodopsin Schiff base is caused by a decrease of the pKa of Asp-96 which follows a protein backbone conformational change

In the bacteriorhodopsin photocycle the transported proton crosses the major part of the hydrophobic barrier during the M to N reaction; in this step the Schiff base near the middle of the protein is reprotonated from D96 located near the cytoplasmic surface. In the recombinant D212N protein at pH > 6, the Schiff base remains protonated throughout the photocycle [Needleman, Chang, Ni, Váró, Fornés, White, & Lanyi (1991) J. Biol. Chem. 266, 11478-11484]. Time-resolved difference spectra in the visible and infrared are described by the kinetic scheme BR-->K<==>L<==>N (-->N')-->BR. As evidenced by the large negative 1742-cm-1 band of the COOH group of the carboxylic acid, deprotonation of D96 in the N state takes place in spite of the absence of the unprotonated Schiff base acceptor group of the M intermediate. Instead of internal proton transfer to the Schiff base, the proton is released to the bulk, and can be detected with the indicator dye pyranine during the accumulation of N'. The D212N/D96N protein has a similar photocycle, but no proton is released. As in wild-type, deprotonation of D96 in the N state is accompanied by a protein backbone conformational change indicated by characteristic amide I and II bands. In D212N the residue D96 can thus deprotonate independent of the Schiff base, but perhaps dependent on the detected protein conformational change. This could occur through increased charge interaction between D96 and R227 and/or increased hydration near D96. We suggest that the proton transfer from D96 to the Schiff base in the wild-type photocycle is driven also by such a decrease in the pKa of D96.     

Free-energy simulations of the retinal cis --> trans isomerization in bacteriorhodopsin

Free-energy profiles for ground-state cis --> trans isomerization of retinal in vacuum, in solution, and in the protein bacteriorhodopsin are calculated using free-energy simulations. The free-energy barriers in the protein were 9 kcal/mol for ionized Asp85 and 14 kcal/mol for neutral Asp85, significantly lower than those found in solution (18 kcal/mol) or vacuum (19 kcal/mol). Therefore, bacteriorhodopsin can be said to act as a catalyst in the isomerization. The barrier in the protein is due mainly to stabilization of the transition state through favorable nonbonded interactions with the protein part of the system, with internal strain and interactions with solvent playing minor roles. The protonated Asp85 simulation models the behavior of the system in the N --> O transition. Our calculated 14 kcal/mol barrier and 4-ms relaxation time for this process are in excellent agreement with experimentally measured values of 12 kcal/mol and 5 ms, respectively. The ionized Asp85 simulation models two hypothetical processes: the N --> O transition with a proton removed from Asp85 and the initial BR568 --> L transition on the ground-state energy surface. The cis-trans isomerization barrier in this system is 9 kcal/mol, the lowest of all the studied cases. The presence of the charged carboxylate group in the ionized Asp85 system leads to strong stabilization of the transition state by interactions with the surroundings and changes the distance between Asp85 and the Schiff base proton compared to the corresponding distance in the neutral Asp85 system. This suggests that the protonation of Asp85 plays an important role in regulating access to the Schiff base proton. For both Asp85 ionization states the calculated cis-trans free-energy difference was close to 0, indicating that the protein can accommodate both retinal isomers equally well. The computed negligible difference between the N and O free-energy levels is in accord with experimental data.     

Transient channel-opening in bacteriorhodopsin: an EPR study

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.     

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein-retinal coupling

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 --> Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.     

Arginine activity in the proton-motive photocycle of bacteriorhodopsin: solid-state NMR studies of the wild-type and D85N proteins

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the epsilon position, an upfield shift of 4 ppm is observed while the eta nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg-82 on the basis of the SSNMR investigations of the alkaline and neutral dark-adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of eta signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg-134. The results are discussed in relation to existing models of bR structure and function.     

Tyrosine structural changes detected during the photoactivation of rhodopsin

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho --> MII difference spectra were obtained at 10 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --> M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates alpha-helix movement.     

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.     

Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over a wide pH range

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.     

A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in solution

An attenuated total reflectance Fourier transform infrared method has been developed that allows collection of spectra from proteins in solution. This method eliminates any structural perturbations induced by the internal reflection element (IRE), and thus the spectra reflect the solution conformation of the protein. A key feature of the method is subtraction of the signal from any protein adsorbed to the IRE. The advantages of this method include the small amount of sample required and the high sampling rate. Attenuated total reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) is more versatile than transmission FTIR because it is possible to collect spectra of nontransparent samples, to use samples of very low protein concentration (< or = 0.3 mg/ml), and to study proteins in the presence of strongly absorbing solutes (such as denaturants). The experimental procedures and data processing routines developed were evaluated by collecting spectra from a set of 13 proteins and evaluating their accuracy with a partial least-squares analysis. The relative mean and standard deviation errors for the basis set analysis were 6.3% for alpha-helix, 5.9% for beta-sheet/extended structure, and 4.4% for turn, which are similar to values from comparable analyses of transmission FTIR spectra. In addition, a detailed comparison between this solution ATR method and the hydrated thin-film ATR technique is presented.     

Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides

Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.     

The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II

The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.     

Time and pH dependence of the L-to-M transition in the photocycle of bacteriorhodopsin and its correlation with proton release

The pH dependence of the L-to-M transition in the photocycle of bacteriorhodopsin was studied in pump-probe resonance Raman (RR) flow experiments in the range pH 3.5-7.8 on a time scale of 0-700 micros. For pH < 5, following the initial decay of L to M, the two intermediates approach nearly constant levels. From a specially designed perturbation-relaxation experiment at pH 4.6, in which the composition of L and M is perturbed by photoreversal of M, it could be concluded that the incomplete decay of L is due to an intermediate equilibration between L and M. It was found, by both RR and optical transient spectroscopy, that the maximum level of M (approximately 500 micros) increases with pH according to a pKa of 5. 6 (150 mM Na+). Since the proton release from an internal group XH to the extracellular surface is determined by nearly the same pKa of 5.7 [Zimanyi, L., Varo, G., Chang, M., Ni, B., Needleman, R., and Lanyi, J. K. (1992) Biochemistry 31, 8535-8543], it is concluded that this increase is controlled by the dissociation of XH. From the analysis of the perturbation-relaxation experiments and the multiexponential rise of M, a kinetic scheme with two sequential L-M equilibria is proposed for the L-M transition. By comparison with the time behavior of proton release [Heberle, J., and Dencher, N. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5996-6000], it is suggested that it is the second equilibrium which is further shifted toward the M state by the dissociation of XH. From the magnitude of this shift, it is concluded that the L-M transition and proton release are not as strongly coupled as is generally assumed. Instead, it is proposed that structural changes during the photocycle are the dominating factors which reduce the pKa of XH to approximately 5.7 so that proton release becomes possible under normal conditions.     

Resonance Raman and optical transient studies on the light-induced proton pump of bacteriorhodopsin reveal parallel photocycles

The photocycle of bacteriorhodopsin (bR) was studied at ambient temperature in aqueous suspensions of purple membranes using time-resolved resonance Raman (RR) and optical transient spectroscopy (OTS). The samples were photolyzed, and the fractional concentrations of the retinylidene chromophore in its parent state, BR570, and in the intermediate states L550, M412, N560, and O640 were determined in the time domain 20 microseconds-1 s and in the pH range 4-10.5. Two kinetically different L components could be identified. At pH 7 one fraction of L (approximately 65%) decays in 80 microseconds to M (deprotonation of the Schiff base), whereas the residual part is converted in approximately 0.5 ms to N. The RR spectra reveal only minor structural changes of the chromophore in the L-->N transition. These were attributed to a conformational change of the protein backbone [Ormos, P., Chu, K., & Mourant, J. (1992) Biochemistry 31, 6933]. With decreasing pH the L-->N transition is delayed to > 2 ms following a titration-like function with pKa approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (Mf, tau f; Ms, tau s; f = fast, s = slow). Both Mf and Ms consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). Mf occurs in the reaction sequences L-->Mf-->N-->BR and L-->Mf-->O-->BR. The population of the first sequence, in which N is formed with the time constant tau f (approximately 2-4 ms, pH 6-10.5), increases with pH. Ms is also found in two different reaction sequences of the form L-->Ms-->BR. The quantitative analysis reveals that each "titration effect" can be related to a certain fraction of bR. It is proposed that each fraction can be identified with a "subspecies" of bR which undergoes an independent and individual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from the pH dependence of the lifetimes of Ms and N that the reconstitution of BR570 in the reaction steps Ms-->BR and N-->BR requires the uptake of a proton from the external phase. It is argued that this proton catalyzes the reisomerization of retinal, whereas the Schiff base is internally reprotonated from Asp-85. A model for proton pumping is proposed in which the proton taken up from the external phase to catalyze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.