THE EFFECT OF VISCOSITY ON THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— We study the effect of solvent viscosity on the kinetics of the photocycle of bacteriorhodopsin (bR) from Halobacterium halobium. Solvent viscosity is altered by changing the glycerol concentration from 20 to 80% glycerol by volume. The kinetics of the photocycle are observed after flash photolysis at four wavelengths at several temperatures between 240 and 315 K. Assuming a sequential model, bR → K -→ L → M → O → bR, Arrhenius plots of the rate coefficients determine the activation enthalpies and frequency factors for each step. Kinetic data from all solvents are considered together and studied as a function of temperature for fixed solvent viscosities. The early steps of the cycle are insensitive to solvent viscosity, →; the later steps are retarded with increasing viscosity. Activation enthalpies are independent of viscosity; the frequency factors are proportional to η^−K, where the exponent k 0.25 for the transition K → L, 0.0 for L → M, 0.8 for M → O and 0.5 for O → bR.     

BRANCHING PATHWAYS IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— The pulsed laser photolysis of light-adapted bacteriorhodopsin (BR₅₇₀) is carried out over the temperature range between 25°C and—92°C in neutral and alkaline water-glycerol solutions. The results arc indicative of considerable complexity, introduced by two temperature dependent branching reactions associated with the intermediates K₆₁₀, L₅₅₀ and M₄₁₂, of the BR₅₇₀ photocycle. (a) At relatively low temperatures the primary photoproduct K-₆₁₀ equilibrates with a blue-shifted species, K_p. Both K₆₁₀ and the new intermediate subsequently decay into another species, K'_r, in a process which competes with the formation of L₅₅₀. Finally, K'_p converts very slowly to L₅₅₀. This branched pathway delays the formation of L₅₅₀ and thus of M₄₁₂, without affecting the final yield of either species, (b) A thermal back-reaction regenerating BR₅₇₀ takes place at the stage of L₅₅₀, inhibiting the formation of M₄₁₂. The reaction which also predominates at low temperatures, is relatively inefficient at high pH when the forward L₅₅₀→ M₄₁₂ step is highly catalyzed. It is the superposition of both branching mechanisms, (a) and (b), which accounts for the complex effects of temperature and pH on the photo-cycle of BR₅₇₀. Mechanism (b) is accounted for by a molecular scheme in which deprotonation of a tyrosine moiety at the stage of L₅₅₀ constitutes a prerequisite for deprotonation of the retinal-lysine schiff-base as required for forming M₄₁₂. This scheme appears to be directly related to the proton pump. Mechanism (a) introduces additional complexity in the photocycle at low temperatures but its molecular aspects are still unclear.     

THE EFFECT OF IONIC STRENGTH AND pH ON THE PROTONATED SCHIFF BASE AND TYROSINE DEPROTONATION KINETICS DURING THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— The deprotonation kinetics of tyrosine and the protonated Schiff base during the bacteriorho-dopsin photocycle were studied under different perturbations by transient absorption spectroscop Native purple membrane, as well as samples which were deionized (blue) then restored with Na⁺ or La³⁺ were used at pH's ranging from 7 to 10 at very low salt concentrations. The results were compared with previous studies at higher ionic strength. The important conclusions can be summarized as follows: (a) The rate constants of both the Schiff base and tyrosine deprotonation are not very sensitive to the changes of conditions. (b) An almost linear relationship is observed between the relative amplitudes of the tyrosine deprotonated during the cycle and the slow component of the Schiff base deprotonation under the different perturbations studied. This was taken to support the two site model for the protonated Schiff base, one near tyrosine and the other near its ionized form. (c) The pK_a value determined from the ratio of the amplitude of the fast to the slow component of the Schiff base deprotonation is found to decrease with increasing ionic strength of the medium. At extremely low ionic strength, it was found to equal that of the tyrosine phenolic group in solution.     

TIME-RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition.     

EFFECTS OF METAL CATIONS, RETINAL, AND THE PHOTOCYCLE ON THE TRYPTOPHAN EMISSION IN BACTERIORHODOPSIN

Abstract— The picosecond fluorescence kinetics of tryptophan residues in bacteriorhodopsin and some perturbed analogs are measured to study the different tryptophan environments and their changes upon metal cation removal, retinal removal, and M₄₁₂ trapping. In bacteriorhodopsin, the emission shows four decay components designated Or, C2r, C3r, and C4r in order of increasing lifetimes. The emission wavelength of C3r and C4r is near that found in aqueous solution, while that of C1r is the shortest. The removal of retinal triples the total emission intensity and reduces the number of components to two, suggesting that the observed variation of the lifetimes in bacteriorhodopsin results from the variation of the energy transfer efficiency between different tryptophans and retinal. We conclude that the Or and C2r emission is from the closest tryptophans to the retinal. The quenching of the C3r emission by all metal cations, including those that cannot act as energy acceptors, e.g. Ca²⁺, is attributed to protein conformation changes caused by metal cation binding which leads to a stronger energy transfer coupling between tryptophans and retinal. The additional quenching of the C2r emission in Eu³⁺bound bacterioopsin is proposed to result from direct energy transfer between tryptophans and Eu³⁺.     

EVIDENCE FOR BRANCHING IN THE PHOTOCYCLE OF BACTERIORHODOPSIN AND CONCENTRATION CHANGES OF LATE INTERMEDIATE FORMS

Abstract— Flash photolysis transients of bacteriorhodopsin were recorded with a spectrograph -multielement photodiode array combination and the recordings were analyzed to determine the concentrations of bacteriorhodopsin intermediates "M" and "O" relative to the amount of "bR" cycling (pH 7.1,10–40°C). Estimated concentration time courses were simulated with solutions to two kinetic decay models which could account for photocycle temperature dependence. A unidirectional unbranched decay model overpredicts our estimated levels of [O(r)], whereas a model branched at the "M" intermediate describes each of the later intermediate levels well (with no evidence for an independent "N" form). Our results are consistent with "M" decay regulating the level and rates of change of [bR (t)] and (bR(f)]- and also suggest that two temperature-dependent pathways form "bR" from "M", one directly, and the other indirectly through "O".     

TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY OF THE BACTERIORHODOPSIN PHOTOCYCLE ON THE PICOSECOND AND NANOSECOND TIME SCALES

Abstract— Resonance Raman spectra of the picosecond bacteriorhodopsin intermediate(s) have been obtained by microbeam, flow and subtraction techniques using a synchronously pumped, cavity-dumped dye laser. Nanosecond spectra also were measured with this laser by cavity dumping without mode-locking. The picosecond spectra in the fingerprint region, which is sensitive to the configuration of the retinal chromophore, differ from spectra of the parent bR₅₇₀ but could be correlated to the spectrum of bR^DA₅₅₀ , a “13-cis” species which has been determined from spectra of bR₅₇₀ and bR^DA₅₆₀. The picosecond transient and bR^DA₅₅₀ also are similar in the 950–1050 cm^-1“deuteration fingerprint” region when the medium is changed from H₂O to D₂O. These results suggest that trans—cis isomerization occurs during the 40-ps pulse duration. The shift relative to the parent bR₅₇₀ in the ethylenic stretch region suggests that the picosecond and nanosecond transients absorb at wavelengths longer than 570 nm. The C band at 1646 cm^-1 is found to shift or to broaden upon photolysis in the picosecond time scale. This might suggest a change in the electronic structure of the group and its environment on the picosecond time domain. The nanosecond spectra obtained in this work (with 15-ns pulses) are similar to the spectra previously observed on the 100-ns time scale but are slightly different from the picosecond spectrum. These data suggest that more than one transient species appears on the picosecond-to-nanosecond time scale. The temporal evolution of Raman bands in the fingerprint as well as the low energy (950–1050 cm^-1) region and its implications are discussed.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP

Abstract
Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The p K values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

EFFECTS OF MUTAGENETIC SUBSTITUTION OF PROLINES ON THE RATE OF DEPROTONATION and REPROTONATION OF THE SCHIFF BASE DURING THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions ofPro–50,Pro–91 andPro–186 on the deprotonation and reprotonation kinetics of the Schiffbase (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate thatPro–50 andPro–91, as well asPro–186, are important for the proton-pumping function of bR.     

THE ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP*

Abstract— Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The pK values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

A DETAILED RESONANCE RAMAN STUDY OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— Resonance Raman spectra of various M₄₁₂ species associated with the bacteriorhodopsin photocycle have been obtained. These correspond to the two forms observed during the formation of M₄1₂ and the two forms that are observed during its decay in absorption experimeents. We do not see any significant difference between the Raman spectra of any of these forms. We therefore conclude that the differences in these species are due to the differences in the protein structure and not in the chromophore.     

KINETIC MODEL OF BACTERIORHODOPSIN PHOTOCYCLE: PATHWAY FROM M STATE TO bR

A model of the last parts of the bacteriorhodopsin (bR) photocycle is proposed on the basis of experimental data for the kinetic behavior of the 'O' intermediate during a temperature pulse in distilled water suspension. The model includes the previously proposed (but not well characterized) intermediate 'N' between the 'M' and 'O' states of bR. This intermediate exists in fast temperature-dependent quasi-stationary equilibrium with the red-shifted intermediate 'O' and has a maximum of absorption close to the bR spectrum.     

EFFECT OF MELITTIN ON PHOTOCYCLE AND PHOTOPOTENTIAL OF PURPLE MEMBRANE: SITES OF INTERACTION BETWEEN BACTERIORHODOPSIN AND MELITTIN

Abstract— Purple membrane (PM) suspension and artificial bilayer lipid membranes (BLM) containing PM sheets were treated with melittin. Both the decaying of the photocycle intermediate M412 and proton translocation were inhibited by melittin: The yields and rate of the slow-decaying component of M412 (M412s) together with the proton release and its uptake rate were significantly decreased, but the rate of the fast-decaying component of M412 (M4120 had only slight changes. Relatively high concentrations of melittin could cause aggregation in PM suspensions. Addition of melittin to a BLM solution increased the continuous photopotential signal but decreased the transient signal. We suggest that there might exist strong interactions between melittin and bacteriorhodopsin in addition to the melittin–lipid action. On the other hand, the results also indicate that proton translocation was more likely to be coupled with M412s and both were more sensitive to the changes caused by the melittin–PM interaction than was M412f.     

ELECTRONIC EFFECTS ON THE FLUORESCENCE OF TYROSINE IN SMALL PEPTIDES

Abstract— It is shown for a series of tyrosine-derivatives and tyrosine-containing peptides that the amide group in combination with electron-withdrawing substituents quenches the fluorescence of the phenol moiety. The ammonium group has the strongest electron-withdrawing effect and thus the largest influence on the quenching rate. The peptide group itself does not quench the fluorescence. In a series of peptides with an increasing number of alanines the decreasing quenching efficiency or the peptide group due to the greater distance of the ammonium group is demonstrated. In tyrosine-containing di- and tripeptides a linear correlation between the ¹³C-NMR chemical shift δ of the C₂ atom of various aliphatic amino acids and the fluorescence-quenching constant confirms the hypothesis that electron-withdrawing and donating groups are modulating the fluorescence-quenching efficiency of the peptide group. In small peptides the fluorescence lifetime of tyrosine is characteristic for the neighboring amino acids. Using model substances the redox properties of a peptide group and the phenol ring were studied electrochemically. The highest occupied molecular orbital of the tyrosine (1.4 V vs saturated calomel electrode [SCE]) and the lowest unoccupied molecular orbital of the peptide group (-3.12 V vs SCE) have appropriate energies for a photoinduced electron transfer reaction. For solute-quenching experiments quencher molecules can be systematically selected.     

ON THE TYROSINATE INVOLVEMENT IN THE SCHIFF BASE DEPROTONATION IN THE PROTON PUMP CYCLE OF BACTERIORHODOPSIN

Abstract— The ultraviolet transient absorption assigned to the tyrosinate species in bacteriorhodopsin is followed in time and as a function of pH. Both its rise time and titration curve closely resemble those observed for the production of the M₄₁₂ intermediate. These results may support a recently proposed mechanism that couples tyrosinate production to the Schiff base deprotonation in the proton pump of bacteriorhodopsin.     

THE TWO CONSECUTIVE M SUBSTATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN ARE AFFECTED SPECIFICALLY BY THE D85N AND D96N RESIDUE REPLACEMENTS

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.     

AGGREGATION OF COLLAGEN EXPOSED TO UVA IN THE PRESENCE OF RIBOFLAVIN: A PLAUSIBLE ROLE OF TYROSINE MODIFICATION

Riboflavin-sensitized photodynamic modification of collagen led to significant formation of cross-linked molecules. Sodium azide or l,4-diazabicyclo(2,2,2)octane, which are known to be singlet oxygen quenchers, and catalase could not inhibit the modification. Surprisingly, the collagen modification was accelerated in the presence of superoxide dismutase. The aggregation was accompanied by the loss of tyrosine and histidine residues in the collagen. An inhibitory effect of dissolved oxygen on the modification of collagen was observed. Similarly, the loss of tyrosine residues in the irradiated collagen was inhibited in the presence of dissolved oxygen. Dityrosine formation was also observed with the loss of tyrosine. These results indicate that photodynamic modification of tyrosine probably contributes to the riboflavin-sensitized cross-linking of collagen through the formation of dityrosine.     

THE EFFECT OF CROSS-LINKING ON PHOTOCYCLING ACTIVITY OF BACTERIORHODOPSIN

Abstract— Purple membrane preparations from Halobacterium halobium were chemically modified with imidoesters. Dimethyl adipimidate (8.3 Å chain length) amidinates about five of the six free lysine residues whereas dimethyl suberimidate (11.3 Å) under the same conditions reacts with only 2–3 residues. Gel electrophoresis showed that the shorter chain length imidoesters were less effective than dimethyl suberimidate in oligomer formation. However, dimethyl adipimidate resulted in a more marked inhibition of the photoreaction activity. Monofunctional imidates, methyl acetimidate and methyl butyrimi-date, at comparable degrees of amidination, did not appreciably affect activity indicating that the presence of bulky groups on the exposed lysine residues does not cause the effects observed. Hence, the introduction of molecular mobility constraints by intramolecular cross-linking slows photocycling, and, therefore, inhibits proton pumping activity of bacteriorhodopsin. This indicates that conforma-tional changes of the protein moiety of bacteriorhodopsin occur during photocycling activity.