TIME-RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185→E: ASP-96 REPROTONATES DURING O FORMATION; ASP-85 AND ASP-212 DEPROTONATE DURING O DECAY

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.     

Water as a cofactor in the unidirectional light-driven proton transfer steps in bacteriorhodopsin

Recent evidence for involvement of internal water molecules in the mechanism of bacteriorhodopsin is reviewed. Water O-H stretching vibration bands in the Fourier transform IR difference spectra of the L, M and N intermediates of bacteriorhodopsin were analyzed by photoreactions at cryogenic temperatures. A broad vibrational band in L was shown to be due to formation of a structure of water molecules connecting the Schiff base to the Thr46-Asp96 region. This structure disappears in the M intermediate, suggesting that it is involved in transient stabilization of the L intermediate prior to proton transfer from the Schiff base to Asp85. The interaction of the Schiff base with a water molecule is restored in the N intermediate. We propose that water is a critical mobile component of bacteriorhodopsin, forming organized structures in the transient intermediates during the photocycle and, to a large extent, determining the chemical behavior of these transient states.     

Bacteriorhodopsin photocycle kinetics analyzed by the maximum entropy method

A maximum entropy method (MEM) was developed for the study of the bacteriorhodopsin photocycle kinetics. The method can be applied directly to experimental kinetic absorption data without any assumption for the number of the intermediate states taking part in the photocycle. Though this method does not give a specific kinetics, its result is very useful for selection between possible photocycle kinetics. Using simulated data, it is shown that MEM gives correct results for the number of the intermediate states and the amplitude distributions around the characteristic lifetimes. Analyzing experimental absorption data at five different wavelengths, MEM gives seven or eight characteristic lifetimes, which means that at least so many distinct intermediate states exist during the photocycle. Many possible photocycle kinetic models were studied and compared with the MEM result. The best agreement was found with a branching photocycle model of eight intermediate states (K, L, M(1), M(2), M(3), M(4), N, O). The branching occurs at the L intermediate state (M(1) and M(2) being in one branch and M(3) and M(4) in the other branch), but at high pH it occurs already at the K state.     

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.     

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.     

The influence of polytypic structures on the M011-->M101 solid-solid phase transition of n-C36H74: an application of the oblique infrared transmission method

The molecular displacements on the M011-->M101 phase transition of n-hexatriacontane (n-C36H74) have been investigated with an IR microscope designed for the oblique infrared transmission method. It has been clarified that two polytypic structures of the M011 modification, single-layered structure (Mon) and double-layered one (Orth II), both transform to the M101 modification of single-layered structure with their respective mechanisms. On the M011(Mon)-->M101 transition, the inclination direction of molecular axis is changed by 90 degrees through an intermediate state in which the molecular chain is set perpendicular to the basal plane of the single crystal. On the other hand, a polymorphic-polytypic composite structural change on the M011(Orth II)-->M101 transition is accomplished through two kinds of molecular displacements occurring alternately along the stacking direction of molecular layers.     

Computational analysis of the proton translocation from Asp96 to schiff base in bacteriorhodopsin

The potential energy change during the M --> N process in bacteriorhodopsin has been evaluated by ab initio quantum chemical and advanced quantum chemical calculations following molecular dynamics (MD) simulations. Many previous experimental studies have suggested that the proton transfer from Asp96 to the Schiff base occurs under the following two conditions: (1) the hydrogen bond between Thr46 and Asp96 breaks and Thr46 is detached from Asp96 and (2) a stable chain of four water molecules spans an area from Asp96 --> Schiff base. In this work, we successfully reproduced the proton-transfer process occurring under these two conditions by molecular dynamics and quantum chemical calculations. The quantum chemical computation revealed that the proton transfer from Asp96 to Shiff base occurs in two-step reactions via an intermediate in which an H(3)O(+) appears around Ala215. The activation energy for the proton transfer in the first reaction was calculated to be 9.7 kcal/mol, which enables fast and efficient proton pump action. Further QM/MM (quantum mechanical/molecular mechanical) and FMO (fragment molecular orbital) calculations revealed that the potential energy change during the proton transfer is tightly regulated by the composition and the geometry of the surrounding amino acid residues of bacteriorhodopsin. Here, we report in detail the Asp96 --> Schiff base proton translocation mechanism of bacteriorhodopsin. Additionally, we discuss the effectiveness of combining quantum chemical calculations with truncated cluster models followed by advanced quantum chemical calculations applied to a whole protein to elucidate its reaction mechanism.     

A role for internal water molecules in proton affinity changes in the Schiff base and Asp85 for one-way proton transfer in bacteriorhodopsin

Light-induced proton pumping in bacteriorhodospin is carried out through five proton transfer steps. We propose that the proton transfer to Asp85 from the Schiff base in the L-to-M transition is accompanied by the relocation of a water cluster on the cytoplasmic side of the Schiff base from a site close to the Schiff base in L to the Phe219-Thr46 region in M. The water cluster present in L, formed at 170 K, is more rigid than that at room temperature. This may be responsible for blocking the conversion of L to M at 170 K. In the photocycle at room temperature, this water cluster returns to the site close to the Schiff base in N, with a rigid structure similar to that of L at 170 K. The increase in the proton affinity of Asp85, which is a prerequisite for the one-way proton transfer in the M-to-N transition, is suggested to be facilitated by a structural change which disrupts interactions between Asp212 and the Schiff base, and between Asp212 and Arg82. We propose that this liberation of Asp212 is accompanied by a rearrangement of the structure of water molecules between Asp85 and Asp212, stabilizing the protonated Asp85 in M.     

FORMATION AND DECAY KINETICS OF BACTERIORHODOPSIN'S N INTERMEDIATE: SOFTENING THE PROTEIN CONFORMATION WITH ALCOHOLS AFFECTS INTRA-PIROTEIN PROTON TRANSFER AND RETINAL ISIOMERIZATION

Abstract— The trans photocycle of bacteriorhodopsin was investigated in the presence of organic solvents with a hydrogen-bonding group; i.e . methanol, ethanol, 1-propanol and so on. These alcohols scarcely or only slightly affected the L→M and O₅₇₀ transitions, but they perturbed the M→N and N→O transitions greatly. The rate of the M→N transition increased linearly with increasing alcohol concentration and, at maximal alcohol concentrations under which the native protein conformation was retained, the M→N transition was accelerated by a factor of ∼5. This alcohol effect was reversible. It is suggested that a long-distance proton transfer involved in the M→N transition (Asp.96→retinal) becomes easier when the protein conformation is softened bv partially breaking hydrogen-bonding networks in the protein. Another significant effect of alochol is inhibition of the N→O transition at weakly acidic pH, which was slowed down maximally by a factor of ˜10. This alcohol effect was less significant at alkaline pH, where reprotonation of Asp-96 from the cytoplasmic membrane surface is a rate-limiting reaction. It is suggested that, at acidic pH, thi: cis-to-trans isomerization involved in the N→O transition is a rate-limiting reaction and that this step is inhibited in the presence of a high concentration of alcohols.     

Reorientational Motions of the D96N and T46V/D96N Mutants of Bacteriorhodopsin in the Purple Membrane

Abstract— The reorientational motions of the D96N and T46V/D96N mutants of bacteriorhodopsin in purple membrane have been investigated by time-resolved linear dichroism measurements. The reorientations in the early stages of the photocycle are identical to those observed in wild-type bacteriorhodopsin: anisotropics of photocycle intermediates in both D96N and T46V/D96N are r_K= 0.38±0.01, r_L= 0.35±0.01, r_M= 0.35±0.01. The anisotropy of the initial state, r_BR, exhibits decays to zero in D96N and to negative values in T46V/D96N on the time scale of tens of milliseconds. This anisotropy decay can be explained by a model that involves the motion of unexcited or spectator proteins adjacent to a photocycling protein. The amplitude and time constants of spectator reorientational motion are similar to those that have been observed in the wild type. Contributions from the anisotropy of the N-state were detected in measurements of the T46V/D96N mutant, in which a large N-state population accumulates. The value of r_N is estimated to be 0.30±0.05 in T46V/D96N.     

Photoelectrochemical Verification of Proton-Releasing Groups in Bacteriorhodopsin

Light-induced transient currents of bacteriorhodopsin (bR) and its mutants (D96N, D85N, E204Q and E194Q) were measured at the interface of an electrode and the aqueous solution in an electrochemical cell. The transient positive (cathodic) and negative (anodic) photocurrents generated upon the onsets of continuous illumination and of turning off light, respectively, were investigated at different electrolyte pH. The wild type exhibits both positive and negative responses, with the sign of the response inverted at pH lt 5. In D85N the response is entirely suppressed, while D96N lacks the negative response. Laser pulse excitation (532 nm) of bR and the mutants showed the response rise time of the positive transient to be uniformly about 100 μs, indicating that the response is linked to the L to M transition in the photocycle. According to these results the positive and negative signals originate from the proton release and uptake reactions, respectively. Photoexcitation of the mutants E204Q and E194Q that lack protonatable residues at position 204 and 194, respectively, produced a negative response at neutral pH, which indicates that in these proteins proton uptake precedes proton release. This result is consistent with our observations made earlier with pH indicator dyes and demonstrates that Glu-204 and Glu-194 constitute the terminal proton release complex in the extracellular region of bR.     

BIPHASIC M DECAY OF HIGH pH DEHYDRATED PURPLE MEMBRANE STUDIED WITH FOURIER TRANSFORM INFRARED SPECTROSCOPY: A MODEL ACCOUNTING FOR FUNCTIONAL DIFFERENCES BETWEEN DIFFERENT M FORMS

Abstract— A purple membrane film treated with a pH 10 buffer and subsequently dehydrated exhibits an M intermediate that consists of two clearly separable fast-and slow-decaying forms at 275 K; 45% decays thermally within the first 10 min but most of the remaining is stable even after 25 min. Fourier transform infrared spectroscopy is applied to investigate differences in molecular structural changes accompanying thermal decay of the fast-decaying form and photochemical decay (M bacteriorhodopsin back photoreaction) of the slow-decaying form. When difference spectra are taken of the two decay transitions, small but highly reproducible differences are observed. These suggest differences in molecular structural changes accompanying the two decay transitionas. However, the high degree of similarity in the spectra also suggests that these forms of M are not in thermal equilibrium with other intermediates such as N. We propose a model to account for functional differences between two M forms.     

Optical and electric signals from dried oriented purple membrane of bacteriorhodopsins

All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. This property might regulate the activity in the cells when they are exposed in the nature to high light intensity. On the other hand this property is involved in many applications. In this study the ground state and M intermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excited with 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. The decay of the absorption of M intermediate had three components: their lifetimes were very different for laser flash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer for the excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fast negative components and small positive components in the 100 μs time domain. The results are expected to have important implications for bioelectronic applications of bacteriorhodopsin.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments

Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.     

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy†

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent‐solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein–protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm^?1. In rapid‐scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.     

Following evolution of bacteriorhodopsin in its reactive excited state via stimulated emission pumping

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.     

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".