Photoexcitation of the O-intermediate in bacteriorhodopsin mutant L93A

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime approximately 140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.     

Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump

Structural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild-type bR could be a hydroxyl-ion pump. A position between the protonated Schiff base and residue 85 serves as an anion-binding site in the mutant protein, and hydroxyl ions should have access to this site during the O-intermediate of the wild-type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier.     

Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M.

Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.     

The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window.

Halobacterium halobium Flx mutants are deficient in bacteriorhodopsin (bR) and halorhodopsin (hR). Such strains are phototactic and the light signal detectors are two additional retinal pigments, sensory rhodopsins I and II (sR-I and sR-II), which absorb maximally at 587 and 480 nm, respectively. A retinal-deficient Flx mutant, Flx5R, overproduces sR-I-opsin and does not show any photochemical activity other than that of sR-I after the pigment is regenerated by addition of all-trans retinal. Using native membrane vesicles from this strain, we have resolved a new photointermediate in the sR-I photocycle between the early bathointermediate S610 and the later intermediate S373. The new form, S560, resembles the L intermediate of bR in its position in the photoreaction cycle, its relatively low extinction, and its moderate blue shift. It forms with a half-time of approximately 90 microseconds at 21 degrees C, concomitant with the decay of S610. Its decay with a half-time of 270 microseconds parallels the appearance of S373. From a data set consisting of laser flash-induced absorbance changes (300 ns, 580-nm excitation) measured at 24 wavelengths from 340 to 720 nm in a time window spanning 1 microsecond to 8 s we have calculated the spectra of the photocycle intermediates assuming a unidirectional, unbranched reaction scheme.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoelectric properties of a detector based on dried bacteriorhodopsin film

The photoelectric response of a detector using dried bacteriorhodopsin (bR) film as the light sensing material is mathematically modeled and experimentally verified in this paper. The photocycle and proton transfer kinetics of dried bR film differ dramatically from the more commonly studied aqueous bR material because of the dehydration process. The photoelectric response of the dried film is generated by charge displacement and recombination instead of transferring a proton from the cytoplasmic side to the extracellular side of the cell membrane. In this work, the wild-type bR samples are electrophoretically deposited onto an indium tin oxide (ITO) electrode to construct a simple multiple layered photo-detector with high sensitivity to small changes in incident illumination. The light absorption characteristics of the thin bR film are mathematically represented using the kinetics of the bR photocycle and the charge displacement theorem. An electrically equivalent RC circuit is used to describe the intrinsic photoelectric properties of the film and external measurement circuitry to analyze the detector's response characteristics. Simulated studies and experimental results show that the resistance of the dried bR film is in the order of 10(11) Omega. When the input impedance of the measurement circuitry is one order of magnitude smaller than the dried film, the detector exhibits a strong differential response to the original time-varying light signal. An analytical solution of the equivalent circuit also reveals that the resistance and capacitance values exhibited by the dried bR film, in the absence of incident light, are almost twice as large as the values obtained while the material is under direct illumination. Experimental observations and a predictive model both support the notion that dried bR film can be used in simple highly sensitive photo-detector designs.     

Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.     

The residues Leu 93 and Asp 96 act independently in the bacteriorhodopsin photocycle: studies with the leu 93-->Ala, Asp 96-->Asn double mutant.

Previous mutagenesis studies with bacteriorhodopsin have shown that reprotonation of the Schiff's base is the rate-limiting step in the photocycle of the D96N mutant, whereas retinal re-isomerization and return of the protein to the initial state constitute the rate-limiting events in the photocycle of the L93A mutant. Thus, in the D96N mutant, decay of the M intermediate is slowed down by more than 100-fold at pH 7. In the L93A mutant, decay of the O intermediate is slowed down by 250-fold. We report here that in the L93A, D96N double mutant, decay of the M intermediate, as well as the formation and decay of the O intermediate, are slowed down dramatically. The photocycle is completed by the decay of a long-lived O intermediate, as in the L93A mutant. The decay of the M and O intermediates in the double mutant parallels the behavior seen in the single mutants over a wide temperature and pH range, arguing that the observed independence is an intrinsic property of the mutant. The slow decay of the M and O intermediates can be selectively and independently reversed under conditions identical to those used for the corresponding intermediates in the D96N and L93A single mutants. Because the effects of the two individual mutations are preserved in the double mutant and can be independently reversed, we conclude that residues Asp 96 and Leu 93 act independently and at different stages of the bacteriorhodopsin photocycle. These results also show that formation of the O intermediate only requires protonation of the Schiff's base and is independent of the protonation of Asp 96 from the aqueous medium.     

Anomalous acceleration of the photocycle in photosynthetic reaction centers inhibited on the acceptor side

The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides. The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c(2+)/c(3+) exchanges, or proton delivery to the secondary quinone. The photocycle was driven by high light intensity of a laser diode (5 W/cm(2) at 808 nm) to avoid light limitation of the observed rate. The fast turnover of the reaction center (up to 10(3) s(-1)) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd(2+) and Ni(2+)), by mutation of a key protonatable group (L213Asp --> Asn), or by use of low-affinity ubiquinone (UQ(0)) to the secondary quinone binding site. Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry, marked light intensity dependence was observed. The rate of the photocycle increased from 7 s(-1) (Ni(2+), low light intensity) to 27 s(-1) (high light intensity) at pH 8.4. The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation. We argue that the continuous excitation of the protein trapped in the reduced acceptor (Q(A)(-)Q(B)(-)) state produces short-lived reduced bacteriopheophytin (I(-)) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant.     

Positioning proton-donating residues to the Schiff-base accelerates the M-decay of pharaonis phoborhodopsin expressed in Escherichia coli.

Phoborhodopsin (also called sensory rhodopsin II, sR-II) is a receptor for the negative phototaxis of Halobacterium salinarum (pR), and pharaonis phoborhodopsin (ppR) is the corresponding receptor of Natronobacterium pharaonis. pR and ppR are retinoid proteins and have a photocycle similar to that of bacteriorhodopsin (bR). A major difference between the photocycle of the ion pump bR and the sensor pR or ppR is found in their turnover rates which are much faster for bR. A reason for this difference might be found in the lack of a proton-donating residue to the Schiff base which is formed between the lysine of the opsin and retinal. To reconstruct a bR-like photochemical behavior, we expressed ppR mutants in Escherichia coli in which proton-donating groups have been reintroduced into the cytoplasmic proton channel. In measurement of the photocycle it could be shown that the F86D mutant of ppR (Phe86 was substituted by Asp) showed a faster decay of M-intermediate than the wild-type, which was even accelerated in the F86D/L40T double mutant.     

Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96]

It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.     

Two substates in the O intermediate of the light-driven proton pump archaerhodopsin-2

The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all-trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13-cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101^aR2, Asp96^bR) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This implies that the structural property of O1 in the aR2 photocycle can be investigated over a wide pH range.     

Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH: Origin of the parallel photocycles of trans-bacteriorhodopsin

At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is ~8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pK_a ≈ 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, “neutral” and “alkaline,” bR ↔ bR_a, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's ~ 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bR_a in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

     

M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH

The dependence of the bacteriorhodopsin (bR) photocycle on the intensity of the exciting flash was investigated in purple membranes. The dependence was most pronounced at slightly alkaline pH values. A comparison study of the kinetics of the photocycle and proton uptake at different intensities of the flash suggested that there exist two parallel photocycles in purple membranes at a high intensity of the flash. The photocycle of excited bR in a trimer with the two other bR molecules nonexcited is characterized by an almost irreversible M --> N transition. Excitation of two or three bR in a trimer induces the N --> M back reaction and accelerates the N --> bR transition. Based on the qualitative similarity of the pH dependencies of the photocycles of solubilized bR and excited dimers and trimers we proposed that the interaction of nonexcited bR in trimers alters the photocycle of the excited monomer as compared to solubilized bR and the changes in the photocycles in excited dimers and trimers are the result of decoupling of this interaction.     

Interrelations of M-intermediates in bacteriorhodopsin photocycle.

The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, tau 1 = 65 and tau 2 = 250 microseconds) for the wild-type bR and three exponents (tau 1 = 55 microseconds, tau 2 = 220 microseconds and tau 3 = 1 ms) for the D96N mutant of bR. A component with tau = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96-->Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M-->M' conformational transition. A model for M rise in the bR photocycle is proposed.     

Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles

A rapid reconstitution procedure has been developed to insert deoxycholate-purified bacteriorhodopsin (bR) into asolectin vesicles. The procedure relies on the ability of the hydrophobic resin Bio-Beads SM-2 to remove octyl glucoside from a mixture of deoxycholate-purified bR, asolectin, and the detergent. Light-dependent acidification of the vesicle interior is observed with the reconstituted preparations as judged by the fluorescence quenching of an entrapped pH indicator, pyranine. Inhibition of proton pumping by the addition of LaCl3 to the external medium indicates that approximately 90% of the bR is oriented such that it pumps protons into the vesicles. Phase-lifetime spectrophotometry was used to study the relaxation processes associated with the intermediate in the photocycle of the reconstituted bR which absorbs at 410 nm. Amplitude spectra indicate that these absorbance changes are associated with the M intermediate in the bR photocycle. Two relaxation processes are observed. One is characterized by a relaxation time of approximately 4 ms and is independent of pH over the range 4.4-9.4. The longer relaxation time varies from 4 to 200 ms in the same pH range. By digitization of transients, which are observable when the actinic source is modulated at a low frequency, information about the dependence of the slower process on the light intensity and carbonyl cyanide m-chlorophenylhydrazone was obtained. The results can be interpreted in terms of two different forms of the M intermediate that decay on parallel kinetic paths. To explain the pH dependence of the decay rate, the slower decaying form must have three coupled protonation states, each with a different decay rate.     

深红红螺菌吸氢酶缺失突变株在管式光合反应器中的产氢

本研究设计了一种2 L分体式管式光合反应器, 并研究了深红红螺菌(Rhodospirillum rubrum)吸氢酶缺失突变株在该反应器中分别利用人工光源(持续光照与光暗交替)和自然光的产氢规律。结果表明在人工光照条件下R. rubrum的产氢可维持5 d, 持续光照和光暗交替条件下(12 h: 12 h)的氢产量可分别达到5752 mL/PBR ± 158 mL/PBR和5012 mL/PBR ± 202 mL/PBR; 自然光条件下, 最适产氢光照强度为30000 Lux~40000 Lux; 在此光照条件下, R. rubrum产氢可维持6 d~ 10 d, 最高氢产量可达到2800 mL/PBR。尽管利用自然光的氢产量比利用人工光源氢产量低, 但是利用自然光的产氢比较经济, 并且该光合产氢系统操作简单, 该工艺有望开发为低成本的光合细菌产氢技术。     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Theoretical modeling of the O-intermediate structure of bacteriorhodopsin

Bacteriorhodopsin is a prototype of efficient molecular machinery functioning as a light-activated proton pump. Among the five distinct intermediates (K, L, M, N, and O) of the photocycle, there is less structural information on the later stages compared with the early intermediates. Here, we report the structural modeling of the O-intermediate for which the determination of experimental structure remains difficult. Hypothetical conformational change of the molecule from the light-adapted state to the O-intermediate state was simulated by gradually changing the protonation state of two residues. To achieve accurate molecular modeling, we carefully constructed a realistic system of the native purple membrane. The modeled structure of the O-intermediate has some implications about proton transfer in the later stages of the photocycle and the structural response of bacteriorhodopsin to the inner charge distribution.     

Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra

Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsins and the effects of R82 mutations on the photocycle and proton release. However, the nature of interactions between R82 and other residues of the protein has remained difficult to decipher. Recent NMR studies showed that the two terminal nitrogens of R82 experience a highly perturbed asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature FT-IR spectra of wild-type and mutant bR samples have demonstrated effects of R82 on vibrations of other amino acid side chains, no bands in these spectra were assignable to vibrations of R82 itself. We have now measured time-resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without thioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope substitution of the thioethylguanidinium attached to R82C and on their disappearance in the R82A spectrum. The frequencies and intensities of these IR bands support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies attributable to R82 guandino group vibrations in M, approximately 1640 and approximately 1545 cm(-)(1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its ionization state.