Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine

Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H(+) release, an important step in the normal light-induced H(+) transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H(+)-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H(+) release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.     

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.     

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.     

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

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

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.     

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

Fourier transform infrared study of the halorhodopsin chloride pump

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.     

Late events in the photocycle of bacteriorhodopsin mutant L93A

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.     

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.     

Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle

In the photocycle of bacteriorhodopsin (bR), light-induced transfer of a proton from the Schiff base to an acceptor group located in the extracellular half of the protein, followed by reprotonation from the cytoplasmic side, are key steps in vectorial proton pumping. Between the deprotonation and reprotonation events, bR is in the M state. Diverse experiments undertaken to characterize the M state support a model in which the M state is not a static entity, but rather a progression of two or more functional substates. Structural changes occurring in the M state and in the entire photocycle of wild-type bR can be understood in the context of a model which reconciles the chloride ion-pumping phenotype of mutants D85S and D85T with the fact that bR creates a transmembrane proton-motive force.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

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

Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin

The role of proline residues in the photocycle of bacteriorhodopsin (bR) is addressed using solid-state NMR. (13)C and (15)N chemical shifts from X-Pro peptide bonds in bR are assigned from REDOR difference spectra of pairwise labeled samples, and correlations of chemical shifts with structure are explored in a series of X-Pro model compounds. Results for the three membrane-embedded X-Pro bonds of bR indicate only slight changes in the transition from the resting state of the protein to either the early or late M state of the protonmotive photocycle. These results suggest that the buried prolines serve a principally structural role in bR.     

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.     

Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle.

The rates are determined for the deprotonation and reprotonation of the protonated Schiff base (PSB) as well as of formation and decay of the UV transient in the photocycle of seven bacteriorhodopsin (bR) mutants in which Arg-7, 82, 164, 175, 225, or 227 are replaced by glutamine and Arg-134 by cysteine. The results show that all these mutations increase the rate of deprotonation of the PSB compared to ebR, (wild-type bacteriorhodopsin expressed in Escherichia coli) greatly increase the rate of the reprotonation of the SB (Schiff base) in the case of the Arg-164 and Arg-175 mutations and dramatically decrease this rate in the case of the Arg-227 mutation. Temperature studies on the latter mutant suggest that the observed change in its rate of reprotonation is due to large decrease in the energy and entropy of activation, similar to those observed for Asp-96 mutations (Miller, A. and D. Orsterhelt. 1990. Biochim. Biophys. Acta. 1020:57-64). These results suggest that the reprotonation process is changed to a proton diffusion-controlled mechanism in the Arg-227 mutant due to a change in the structure of the proton channel. The absorption intensity ratio (AUV/AMslow) of each arginine mutant relative to that of ebR is found to be similar to that for native purple membrane (PM) except for the Arg-227 mutant where it is greatly reduced, and for the Arg-82 mutant where it is not observed, suggesting that both Arg-227 and Arg-82 residues somehow play roles in inducing the UV transient absorption. All the above results are discussed in terms of the model for the structure of bR proposed by Henderson, R., J.M. Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann, and K.H. Downing. (1990. J. Mol. Biol. 213:899-929).     

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Procedure for testing kinetic models of the photocycle of bacteriorhodopsin.

Given some simple kinetic models of the photocycle of bacteriorhodopsin (bR) and data taken at many wavelengths and under conditions that avoid photoselection and steady-state cycling complications, it is shown how to extract the apparent rate constants and the spectra of the intermediates. Special consideration was given to establishing the range of error of these results. There are many criteria, which we explicitly discuss, that the spectra should satisfy in order that the kinetic model be acceptable. New data for the photocycle of purple membrane fragments in dilute buffer at pH 7.0 has been obtained at 15 measuring wavelengths and four temperatures. The procedure, which can be generalized to more complex models, has been applied to these data to test two kinds of kinetic models: the unidirectional unbranched model and the undirectional model with simple branching straight back to bR from any intermediate. In these models the spectrum of the O intermediate is highly temperature sensitive, even with branching, and/or has two broad maxima. Moreover, the spectrum of the M intermediate has a secondary maximum and two M-like states appear to be required. Thus, neither model satisfies the physical criteria.     

Protonation of a novel intermediate P is involved in the M → bR step of the bacteriorhodopsin photocycle

A novel intermediate (P) of the bacteriorhodopsin (bR) photocycle, appearing between M412 and bR is described. Like bR, intermediate P shows an absorption maximum at 560–570 nm. However, the extinction coefficient of P is somewhat lower than that of bR. Moreover, there are some differences in spectra of bR and P at wavelengths shorter than 450 nm. The P → bR transition correlates with the absorption of H⁺ from the water medium. The following conditions proved to be favourable for the detection of the new intermediate: a high salt concentration, low light intensity and low temperature (0.5°C). The P → bR transition is strongly decelerated by a small amount of Triton X-100. Illumination of P does not produce M412 before bR is formed. It is assumed that M412 converts to P when the Schiff base is protonated by a proton transferred from a protein protolytic group which participates in the inward H⁺-conductivity pathway. Reprotonation of this group results in the conversion of P to bR. No more than 1 H⁺ is transported per bR photocycle.     

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.     

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.