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

Chernavskii, D.S.; Chizhov, Igor; Lozier, Richard H.; Murina, T. M.; Prokhorov, A M; Zubov, B. V.

doi:10.1111/j.1751-1097.1989.tb08437.x

Cited by 49 publications

(28 citation statements)

References 17 publications

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“….) (22,28,31,32); (ii) at least two parallel photocycles of slightly different bR molecules (23,28,(33)(34)(35). The present results cannot unequivocally distinguish between the two proposed models.…”

Section: Discussioncontrasting

confidence: 50%

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

Gerwert¹,

Souvignier²,

Hess³

1990

Proc. Natl. Acad. Sci. U.S.A.

274

203

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Absorbance changes in the infrared and visible spectral range were measured in parallel during the photocycle oflight-adapted bacteriorhodopsin, which is accompanied by a vectorial proton transfer. A global fit analysis yielded the same rate constants for the chromophore reactions, for protonation changes of protein side groups, and for the backbone motion. From this result we conclude that all reactions in various parts of the protein are synchronized to each other and that no independent cycles exist for different parts. The carbonyl vibration of Asp-85, indicating its protonation, appears with the same rate constant as the Schiff base deprotonation. The carbonyl vibration of Asp-96 disappears, indicating most likely its deprotonation, with the same rate constant as for the Schiff base reprotonation. This result supports the proposed mechanism in which the protonated Schiff base, a deprotonated aspartic acid (Asp-85) on the proton-release pathway, and a protonated aspartic acid (Asp-96) on the proton-uptake pathway act as internal catalytic proton-binding sites.Recently, the structure of two membrane proteins, the photosynthetic reaction center and bacteriorhodopsin (bR), have been determined at near-atomic and molecular resolution, respectively (1, 2). In order to understand the structurefunction relationship on an atomic level, time-resolving methods have to be applied to yield insight into the intramolecular reactions. Here, the light-driven proton pump bR (3) is investigated by time-resolved Fourier-transform infrared (FTIR) difference spectroscopy (4, 5).The proton-transfer reactions of bR are initiated by a light-induced isomerization reaction of the chromophore retinal. This reaction is followed by protonation changes of the protonated Schiff base binding site between chromophore and protein and internal aspartic acids of the protein (6, 7). Based on these results, it has been proposed that the interplay between the protonated Schiff base and a deprotonated and a protonated internal aspartic acid describes the principal features of the pump mechanism (5, 6). By using mutant proteins in which internal aspartic acids were altered, these two residues were identified as Asp-85 and Asp-96 by static FTIR difference spectroscopy (8, 9). Substitution of these two residues results in defective proton pumping by the mutated proteins (10, 11).To determine simultaneously the light-induced kinetics in various parts of the protein, including the protonation changes of Asp-85 and Asp-96 relative to the Schiff base, we performed time-resolved FTIR experiments and, in parallel, measured absorbance changes in the visible spectral range. MATERIALS AND METHODSPurple membrane was isolated as described (12). Wet, highly concentrated purple membrane pellets in distilled water were squeezed between two CaF2 windows, separated by a 2.5-,um spacer in a homemade sample chamber. The final OD was -0.5 and the pH -6. No salt was added. IR spectra were recorded on a Bruker IFS 88 instrument with the modifications and addition...

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Section: Discussioncontrasting

confidence: 50%

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

Gerwert¹,

Souvignier²,

Hess³

1990

Proc. Natl. Acad. Sci. U.S.A.

274

203

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“…Additionally, back-reactions were considered which were placed in the last part of the photoreaction cycle (Kouyama et al 1988, Fodor et al 1988). Similar conclusions were drawn by Chernavskii et al (1989) from temperature pulse experiments which indicated that the back-reactions from N to M and O to N cannot be neglected (see also Vfir6 and Lanyi 1990;Vfir6 et al 1990; Ames and Mathies 1990). Additionally, a possible branching of the photocycle was discussed.…”

Section: Introductionsupporting

confidence: 57%

“…However, discrete first order processes seem to be sufficient to describe the data if branching and back reactions are introduced (e.g. Maurer et al 1987 a, b;Kouyama et al 1988;Chernavskii et al 1989;Vfir6 and Lanyi 1990). To allow a comparison with data from the literature in the present study, a sum of first order exponentials is used to evaluate the time course of the reaction cycle.…”

Section: Data Evaluationmentioning

confidence: 99%

“…Maurer et al 1987 b;Kouyama et al 1988;Chernavskii et al 1989;Vfir6 and Lanyi 1990). For example, the accumulated evidence seems to indicate that the M decay is characterized by an equilibrium between the intermediates M and N and between N and O (Kouyama et al 1988;Chernavskii et al 1989) (see also Diller et al 1988 andButt et al 1989a). Applying these models, the rates determined in this study can only be considered as apparent kinetic constants composed of the intrinsic rates.…”

Section: Kinetic Description Of the Reaction Cyclementioning

confidence: 99%

“…These exponentials reflect a part of the photocycle whose mechanism is still controversial (e.g. Vfir6 et al 1990;V/tr6 and Lanyi 1990;Ames and Mathies 1990;Chernavskii et al 1989). The discussion involves the number of intermediates and the position of these intermediates in the photocycle.…”

Section: Apparent Rate Constants and Photocycle Intermediatesmentioning

confidence: 99%

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The reaction cycle of bacteriorhodopsin: an analysis using visible absorption, photocurrent and infrared techniques

Miller¹,

Butt

Bamberg

et al. 1991

Eur Biophys J

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The light activated absorbance changes and photo-electric events of bacteriorhodopsin (bR) were simultaneously measured. The results were compared with the kinetics of the time resolved infrared signals which are characteristic for protonation changes of Asp residues, chromophore vibrations, and amide I vibrations. Each data set was analyzed separately. Assuming first order reactions the experimental curves in the time range from L back to bR could be fitted by a sum of five exponentials. However, for the photocurrent signal only four exponentials were necessary. The corresponding half-life times were of the same order of magnitude. Simultaneous fits of the traces from absorption changes in the visible range and the photocurrent signal provided evidence that the photocurrent data could also be described by the same sum of exponentials as the data obtained in the visible range. The rate constants obtained from the different methods applied were, within the limits of error, identical. These results demonstrate that retinal monitors not only charge displacements but also conformational movements of the protein moiety. The reprotonation of the Schiff base occurs synchronously with a protonation change of an internal aspartic acid which absorbs at 1755 cm -1. From the IR-signals, amplitude spectra could be derived which provided evidence that Asp-residues absorbing at 1765cm -1 (Asp85) and 1755 cm 1 are still protonated in the O-intermediate. Major conformational changes of the peptide back bone occur in the time range of the L~ M transition and with opposite sign during the decay of the O-intermediate.

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The Photocycles of Bacteriorhodopsin

Lanyi

Váró

1995

Israel Journal of Chemistry

147

133

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The photoisomerization of all-trans-retinal of bacteriorhodopsin to 13-cis gives rise to a series of unstable states that thermally interconvert on the picosecond to millisecond timescale, and ultimately decay back to the initial state. Since this "photocycle" drives the translocation of a proton from the cytoplasmic to the extracellular side of the membrane, its detailed description is essential to understand the mechanism of the ion transport. The interconversions of the intermediates of the cycle, K 61O , L 550 , M 41O , N 560 , and 0 640 , proceed with complex multiphasic kinetics, and there is much disagreement over the interpretation of this. However, many groups have described the photocycle as a single reaction sequence with several reversible reactions, some pH-dependent. Through the use of time-resolved spectroscopy and site-specific mutations, such a scheme provides a reasonable model for how the retinal Schiff base first transfers its proton to Asp-85, followed by release of a proton to the extracellular side; then the Schiff base is reprotonated from Asp-96, and proton uptake from the cytoplasmic surface restores the initial protonation state of Asp-96, while the retinal reisomerizes to all-trans.

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

Cited by 49 publications

References 17 publications

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

The reaction cycle of bacteriorhodopsin: an analysis using visible absorption, photocurrent and infrared techniques

The Photocycles of Bacteriorhodopsin

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