Influence of an electrical potential on the charge transfer kinetics of bacteriorhodopsin

Kleinschmidt, Christoph; Hess, Benno

doi:10.1016/s0006-3495(90)82408-5

Cited by 4 publications

(3 citation statements)

References 19 publications

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“…The millisecond kinetics of the charge motion induced in the remaining unphotolyzed population by the second flash is therefore slowed down. This interesting phenomenon of the effect of an electric potential on the charge transfer kinetics has also been discussed elsewhere (Kleinschmidt and Hess, 1990;Nieto-Frausto et al, 1992) and explains the following two experimental observations. The amplitude of the millisecond electrical component induced by the second flash is quite small (Fig.…”

Section: Discussionsupporting

confidence: 70%

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

Dickopf¹,

Heyn²

1997

Biophysical Journal

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The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pK's of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.

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Section: Discussionsupporting

confidence: 70%

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

Dickopf¹,

Heyn²

1997

Biophysical Journal

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“…In the case of the DEM and related systems for measuring voltages and currents, however, this simple demonstration is insufficient because uncouplers will also lower the RC time constant and therefore the electrical time response of the system. An early indication that back‐pressure may be operative in the formation and dissipation of voltage in PM was that of Kleinschmidt and Hess [7]. They studied current flow in PM fixed to a black lipid membrane (BLM).…”

Section: Discussionmentioning

confidence: 99%

“…In the current paper, which utilizes the uncouplers carbonyl cyanide m ‐chlorophenylhydrazone (CCCP) and valinomycin, we demonstrate that the kinetics of voltage generation in purple membranes (PM) from Halobacterium salinarium are strongly affected by Δµ˜ H+ . An earlier observation that indicated the possible influence of Δµ˜ H+ on voltage formation and dissipation was that of Kleinschmidt and Hess [7]. However, the conclusion, based on experiments utilizing uncouplers, that Δµ˜ H+ can directly influence the kinetics of voltage transitions of photocycle intermediates through back‐pressure effects is not readily deducible because of a possible alternative explanation based on the RC time constant of the system.…”

mentioning

confidence: 99%

On the kinetics of voltage formation in purple membranes of Halobacterium salinarium

Hendler

Drachev

Bose

et al. 2000

European Journal of Biochemistry

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Skulachev, V.P. (1974) Nature 249, 321±324 are sixfold slower than the kinetics obtained in optical studies with suspensions of purple membrane patches. In this study, we have investigated the reasons for this discrepancy. In the presence of the uncouplers carbonyl cyanide m-chlorophenylhydrazone or valinomycin, the rates in the DEM system are similar to the rates in suspensions of purple membrane. Two alternative explanations for the effects of uncouplers were evaluated: (a) the`back-pressure' of the Dm Ä H1 slows the kinetic steps leading to its formation, and (b) the apparent difference between the two systems is due to slow major electrogenic events that produce little or no change in optical absorbance. In the latter case, the uncouplers would decrease the RC time constant for membrane capacitance leading to a quicker discharge of voltage and concomitant decrease in photocycle turnover time. The experimental results show that the primary cause for the slower kinetics of voltage changes in the DEM system is thermodynamic back-pressure as described by Westerhoff, H. Previous studies [6], using the same current clamp methods as we use here, have shown similar kinetics for voltage transitions as found by optical transitions in the closed vesicles cited above, and as we report here using the direct electrical measurement (DEM) system. In the current paper, which utilizes the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, we demonstrate that the kinetics of voltage generation in purple membranes (PM) from Halobacterium salinarium are strongly affected by Dm Ä H1 . An earlier observation that indicated the possible influence of Dm Ä H1 on voltage formation and dissipation was that of Kleinschmidt and Hess [7]. However, the conclusion, based on experiments utilizing uncouplers, that Dm Ä H1 can directly influence the kinetics of voltage transitions of photocycle intermediates through back-pressure effects is not readily deducible because of a possible alternative explanation based on the RC time constant of the system. In this paper, we describe how effects resulting from by changes to RC time constants can be separated from those due to back-pressure. We find that back-pressure is the predominant factor in the slow kinetics of voltage generation and dissipation that we have measured. E X P E R I M E N T A L P R O C E D U R E S Direct electrical measurementsThe apparatus used for the DEM [8] and the use of a nonbiological membrane as a support for the biological membrane under study [9] have been previously described. In our work, we tried both a collodion film (0.2 m thick) and a porous Teflon film, impregnated with phospholipids dissolved in decane as the supporting membrane. The Teflon film was prepared from plumber's polytetrafluoroethylene thread seal Teflon tape [10]. Our best results were obtained with tape manufactured in Malaysia, which is readily available in hardware stores. The tape was heated to 90±100 8C, then held by clamps and stretched to about 20 times its width. We ...

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Kinetics of Light‐Induced Intramolecular Charge Transfer and Proton Release in Bacteriorhodopsin

Moltke¹,

Alexiev²,

Heyn³

1995

Israel Journal of Chemistry

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Abstract. Recent advances in understanding the intramolecular charge transfer and proton release by the light-driven proton pump bacteriorhodopsin (bR) are critically reviewed. The focus is on the time-resolved electrical methods, i.e., photocurrent and photovoltage measurements, and on transient absorption experiments with pH-sensitive dyes. Particular attention is paid to the following topics: charge translocation in the low-pH forms of bR (acid-blue and acid-purple); electrogenicity of the 13-cis cycle; results with bR mutants; surface-bound dyes to detect the proton release at specific sites on either side of the protein; the question of kinetic coupling between the deprotonation of the Schiff base and proton release; and rapid long-range migration of protons along the surface of the purple membrane.

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Influence of an electrical potential on the charge transfer kinetics of bacteriorhodopsin

Cited by 4 publications

References 19 publications

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

On the kinetics of voltage formation in purple membranes of Halobacterium salinarium

Kinetics of Light‐Induced Intramolecular Charge Transfer and Proton Release in Bacteriorhodopsin

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