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

Moltke, Stephan; Alexiev, Ulrike; Heyn, Maarten P.

doi:10.1002/ijch.199500039

Cited by 11 publications

(17 citation statements)

References 82 publications

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“…The amplitude of the slow electrical component u2 never exceeds 40% of the total amplitude, whereas the slow absorption amplitude a2 is largest in all cases, except for delays shorter than 200 ,us. The interpretation of the electrical amplitudes is more complicated than that of the optical data, because the voltage associated with a transition is proportional to the distance the charge moved as well as inversely proportional to the local instantaneous dielectric constant (Trissl, 1990;Moltke et al, 1995). The electrical amplitudes are therefore not related to the optical ones in a simple way.…”

Section: Discussionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

“…The time-resolved photovoltage measurements were performed with the method of capacitative coupling, in which purple membranes are adsorbed to a lipid-impregnated support foil, as described Moltke et al, 1995). The experimental setup for the electrical double-flash experiments is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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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“…Proton movement during the bacteriorhodopsin photocycle has been investigated using both photocurrent and photovoltage measurements, on bR oriented within lipid bilayers, upon a substrate, or within gels (reviewed in Trissl, 1990;Läuger, 1991;Moltke et al, 1995). Individual charge movements coincide with transitions between the photointermediates of the photocycle.…”

Section: Introductionmentioning

confidence: 99%

Contribution of Proton Release to the B2 Photocurrent of Bacteriorhodopsin

Misra

1998

Biophysical Journal

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The contribution of proton release from the so-called proton release group to the microsecond B2 photocurrent from bacteriorhodopsin (bR) oriented in polyacrylamide gels was determined. The fraction of the B2 current due to proton release was resolved by titration of the proton release group in M. At pH values below the pKa of the proton release group in M, the proton release group cannot release its proton during the first half of the bacteriorhodopsin photocycle. At these pH values, the B2 photocurrent is due primarily to translocation of the Schiff base proton to Asp85. The B2 photocurrent was measured in wild-type bR gels at pH 4.5-7.5, in 100 mM KCl/50 mM phosphate. The B2 photocurrent area (proportional to the amount of charge moved) exhibits a pH dependence with a pKa of 6.1. This is suggested to be the pKa of the proton release group in M; the value obtained is in good agreement with previous results obtained by examining photocycle kinetics and pH-sensitive dye signals. In the mutant Glu204Gln, the B2 photocurrent of the mutant membranes was pH independent between pH 4 and 7. Because the proton release group is incapacitated, and early proton release is eliminated in the Glu204Gln mutant, this supports the idea that the pH dependence of the B2 photocurrent in the wild type reflects the titration of the proton release group. In wild-type bacteriorhodopsin, proton release contributes approximately half of the B2 area at pH 7.5. The B2 area in the Glu204Gln mutant is similar to that in the wild type at pH 4.5; in both cases, the B2 current is likely due only to movement of the Schiff base proton to Asp85.

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“…Proton release and uptake were detected in the aqueous bulk medium of the purple membrane suspension, containing 4 -15 M bR in 150 mM KCl, by calculating the difference of the measured flash-induced absorbance changes at 450 nm between samples with and without 45 M pyranine at pH 7.3 and 22°C (25,29). The light-induced proton concentration changes, detected in samples with fluorescein attached to cysteine residues in bR, were determined by calculating the measured flash-induced absorbance difference at 495 nm between samples with and without 10 mM Tris or MOPS buffer, pH 7.3, in 150 mM KCl at 22°C (25,29).…”

mentioning

confidence: 99%

“…The light-induced proton concentration changes, detected in samples with fluorescein attached to cysteine residues in bR, were determined by calculating the measured flash-induced absorbance difference at 495 nm between samples with and without 10 mM Tris or MOPS buffer, pH 7.3, in 150 mM KCl at 22°C (25,29).…”

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confidence: 99%

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Alexiev¹,

Mollaaghababa²,

Khorana³

et al. 2000

Journal of Biological Chemistry

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Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/ G231C. (i) In the double mutants R82A/G72C and R82A/ A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay ( ϳ 8 ms), in R82A it occurs already during the first phase of the M-rise ( < 1 s). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical Gprotein-coupled receptors.Allosteric interactions are well known and of great importance for water soluble enzymes, where ligand or substrate binding to a specific site alters the conformation and changes the affinity at a different site of the protein (for reviews see Refs. 1 and 2). For membrane-bound G-protein-coupled receptors analogous effects are expected to be of considerable functional significance. For this class of proteins ligand binding occurs mostly on the extracellular surface resulting in the active form of the receptor with binding and activation of the G-protein at the opposite cytoplasmic surface of the protein.The membrane protein bacteriorhodopsin (bR) 1 shares the heptahelical bundle motif with G-protein-coupled receptors. In this report, we present evidence for long range interactions between the extracellular and cytoplasmic parts of bR based on evidence from the mutant R82A and its second site revertant R82A/G231C.Bacteriorhodopsin acts as a light-driven proton pump in the plasma membrane of the archaebacterium Halobacterium salinarium. A retinylidene chromophore is bound via a protonated Schiff base linkage to Lys-216. Upon flash excitation bR undergoes a cyclic photoreaction with distinct spectroscopic intermediates and proton translocation from one side of the membrane to the other (for reviews see Refs. 3-5). In the first half of this photocycle, the Schiff base becomes deprotonated during the transition to the ...

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

Cited by 11 publications

References 82 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

Contribution of Proton Release to the B2 Photocurrent of Bacteriorhodopsin

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

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