Voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior, The

voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior, The

Geibel, Sven

ABSTRACT The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the ApH versus Atp dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one ApH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The Mz BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at – 160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived Ml* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.


Fendler for numerous valuable discussions.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB472).


Bamberg, E., and A. Fahr. 1980. Photocurrents induced on black lipid membranes by purple membranes: a method of reconstitution and a kinetic study of the photocurrents. Ann. N.Y. Acad. Sci. 358:324-327.

Braun, D., N. A. Dencher, A. Fahr, M. Lindau, and M. P. Heyn. 1988. Nonlinear voltage dependence of the light-driven proton pump current of bacteriorhodopsin. Biophys. J. 53:617-621.

Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7:2745-2752.

Dancshdzy, Zs., S. L. Helgerson, and W. Stoeckenius. 1983. Coupling between the bacteriorhodopsin photocycle kinetics and the protonmotive force. 1. Single flash measurements in Halobacterium halobium cells. Photobiochem. Photobiophys. 5:347-357.

Druckmann, S., M. Ottolenghi, A. Parade, J. Parade, and R. H. Callender. 1982. Acid-base equilibrium of the Schiff base in bacteriorhodopsin. Biochemistry. 21:4953-4959.

Essen, L., R. Siegert, W. D. Lehmann, and D. Oesterhelt. 1998. Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex. Proc. Natl. Acad. Sci. U.S.A. 95: 11673-11678.

Groma, G. L, S. L. Helgerson, P. K. Wolber, D. Beece, Z. Dancshazy, L. Keszthelyi, and W. Stoeckenius. 1984. Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. II. Quantitation and preliminary modeling of the M-bR reactions. Biophys. J. 45:985-992.

Grygorczyk, R., P. Hanke-Baier, W. Schwarz, and H. Passow. 1989. Measurement of erythroid band 3 protein-mediated anion transport in mRNA-injected oocytes of Xenopus laevis. Methods Enzymol. 173: 453-466.

Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391:85-100.

Henderson, R., J. M. Baldwin, T. A. Ceska, F. Zemlin, E. Beckmann, and K. H. Downing. 1990. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213: 899-929.

Kaim, G., and P. Dimroth. 1999. ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage. EMBO J. 18: 4118-4127.

Landau, E. M., and J. P. Rosenbusch. 1996. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 93:14532-14535.

Luecke, H., B. Schobert, H. T. Richter, J. P. Cartailler, and J. K. Lanyi. 1999. Structure of bacteriorhodopsin at 1.55 A resolution. J. Mol. Biol. 291:899-911.

Michel, H., and D. Oesterhelt. 1976. Light-induced changes of the pH gradient and the membrane potential in Halobacterium halobium. FEBS Lett. 65:175-178.

Nagel, G., B. Kelety, B. Mockel, G. Biildt, and E. Bamberg. 1998. Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of Ml to M2. Biophys. J. 74:403-412.

Nagel, G., B. Mockel, G. Biildt, and E. Bamberg. 1995. Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H+ pumping. FEBS Lett. 377: 263-266.

Oesterhelt, D., and B. Hess. 1973. Reversible photolysis of the purple complex in the purple membrane of Halobacterium halobium. Eur. J. Biochem. 37:316-326.

Ormos, P., Z. Dancshazy, and B. Karvaly. 1978. Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. Biochim. Biophys. Acta. 503:304-315.

Quintanilha, A. T. 1980. Control of the photocycle in bacteriorhodopsin by electrochemical gradients. FEBS Lett. 117:8-12.

Sheves, M., A. Albeck, N. Friedman, and M. Ottolenghi. 1986. Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues. Proc. Natl. Acad. Sci. U.S.A. 83:3262-3266.

Shull, G. E. 1990. cDNA cloning of the beta-subunit of the rat gastric H, K-ATPase. J. BioL Chem. 265:12123-12126.

Westerhoff, H. V., and Zs. Dancshdzy. 1984. Keeping a light-driven proton pump under control. TIBS. 9:112-117.

Sven Geibel,* Thomas Friedrich,* Pal Ormos,t Phillip G. Wood,* Georg Nagel,* and Ernst Bamberg*

*Max Planck Institut fiir Biophysik, D-60596 Frankfurt am Main, Germany; and tinstitute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary

Received for publication 30 October 2000 and in final form 15 June 2001. Address reprint requests to Dr. Ernst Bamberg, Max Planck Institut far Biophysik, Kennedyallee 70, D-60596 Frankfurt am Main, Germany. Tel.: 49-69-6303-300/301; Fax: 49-69-6303-305; E-mail: bamberg@ biophys.mpg.de.

Copyright Biophysical Society Oct 2001

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