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.
INTRODUCTION
Fendler for numerous valuable discussions.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB472).
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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.
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