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Photoexcitation of the O-Intermediate in Bacteriorhodopsin Mutant L93A

R. Tóth-Boconádi ^*, L. Keszthelyi ^* and W. Stoeckenius 

^* Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary; and Department of Biochemistry and Cardiovascular Research Institute, University of California, San Francisco, California, and Department of Chemistry, University of California at Santa Cruz, Santa Cruz, California USA

Correspondence: Address reprint requests to Dr. Lajos Keszthelyi, Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Temesvari krt. 62, Hungary. Tel.: 36-62-599-615; Fax: 36-62-433-133; E-mail: kl{at}nucleus.szbk.u-szeged.hu.

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime 140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.

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