THE PHOTORECEPTOR SENSORY RHODOPSIN-I AS A 2-PHOTON DRIVEN PROTON PUMP

Proton translocation experiments with intact cells of Halobacterium salinarium overproducing sensory rhodopsin I (SRI) revealed transport activity of SRI in a two-photon process, The vectoriality of proton translocation depends on pH, being outwardly directed above, and inwardly directed below, pH 5.7. Activation of the transport cycle requires excitation of the initial dark state of SRI, SRI(590), to form the intermediate SRI(380). Action spectra identify the photocycle intermediates SRI(380) and SRI(520) as the two photochemically reactive species in the outwardly directed transport process. As shown by flash photolysis experiments, SRI(520) undergoes a so-far unknown photochemical reaction to SRI(380) With a half-time of <200 mu s. Mutation of SRI residue Asp-76, the residue which is equivalent to the proton acceptor Asp-85 in bacteriorhodopsin, to asparagine leads to inactivation of proton translocation. This demonstrates that the underlying mechanisms of proton transport in both retinal proteins share similar features, However, SRI is to our knowledge the first case where photochemical reactions between two thermally unstable photoproducts of a retinal protein constitute a catalytic ion transport cycle.