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

Proton translocation experiments with intact cells of Halobacterium sa linarium overproducing sensory rhodopsin I (SRI) revealed transport ac tivity of SRI in a two-photon process, The vectoriality of proton tran slocation depends on pH, being outwardly directed above, and inwardly directed below, pH 5.7. Activation of the transport cycle requires exc itation of the initial dark state of SRI, SRI(590), to form the interm ediate SRI(380). Action spectra identify the photocycle intermediates SRI(380) and SRI(520) as the two photochemically reactive species in t he outwardly directed transport process. As shown by flash photolysis experiments, SRI(520) undergoes a so-far unknown photochemical reactio n to SRI(380) With a half-time of <200 mu s. Mutation of SRI residue A sp-76, the residue which is equivalent to the proton acceptor Asp-85 i n bacteriorhodopsin, to asparagine leads to inactivation of proton tra nslocation. This demonstrates that the underlying mechanisms of proton transport in both retinal proteins share similar features, However, S RI is to our knowledge the first case where photochemical reactions be tween two thermally unstable photoproducts of a retinal protein consti tute a catalytic ion transport cycle.