PHOTOREGENERATION OF BOVINE RHODOPSIN FROM ITS SIGNALING STATE

In rhodopsin, 11-cis-retinal is bound by a protonated Schiff base and acts as a strong antagonist, which holds the receptor in its inactive ground state conformation, Light induces cis-/trans-retinal isomerization and a sequence of thermal transitions through intermediates. The active conformation that catalyzes GDP/GTP exchange in the G-protein (G(t)) is generated from the metarhodopsin II intermediate (MII) and mediated by Schiff base proton translocation and proton uptake from the aqueous phase. In the stable nucleotide-free MII-G(t) complex, any thermal transition of MII into other forms of rhodopsin is blocked, We have now studied how G(t) affects flash-induced photochemical conversions of MII, Difference spectra from measured absorption changes show that MII photolyzes through two parallel pathways, with fast (1 ms) and slow (50 ms) kinetics (12 degrees C, pH 6). The slow pathway regenerates rhodopsin (9- or 11-cis) via Schiff base reprotonation and proton release. We infer a cis-isomerized early photoproduct (reverted meta, RM) preceding these thermal transitions. When MII is photolyzed in the MII-G(t) complex, the slow absorption change is abolished, indicating that G(t) blocks the completion of the regeneration process, This is due to the formation of a stable RM-G(t) complex, as shown by successive photolysis of MII, RM, and ground state rhodopsin, and the application of GTP gamma S at different stages. The complex dissociates with GTP gamma S, and rhodopsin relaxes to the ground state, The results indicate that cis-retinal and G(t) can bind to the receptor at the same time. We discuss the result that the protonations in the meta II state uncouple retinal geometry from G(t) interaction.