TRANSDUCER PROTEIN HTRI CONTROLS PROTON MOVEMENTS IN SENSORY RHODOPSIN-I

Sensory rhodopsin I (SR-I lambda(max) 587 nm) is a phototaxis receptor in the archaeon Halobacterium salinarium. Photoisomerization of retin al in SR-I generates a long-lived intermediate with lambda(max) 373 nm which transmits a signal to the membrane-bound transducer protein Htr I. Although SR-I is structurally similar to the electrogenic proton pu mp bacteriorhodopsin (BR), early studies showed its photoreactions do not pump protons, nor result in membrane hyperpolarization. These stud ies used functionally active SR-I, that is, SR-I complexed with its tr ansducer HtrI. Using recombinant DNA methods we have expressed SR-I pr otein containing mutations in ionizable residues near the protonated S chiff base, and studied wild-type and site-specifically mutated SR-I i n the presence and absence of the transducer protein. UV-Vis kinetic a bsorption spectroscopy, FT-IR, and pH and membrane potential probes re veal transducer-free SR-I photoreactions result in vectorial proton tr anslocation across the membrane in the same direction as that of BR. T his proton pumping is suppressed by interaction with transducer which diverts the proton movements into an electroneutral path. A key step i n this diversion is that transducer interaction raises the pK(a) of th e aspartyl residue in SR-I (Asp76) which corresponds to the primary pr oton-accepting residue in the BR pump (Asp85). In transducer-free SR-I , our evidence indicates the pK(a) of Asp76 is 7.2, and ionized Asp76 functions as the Schiff base proton acceptor in the SR-I pump. In the SR-I/HtrI complex, the pK(a) of Asp76 is 8.5, and therefore at physiol ogical pH (7.4) Asp76 is neutral. Protonation changes on Asp76 are cle arly not required for signaling since the SR-I mutants D76N and D76A a re active in phototaxis. The latent proton-translocation potential of SR-I may reflect the evolution of the SR-I sensory signaling mechanism from the proton pumping mechanism of BR.