MECHANISM OF PROTON ENTRY INTO THE CYTOPLASMIC SECTION OF THE PROTON-CONDUCTING CHANNEL OF BACTERIORHODOPSIN

Bacteriorhodopsin is the light-driven proton-pumping protein of Haloba cterium salinarum that extracts protons from the well-buffered cytopla smic space within the time limits set by the photocycle turnover. The specific mechanism of the proton uptake by the cytoplasmic surface of the protein was investigated in this study by the laser-induced proton pulse technique. The purple membrane preparations were labeled by flu orescein at two residues (36 or 38) of the cytoplasmic surface of the protein, sites that are close to the orifice of the proton-conducting channel. The membranes were pulsed by protons discharged from photoexc ited pyranine [Nachliel, E., Gutman, M., Kiryati, S,, and Dencher, N. A. (1996) Proc, Nar Acad. Sci. U.S.A. 93, 10747-10752). The reaction o f the discharged protons with the pyranine anion and the fluorescein w as measured with sub-microsecond resolution. The experimental signals were reconstructed through numeric integration of differential rate eq uations which quantitated the rates of all proton transfer reactions b etween all reactants present in the system. The interaction of protons with the orifice of the cytoplasmic channel is enhanced by the expose d carboxylates of the protein, A cluster of three carboxylates acts as a strong proton attractor site while one carboxylate, identified as D 36, acts as a mediator that delivers the proton to the channel. The co mbination of these reactions render the surface of the protein with pr operties of a proton-collecting antenna. The size of the collecting ar ea is less than that of the protein's surface.