Mechanism of proton entry into the cytoplasmic section of the proton-conducting channel of bacteriorhodopsin.
Bacteriorhodopsin is the light-driven proton-pumping protein of Halobacterium salinarum that extracts protons from the well-buffered cytoplasmic 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 fluorescein 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 photoexcited pyranine [Nachliel, E., Gutman, M., Kiryati, S., and Dencher, N.A. (1996) Proc. Nat Acad. Sci. U.S.A. 93, 10747-10752). The reaction of the discharged protons with the pyranine anion and the fluorescein was measured with sub-microsecond resolution. The experimental signals were reconstructed through numeric integration of differential rate equations which quantitated the rates of all proton transfer reactions between all reactants present in the system. The interaction of protons with the orifice of the cytoplasmic channel is enhanced by the exposed carboxylates of the protein. A cluster of three carboxylates acts as a strong proton attractor site while one carboxylate, identified as D36, acts as a mediator that delivers the proton to the channel. The combination of these reactions render the surface of the protein with properties of a proton-collecting antenna. The size of the collecting area is less than that of the protein's surface.
Checover
S
S
Department of Biochemistry, Tel Aviv University, Israel.
Nachliel
E
E
Dencher
N A
NA
Gutman
M
M
D001436:Bacteriorhodopsins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D019617:Halobacterium salinarum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D017494:Proton Pumps / Q000378:metabolism*; D011522:Protons; D018717:Purple Membrane / Q000378:metabolism; D013053:Spectrophotometry; D017186:Titrimetry
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007473:Ion Channels; D017494:Proton Pumps; D011522:Protons; D001436:Bacteriorhodopsins; C037041:fluorescein 5-maleimide