A Photoelectrochemical Evidence for the Role of Glutamate at the Extracellular Proton-releasing Residue Site in Bacteriorhodopsin

Abstract

Participation of Glu-194 and Glu-204 in the photo-induced proton releasing reaction at the extracellular surface of bacteriorhodopsin molecule was examined by a photoelectrochemical means in which films of bacteriorhodopsin and its site-displacement mutants are immobilized at the electrode-electrolyte interface. Displacement of glutamate with aspartate at the residue site 194 and 204 suppressed the rate of proton release with distinct pH dependence characteristics, indicating that pKa’s of these acidic residues are crucial for activating the proton release. In pH dependence, the mutant of Glu-194 to Asp-194 displacement showed a pKa of proton release much increased from that of the wild type. Laser pulse excitation of the mutant showed large retardation in proton release rate. Glu-194 is thus regarded to participate in the pKa-controlled proton transfer relay from Schiff base to Asp-85 in the reaction center core and work as a terminal proton releaser at the extracellular surface.