Photovoltage evidence that Glu-204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied, A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown, This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the mild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state (<3) and far below the pK of the terminal proton release group (similar to 6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump. (C) 1998 Federation of European Biochemical Societies.