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 st udied, A two-fold decrease in the magnitude of microsecond photovoltag e generation coupled to M intermediate formation in the E204Q mutant i s shown, This means that deprotonation of E204 is an electrogenic proc ess and its electrogenicity is comparable to that of the proton transf er from the Schiff base to D85. pH dependence of the electrogenicity o f M intermediate formation in the mild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bact eriorhodopsin ground state and transition of the purple neutral form i nto the blue acid form. Thus, the pK of E204 in the M state is close t o the pK of D85 in the bacteriorhodopsin ground state (<3) and far bel ow the pK of the terminal proton release group (similar to 6). It is c oncluded that E204 functions as the intermediate proton donor rather t han the terminal proton release group in the bacteriorhodopsin proton pump. (C) 1998 Federation of European Biochemical Societies.