Time-resolved FT-IR spectroscopic investigation of the pH-dependent protontransfer reactions in the E194Q mutant of bacteriorhodopsin

The photoreaction of the E194Q mutant of bacteriorhodopsin has been investi gated at various pH values by time-resolved step-scan Fourier-transform inf rared difference spectroscopy employing the attenuated total reflection tec hnique. The difference spectrum at pH 8.4 is comparable to the N-BR differe nce spectra of the wild type with the remarkable exception that D85 is depr otonated. Since the retinal configuration is not perturbed by the E194Q mut ation, it is concluded that there is no interaction of D85 with retinal dur ing the lifetime of the N state. At pH 6, a consecutive state to the O inte rmediate is detected in which D212 is transiently protonated. The compariso n with wild-type bacteriorhodopsin reveals that protonation of D212 represe nts an intermediate step during proton transfer from D85 to the proton rele ase group in the final stage of the reaction cycle. The described effects a re more pronounced in the E194Q mutant than in the E204Q mutant demonstrati ng different roles of these two glutamates/glutamic acids at least in the f inal stages of the catalytic cycle of bacteriorhodopsin. (C) 2001 Academic Press.