Aspartic Acid-96 is the Internal Proton Donor in the Reprotonation of the Schiff Base of Bacteriorhodopsin

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.