RELATIONSHIP OF PROTON UPTAKE ON THE CYTOPLASMIC SURFACE AND REISOMERIZATION OF THE RETINAL IN THE BACTERIORHODOPSIN PHOTOCYCLE - AN ATTEMPT TO UNDERSTAND THE COMPLEX KINETICS OF THE PH CHANGES AND THE N AND OINTERMEDIATES

In the bacteriorhodopsin photocycle the recovery of the initial BR sta te from the M intermediate occurs via the N and O intermediates. The m olecular events in this process include reprotonation of the Schiff ba se and the subsequent uptake of a proton from the cytoplasmic side, as well as reisomerization of the retinal from 13-cis to all-trans. We h ave studied the kinetics of the intermediates and the proton uptake. A t moderately low pH little of the N state accumulates, and the O state dominates in the reactions that lead from M to BR. The proton uptake lags behind the formation of O, suggesting the sequence N(0) <-> O(0) + H+ (from the bulk) --> O(+1) --> BR + H+ (to the bulk), where the su perscripts indicate the net protonation state of the protein relative to BR. Together with a parallel study of ours at moderately high pH, t hese results suggest that the sequence of proton uptake and retinal re isomerization depends on pH: at low pH the isomerization occurs first and O accumulates, but at high pH the isomerization is delayed and the refore N accumulates. Although this model contains too many rate const ants for rigorous testing, we find that it will generate most of the c haracteristic pH-dependent kinetic features of the photocycle with few assumptions other than pH dependency for protonation at the proton re lease and uptake steps.