PATHWAY OF PROTON UPTAKE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The time courses of chromophore reactions and proton uptake in the sec ond half of the photocycle of the proton pump bacteriorhodopsin (BR) w ere examined. At pH >8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics af ter perturbation of M with a second, blue flash confirm that M <-> N e quilibration is the only significant cause of the biphasic M decay. Wi th this feature, the analysis of time-resolved difference spectra yiel ds a scheme which contains two sequential N states connected by a near ly unidirectional reaction. The proton uptake from the bulk, as measur ed with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the m odel M2(-1) <-> N(-1) + H+ (from the bulk) <-> N(0) --> BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1) --> N(-1) is reprotonation of the Schiff base from D96, N( -1) + H+ (from the bulk) --> N(0) is uptake of proton from the cytopla smic side, and N(0) --> BR represents 13-cis to all-trans reisomerizat ion of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/ [N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pK(a) o f D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1) <-> N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pK(a) for pro ton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the p roton uptake kinetics that at physiological pH free energy is converte d to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimanyi, L., Varo. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 853 5-8543] but also as protons are taken up on the cytoplasmic side.