Interconversions of the M, N, and O intermediates in the bacteriorhodopsin photocycle

The reaction sequence in the second half of the bacteriorhodopsin (BR) photocycle (i.e., the steps which involve the M, N, and O intermediates) was investigated with a gated multichannel analyzer. The difference spectra, obtained between 0.4 and 25 ms after laser photoexcitation, were converted to absolute spectra of the mixtures of intermediates at each delay time. From these, the time courses of the concentrations of M, N, O, and BR in a single turnover were reconstructed. We found that in 1 M Na2SO4, between pH 4 and 7, the measurements were not complicated by multiple kinetic forms of M; thus, the progressive changes in the rise and decay kinetics of N and O, as well as the recovery of BR, with pH could be followed. The data are inconsistent with a linear sequence but suggest a model in which N is produced directly from M, and returns to BR via two pathways: (a) O in equilibrium N----BR; (b) N in equilibrium O----BR. The individual rate constants of the reactions vary characteristically with pH. Because of these variations, pathway a predominates at pH less than 6 and results in the increased transient accumulation of O by equilibration with N at acidic pH. Pathway b begins to contribute to pH greater than 6 and results in the decreased accumulation of O but the increased accumulation of N at higher pH. Comparison of these results with the initial rate of proton transport between pH 4 and 7 indicates that proton translocation does not require that the BR photocycle pass through O.