Time‐course and stoichiometry of light‐induced proton release and uptake during the photocycle of bacteriorhodopsin

The kinetics and stoichiometry of light‐induced proton release in purple membrane suspensions have been investigated using the pH‐indicator dye pyranine and single‐turnover flash spectroscopy at a time resolution of 0.1 μs. The number of protons detected by pyranine is inversely proportional to the buffering power of the medium and 1.1 ± 0.2 protons are released per photocycling bacteriorhodopsin molecule (pH 7.0, 20°C). At high ionic strength (250 mM KCl) and higher phosphate buffer concentrations (1 mM) the proton release lags slightly behind the formation of the intermediate M‐412 and proton reuptake by the purple membrane parallels the slow decay process of M‐412 or the decay of O‐640.