Phase-lifetime spectroscopy of photocycle processes: proton release and uptake kinetics of purple membrane.

Phase-lifetime spectroscopy was used to measure the kinetics of light-driven proton transients and M intermediate decay of purple membrane. The kinetics were measured in distilled water and in 0.4 M KCl over the pH range of 5.3-8.8. The low ionic strength data showed results that were comparable to previous data. The lifetimes for the decay of the proton transient were intermediate between the two M decay lifetimes. Deviations from this behavior occurred at pH 5.3. The high-salt data showed two proton transients: a release process with a lifetime similar to the fast M decay, and an uptake process with a lifetime similar to the slow M decay. The release process is not seen in flash experiments. This difference is due to differences in the amplitudes of the relaxation processes for the two types of experiments. The high-salt data showed little pH dependence below pH 7.8. Both sets of data were shown to be consistent with a single mechanism that correlates the M kinetics with the proton kinetics. The proposed mechanism consists of the parallel formation and decay of two M intermediates that can interconvert. Only one of the intermediates results in a proton being released from the bacteriorhodopsin. Thus, this mechanism has a nonproductive pathway as well as a productive pathway in which a single proton is pumped. Detailed kinetic expressions are derived for this mechanism for the phase-lifetime and flash kinetic experiment. These results could explain the qualitative features of the distilled water data and provide a quantitative description of the high-salt data. Expressions for the quantum yield and the H(+) to M ratio were derived in terms of the individual rate constants of the mechanism. Conditions are established in which the quantum yield for proton release can vary with only small changes in the M/H(+). This reconciles an apparent conflict in previous evidence from flash experiments and demonstrates the difficulty of determining stoichiometry from kinetic amplitude information.