Phase-lifetime spectroscopy of photocycle processes: kinetic analysis of amplitude dispersion curves.

Phase-lifetime spectroscopy has been recently used to obtain kinetic information on biological photocycles. A simple, general method is presented for deriving the amplitude response function for light-driven processes. These amplitude response functions may be used to analyze the experimental data obtained when driving the photosystem with a mechanically chopped, actinic light source. This analysis allows a comparison of kinetic parameters obtained from modulation methods with those obtained by flash techniques. Typically the experimental data consist of the signal amplitude measured at several chopping frequencies of the actinic light. These amplitude dispersion curves will be dependent on the harmonic sensitivity of the phase-sensitive detector used to measure the signal. This harmonic sensitivity is taken into account by performing a Fourier decomposition on the amplitude response function of the system and weighting each harmonic in a fashion appropriate for the specific amplifier under consideration. The resulting response function obtained for two commonly used amplifiers is derived. In addition to simple photocycles, the analysis of photocycle-coupled processes is also considered. This second relaxation process, which is coupled to a photocycle process, could represent the chemiosmotic coupling of a light-driven ion pump to a second ion transport protein. Conditions are established in which the kinetics of the second process can be resolved from the photocycle process.