Procedure for testing kinetic models of the photocycle of bacteriorhodopsin

Given some simple kinetic models of the photocycle of bacteriorhodopsin (bR) and data taken at many wavelengths and under conditions that avoid photoselection and steady-state cycling complications, it is shown how to extract the apparent rate constants and the spectra of the intermediates. Special consideration was given to establishing the range of error of these results. There are many criteria, which we explicitly discuss, that the spectra should satisfy in order that the kinetic model be acceptable. New data for the photocycle of purple membrane fragments in dilute buffer at pH 7.0 has been obtained at 15 measuring wavelengths and four temperatures. The procedure, which can be generalized to more complex models, has been applied to these data to test two kinds of kinetic models: the unidirectional unbranched model and the undirectional model with simple branching straight back to bR from any intermediate. In these models the spectrum of the O intermediate is highly temperature sensitive, even with branching, and/or has two broad maxima. Moreover, the spectrum of the M intermediate has a secondary maximum and two M-like states appear to be required. Thus, neither model satisfies the physical criteria.