Dissecting the photocycle of the bacteriorhodopsin E204Q mutant from kinetic multichannel difference spectra. Extension of the method of singular value decomposition with self-modeling to five components.

Kinetic multichannel difference spectroscopy in the visible spectral range of the Glu204 --> Gln(E204Q) site-directed mutant of bacteriorhodopsin revealed five spectrally distinct metastable intermediates, as for the wild type. Due to the perturbation of the extracellular proton release cluster, the late O intermediate accumulates in much higher amounts in this mutant, and the photocycle is not complicated by the pH-dependent branching observed in the wild type protein. This mutant is therefore more amenable than the wild type to the determination of the intermediate spectra with the method of singular value decomposition with self-modeling, developed recently for three components (Zimányi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4408-4413, 4414-4419). The method provides the most reliable spectra so far, defining the time evolution of the intermediates essential to the determination of the reaction scheme that describes the photocycle. The analysis confirms published results on this mutant by and large, but revises the locations of the L intermediates in the photocycle. In addition, it allows identification of the pH-dependent transitions of the photocycle, and offers an alternative mechanism for the pH dependence of the yield and kinetics of the late O intermediate.