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 reve aled five spectrally distinct metastable intermediates, as for the wild typ e. 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 i n the wild type protein. This mutant is therefore more amenable than the wi ld type to the determination of the intermediate spectra with the method of singular value decomposition with self-modeling, developed recently for th ree components (Zimanyi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4408 -4413, 4414-4419). The method provides the most reliable spectra so far, de fining the time evolution of the intermediates essential to the determinati on of the reaction scheme that describes the photocycle. The analysis confi rms published results on this mutant by and large, but revises the location s of the L intermediates in the photocycle. In addition, it allows identifi cation of the pH-dependent transitions of the photocycle, and offers an alt ernative mechanism for the pH dependence of the yield and kinetics of the l ate O intermediate.