A priori resolution of the intermediate spectra in the bacteriorhodopsin photocycle: the time evolution of the L spectrum revealed.

Resolution of the spectra of the intermediates in the photocycle of wild-type bacteriorhodopsin (BR) was achieved by singular value decomposition with exponential-fit-assisted self-modeling (SVD-EFASM) treatment of multichannel difference spectra measured at 5 degrees C during the course of the photocycle. New is the finding that two spectrally distinct L intermediates, L(1) and L(2), form sequentially. Our conclusion is that the photocycle is more complex than most published schemes. The dissection of the spectrally different L forms eliminates stoichiometric discrepancies usually appearing as systematically varying total intermediate concentrations before the onset of BR recovery. In addition, our analysis reveals that the red tails in the spectra of K and L(1) are more substantial than those of L(2) and BR. We suggest that these subtle differences in the shapes of the spectra reflect torsional and/or environmental differences in the retinyl chromophore.