Effects of temperature on rhodopsin photointermediates from lumirhodopsin to metarhodopsin II

Abstract

Absorbance changes following the photolysis of mildly sonicated membrane suspensions of bovine rhodopsin are monitored using multichannel detection at 15, 20, 25, 30, and 35 degrees C. Difference spectra collected with microsecond time resolution are analyzed by singular value decomposition and multiexponential fitting. Several kinetic schemes are tested using methods that compare the observed rates and associated spectral amplitudes to the eigenvalues and eigenvectors of kinetic matrices. The time evolution of the spectra is more complex than can be accounted for by the traditional lumi-->metarhodopsin I<-->metarhodopsin II scheme. Above 25 degrees C, the formation of metarhodopsin II is achieved without a large transient accumulation of metarhodopsin I. Within the framework of first-order kinetics, the observations are explained by simple kinetic schemes that lead to the formation of a deprotonated Schiff's base species temporally distinct from metarhodopsin II directly upon the decay of lumirhodopsin.