Protein configuration changes in the photolysis of rhodopsin II. The sequence of intermediates in thermal decay of cattle metarhodopsin in vitro

Abstract

On illumination of solutions of the visual chromoprotein, cattle rhodopsin, there is a sequence of thermal, first-order reactions. Whereas previous work has shown the first two appear to involve a rearrangement of the protein structure in which the solvent plays only a secondary role, the thermal changes beginning with metarhodopsin and terminating with a hydrolysis yielding the protein, opsin and trans-retinal all involve the solvent directly. Our studies show that there are five intermediates in the solvent-dependent sequence (subscripts denote spectral maxima): Previous flash-photolysis studies1 and the present kinetic data are consistent with the notion of three forms of metarhodopsin478 decaying by first-order, pH-dependent processes to metarhodopsin380. This is also borne out by the behavior of the metarhodopsin478 ↔ metarhodopsin380 reaction at different temperatures and pH's. The decay of metarhopopsin380 to metarhodopsin465 has a small temperature coefficient, a large negative ΔS∗, and is slow enough to be rate controlling at physiological temperatures. Metarhodopsin465 (transient orange) decays to N-retinylideneopsin (indicator yellow) by a single first-order process and like metarhodopsin478 → metarhodopsin380 has a large positive ΔH∗ and ΔS∗ rate constant is about io6 times smaller. Irradiation of metarhodopsin465 yields rhodopsin498, isorhodopsin487 and cis-isomers of retinal. The hydrolysis of N-retinylidene-opsin to retinal and opsin is faster than the metarhodopsin465 → N-retinylidene-opsin process at pH 7 but is sufficiently slower and lower and higher pH's to be observed.