Mechanism of Retinal Schiff Base Formation and Hydrolysis in Relation to Visual Pigment Photolysis and Regeneration: Resonance Raman Spectroscopy of a Tetrahedral Carbinolamine Intermediate and Oxygen-18 Labeling of Retinal at the Metarhodopsin Stage in Photoreceptor

Article abstract of DOI:10.1021/ja00258a002

The mechanism of formation and hydrolysis of N-retinylidene-n-butylamine, as a model of the rhodopsin chromophore, has been investigated by a study of the kinetic and equilibrium properties in aqueous anionic, cationic, and neutral detergent micelle systems.The pH dependence of steady-state formation and hydrolysis rate constants is consistent with the classical imine reaction mechanism involving tetrahedral carbinolamine intermediates.Kinetic transients consistent with such intermediates can be seen using rapid stopped-flow techniques.Hydrolysis rates in neutral detergent micelles exhibit general base catalysis, and there are pronounced detergent-specific effects which can be qualitatively interpreted in terms of ionic effects on Schiff base pK_a and micellar hydrogen ion activities.This suggests a rational explanation for the anomalous pK_a and thermodynamic stability of visual pigment chromophores under physiological conditions.The tetrahedral intermediate has been observed directly at room temperature by continuous-flow, pH-jump resonance Raman spectroscopy, and the spectrum of this transient species shows remarkable similarity with the previously reported Raman spectrum of the metarhodopsin II intermediate of bovine rhodopsin photolysis.Isotope-labeling experiments on bovine photoreceptor membranes exposed to oxygen-18 enriched water during bleaching show incorporation of 18O at the retinal aldehyde site during the metarhodopsin I -> II transition.These observations support the hypothesis that the vertebrate Meta I -> Meta II transition involves hydrolytic attack by water on the retinyl-lysine Schiff base linkage of the rhodopsin chromophore.