Solvent isotope effects on retinal cis-trans isomerization in the dark adaptation of bacteriorhodopsin

The solvent isotope effect on the first-order rate constant for dark adaptation of bacteriorhodopsin, near neutral pH (pD) is inverse: kD/kH = 1.24, suggesting that proton movement in the rate controlling step is unlikely. The fit of the isotope effect's dependence on the atom fraction of deuterium in the solvent, to the Gross-Butler equation leads to the conclusion that the proton(s) in motion is(are) less tightly bound in the reactant than in a reactive intermediate, formed prior to the rate-controlling step of cis-trans isomerization. The near unity isotope effect on the equilibrium between bound 13-cis- and all-trans-retinals in the dark adapted state mixture indicates that the isotope effects on the forward and reverse rate constants for isomerization are approximately equal to each other and equal to the isotope effect on the observed rate constant for dark adaptation. The results support a previously proposed mechanism of rate controlling Asp-212-nucleophilic catalysis of retinal cis-trans isomerization in the dark adaptation process