Effects of solvent composition and viscosity on the rates of CO binding to heme proteins.

Abstract

The rate of CO binding to myoglobin increases 4-fold, from 5 X 10(5) M-1 s-1 to 2 X 10(6) M-1 s-1, in going from 0 to 80% glycerol in phosphate buffer at pH 7, 20 degrees C. Under the same conditions, the rate of CO binding to protoheme decreases monotonically from about 1 X 10(8) M-1 s-1 to 2 X 10(7) M-1 s-1. The kinetic behavior of protoheme at neutral pH is that expected for a diffusion-controlled reaction. Increasing solvent viscosity causes a decrease in the observed second order rate constant. In contrast, the behavior of the myoglobin indicates quite clearly that internal, nondiffusive processes are limiting the speed of the reaction. The rate enhancement is due to an increase in the standard chemical potential of the ligand molecule as the polyalcohol concentration is increased. Both types of behavior are observed for ligand binding to protoheme in 0.1 N NaOH; first an increase and then a decrease in rate is observed as the concentration of glycerol is increased. At low glycerol concentrations, the reaction rate is limited by a first order process. At high concentrations, the rate becomes diffusion-controlled and exhibits a dependence on the reciprocal of the solvent viscosity. The data for all these conditions have been analyzed empirically in terms of a single free energy barrier and more specifically in terms of a consecutive reaction scheme.