Role of the relaxation of the iron(III) ion spin states equilibrium in the kinetics of ligand binding to methaemoglobin

Abstract

Temperature-jump experiments of the reaction of the thiocyanate ion with human aquomethaemoglobin have been performed in the presence of a 10-fold excess of inositol hexakisphosphate (inositol-P₆). Two kinetic phases corresponding to the α and β subunits were observed. Kinetic parameters of the reaction were evaluated from the reciprocal relaxation times on the basis of a fast relaxation of the iron(iii) ion spin states equilibrium before binding of the ligand. The association, k_iL, and dissociation, k_–iL, rate constants determined were: k_αL= 225 dm³ mol^–1 s^–1, k_–αL= 1.52 s^–1, k_βL= 2430 dm³ mol^–1 s^–1, k_–βL= 6.51 s^–1 at 27 °C, pH 6.44. There was good agreement between the equilibrium constant of the ligand binding step determined by static methods (K_equ= 204 ± 11 dm³ mol^–1) and that evaluated from kinetic data [(K_αLK_βL)^1/2= 235 ± 12 dm³ mol^–1]. The value k_βL/k_αL= 11 obtained ensured proper separation of the two kinetic phases. Analyses of the subunit relaxation amplitudes, δE_iL, showed that inositol-P₆ perturbed the absorption spectrum of the β subunits. This suggests that in the presence of the organic phosphate, methaemoglobin behaves as a protein with independent binding sites rather than as an allosteric molecule. The kinetic and relaxation amplitude spectral characteristics of the subunits, in the presence of inositol-P₆ have demonstrated that the kinetic dynamics are effectively decoupled in a stable tetramer.