ROLE OF THE RELAXATION OF THE IRON(III) ION SPIN STATES EQUILIBRIUM IN THE KINETICS OF LIGAND-BINDING TO METHEMOGLOBIN

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 alpha and beta 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(alphaL) = 225 dm3 mol-1 s-1, k(-alphaL) = 1.52 s-1, k(betaL) = 2430 dm3 mol-1 s-1, k(-betaL) = 6.51 s-1 at 27-degrees-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(alphaL)K(betaL))1/2 = 235 +/- 12 dm3 mol-1]. The value k(betaL)/k(alphaL) = 11 obtained ensured proper separation of the two kinetic phases. Analyses of the subunit relaxation amplitudes, deltaE(iL), showed that inositol-P6 perturbed the absorption spectrum of the beta 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.