Studies on Cobalt Myoglobins and Hemoglobins: II. KINETIC STUDIES OF REVERSIBLE OXYGENATION OF COBALT MYOGLOBINS AND HEMOGLOBINS BY THE TEMPERATURE JUMP RELAXATION METHOD

Abstract

Abstract The reversible oxygenation equilibrium of cobalt myoglobins and hemoglobins, complexes of apomyoglobin and apohemoglobin with cobaltous proto-, meso-, and deuteroporphyrins IX (proto-, meso-, and deutero-CoMb and -CoHb) has been investigated kinetically by the temperature jump relaxation method. The following association and dissociation rate constants (kon and koff) were obtained in 0.1 m phosphate buffer, pH 7.0, at 22.2° for proto-, meso-, and deutero-CoMbs: kon of 4.0, 3.1, and 6.9 x 107 m-1 s-1 and koff of 2.8, 2.7, and 1.6 x 103 s-1. The values of kon were in the same order of magnitude as those of the corresponding FeMbs, whereas the values of koff were about 102 times larger than those of the corresponding FeMb. The activation energies and preexponential factors were calculated by measuring the temperature dependencies of these rates. The comparison between these rates and those of the related reaction of metalloporphyrins with the rate parameters expected for diffusion-controlled reactions has led to the suggestion of a possible reaction pathway. The kinetic data show the oxygenation reactions of metalloporphyrin systems to be in a range similar to typical enzyme-substrate association-dissociation reactions. The relaxation spectrum of CoHb contains more than two relaxation times while that of CoMb is described by a single one. The relaxation time of the faster (τf) among two main components was shorter than that of CoMb. To a first approximation it was dependent on temperature and oxygen concentration, and independent of pH or addition of organic phosphate. The slower relaxation time (τs) was longer than the single one for CoMb, and was dependent on oxygen concentration. The amplitude of this component decreased with increase of pH or the addition of organic phosphate. The assignment of these relaxations to the special molecular level mechanism is difficult without further detailed knowledge of the properties of CoHb solution and of the CoHb-O2 equilibrium system.