Ascorbate oxidation catalyzed by Bis(histidine)copper(II)

The kinetics of ascorbic acid oxidation by molecular oxygen, catalyzed by bis(histidine)copper(II) (CuL22+), was followed in 0.1 M phosphate buffer at pH 7.0. Saturation of the oxidation rate was observed at increasing O2, ascorbate and CuL22+ concentrations. The oxidation state of the copper ion during the catalysis and the concentration of the ascorbyl radical were followed by ESR and/or by optical spectroscopy. No significant reduction of Cu(II) was observed under vacuum or in the presence of oxygen at ascorbate concentrations <20 mM. Evidence for the binding of ascorbate to CuL22+ was found by ESR, and a stability constant of 20 M-1 was estimated. We suggested a mechanism which is consistent with our experimental findings and explains some of the contradictory data reported in the past by various authors. The saturation of the reaction rate on increasing [CuL22+] is explained in terms of its catalytic effect on ascorbate oxidation and the superoxide dismutase-like activity of this complex. The experimental concentration of the ascorbyl radical, which is an intermediate product, was in good agreement with that calculated on the basis of the proposed mechanism.