Oxidation-reduction potentials of D-amino acid oxidase.

Abstract This paper reports a study of the oxidation-reduction equilibrium of d-amino acid oxidase, a flavoprotein containing FAD. The oxidation-reduction potential at 50% oxidation (E½) is -0.004 volt at pH 7.0 and 20°, and therefore about 180 mv higher than that of the free coenzyme (FAD). This difference in oxidation-reduction potential may be described in terms of relative affinity of the apoenzyme for the reduced and oxidized forms of the coenzyme. On this basis the affinity constant for the binding of reduced FAD to the apoenzyme is about 106 higher than that of oxidized FAD. The curve relating E½ to pH is in the alkaline range consistent with a slope of about -0.058 volt per pH unit which corresponds to the difference of 1 proton between the oxidized and reduced forms of the enzyme. The apparent pK of the oxidation-linke group, which belongs to the oxidized form, is ∼7.1. The shape of the oxidation-reduction equilibrium curve of d-amino acid oxidase is pH dependent, the value of n increasing from about 1 at pH 8.6 to about 3, or more, at pH 6.6. Under these conditions, therefore, one must consider the existence of functional homotropic interactions between at least 2 FAD molecules. The pH dependence of the cooperative oxidation-reduction equilibrium is discussed in the framework of the theory of linked functions