The Preparation and Properties of Deflavo Xanthine Oxidase

Abstract

Abstract The FAD prosthetic group of milk xanthine oxidase has been removed by treatment with high concentrations of CaCl2. This agent has been shown to promote the hydrolysis of FAD to FMN. The deflavoenzyme has been shown to be devoid of xanthine oxygen reductase activity, and can be reconstituted by a short incubation with FAD. Reconstitution of xanthine oxygen reductase activity was also obtained with FMN; however, high concentrations were required. The absorption and circular dichroism spectra of the deflavoenzyme are similar to those of the simple iron-sulfur proteins such as the ferredoxins. Rapid reaction studies have shown that the deflavoenzyme is rapidly reduced by xanthine and glycolaldehyde, and that the loss of xanthine oxygen reductase activity is due to the fact that the reduced deflavoenzyme has a negligible reoxidation rate with O2. Consistent with these findings, the deflavoenzyme is catalytically active in the oxidation of xanthine with acceptors such as ferricyanide and cytochrome c. On the basis of these results, and of inhibition studies with cyanide, pathways of electron transfer in the reaction of xanthine oxidase with various hydrogen donors and acceptors are proposed.