title: The redox centers of xanthine oxidase are on independent structural domains of the enzyme.
abstract: Xanthine oxidase employs four electron transport sites (flavin adenine dinucleotide (FAD), molybdenum, and two FeS centers) in catalyzing a variety of redox reactions. To determine whether the redox sites reside in independent domains of the enzyme, the temperature of heat inactivation of each site's catalytic activity was determined, except that no attempt was made to distinguish between the two FeS sites. In the oxidase form of xanthine oxidase, the order of thermal stabilities was Mo greater than FAD greater than FeS, while after conversion to its dehydrogenase form the above ranking was Mo greater than FeS greater than FAD. The small but reproducible difference in heat inactivation temperatures among the redox sites demonstrated that the sites are located in separate domains of the enzyme. To confirm the above segregation of redox centers, the temperature of heat-induced release of each redox cofactor from its site on the enzyme was examined. These temperatures were found to be different for each redox cofactor and agreed closely with the heat inactivation temperatures measured above. The data thus demonstrate that both heat inactivation and cofactor release derive from thermal unfolding of independent domains. Using a technique termed "thermal digestion analysis," the FAD domain was located in a approximately equal to 42,000-Da tryptic fragment, while the FeS and Mo domains were isolated in a trypsin-resistant 92,000-Da fragment. We conclude that xanthine oxidase is constructed in modular fashion with the redox sites located in independent structural domains.
mesh_terms: D001665:Binding Sites; D003067:Coenzymes; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide; D006358:Hot Temperature; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011489:Protein Denaturation; D014969:Xanthine Oxidase
chemical_list: D003067:Coenzymes; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D014969:Xanthine Oxidase