Structure-function studies of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-containing enzyme that catalyzes the final two steps in human purine catabolism, taking hypoxanthine to xanthine and then on to uric acid. The enzyme exists as a homodimer, with each monomer possessing a catalytic active site that contains one molybdenum atom coordinated to a pterin ring via an enedithiolate side chain. Each monomer also contains two [2Fe2S] clusters and one equivalent of FAD. These centers form an electron transfer chain as electrons are passed sequentially from the molybdenum to the FAD via the iron-sulfur clusters. The molybdenum center cycles from MoVI to MoIV and back during catalysis, passing through an occasional MoV intermediate state. Electrons are passed from the flavin site out of the enzyme to either NAD+ or molecular oxygen. XOR is initially expressed as a dehydrogenase (xanthine dehydrogenase, XDH) with a strong preference for reducing NAD+ to NADH, although it can reduce O2 under conditions of low NAD + concentrations. Under certain conditions, XDH can be converted by oxidation and/or limited proteolysis to an oxidase form (xanthine oxidase, XO) that utilizes O2 exclusively as the terminal electron acceptor, thereby generating superoxide and other reactive oxygen species. The oxidase is formed primarily during hypoxia or ischemia, and the corresponding increase in reactive oxygen species has lead to investigation of the enzyme's role in the pathophysiological mechanism of ischemia-reperfusion. The production of uric acid by XOR makes the enzyme a primary target in hyperuricemia, and it is this therapeutic intervention for which allopurinol has been used for over 60 years. The prevalence of XOR in mechanisms leading to some human diseases and the unique chemistry by which the enzyme catalyzes the production of uric acid makes this enzyme system an interesting subject of biochemical investigation. We have sought here to understand the structure and function of xanthine oxidoreductase, focusing on the nature of the molybdenum-containing active site as well as innate functional differences between the two forms of the enzyme. Our crystallographic and spectroscopic studies provide insight into the mechanism of XOR catalysis as well as the roles of XOR in human pathology.