Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp 2 -hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 Å resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 Å resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg 880 and Glu 802 , previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.Purine oxidation in nature is catalyzed by three distinct classes of enzymes: the molybdenum-containing hydroxylases such as xanthine oxidoreductase (1, 2), the Fe II -and ␣-ketoglutarate-dependent xanthine hydroxylases (3, 4), and a newly described two-component system, HpxDE, consisting of a [2Fe-2S]/flavin-containing reductase and a Rieske/non-heme iron-containing oxygenase (5, 6). The first class is by far the most broadly distributed, with members found throughout the eubacteria, archaea, and eukaryota. The second class is found principally in fungae, including yeasts (Saccharomyces cerevisiae, for example), and the third is identified to date only in Klebsiella oxytoca and Klebsiella pneumoniae.Xanthine oxidoreductases from eukaryotes are homodimers of ϳ290 kDa, with each monomer containing four redox-active sites: an active site molybdenum center, a pair of spinach ferredoxin-like [2Fe-2S] clusters, and FAD (7). The overall catalytic sequence consists of a reductive half-reaction in which substrate is oxidatively hydroxylated at the molybdenum center (reducing it from Mo(VI) to Mo(IV)) and, after intramolecular electron transfer, an oxidative half-reaction in which reducing equivalents are removed from the enzyme via its FAD. In the reductive half-reaction, purine substrates are hydroxylated at a specific carbon position in a reaction initiated by nucleophilic attack of an equatorial Mo-OH group of the metal center whose deprotonation is thought to be facilitate...

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 Å resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 Å resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD ؉ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O 2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3 Å . This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the Mo V transition state.Xanthine oxidoreductase (XOR) 2 is the prototypical member of the molybdenum hydroxylase family of proteins (1, 2). In humans, XOR catalyzes the hydroxylation of hypoxanthine to xanthine as well as xanthine to uric acid, and the mammalian enzyme exists in two alternative forms of the same gene product. Normally, the enzyme exists in a dehydrogenase form (xanthine dehydrogenase) but can be readily converted to an oxidase form (XO) by oxidation of sulfhydryl residues or by limited proteolysis (3). Xanthine dehydrogenase shows a preference for NAD ϩ as the oxidizing substrate (although it is also able to react with O 2 ), whereas XO is unable to react with NAD ϩ and uses O 2 exclusively (3). This conversion of xanthine dehydrogenase to XO is thought to be relevant in the context of ischemia-reperfusion pathology (4). The enzyme is a target of drugs against gout and hyperuricemia and is often targeted in tandem in chemotherapeutic regimens (5). Excellent reviews describing XOR in pharmacology and human pathology are available (6, 7).Like the human enzyme, the bovine enzyme is a 290-kDa homodimer, each monomer having a molybdenum center plus two [2Fe-2S] clusters (with each iron coordinated by a pair of cysteines) and FAD. Oxidative hydroxylation of the purine substrate occurs at the molybdenum center, which results in the two-electron reduction of the enzyme. After internal electron transfer via the [2Fe-2S] centers to the FAD, reducing equivalents are passed to the final electron acceptor (O 2 or NAD ϩ ). The crystal structure of the bovine enzyme has been determined (8), and it shows that the four redox-active centers of each monomer are found in separate, distinctly folding domains.The catalytic sequence of XOR is thought...