Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X‐ray crystallographic and site‐directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO.

TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid) is an extremely potent inhibirespectively. Fluorescence-monitored titration experiments showed that TEI-6720 bound very tightly to both the active and the inactive desulfo-form of the enzyme. The dissociation constant determined for the desulfoform was 2 ؎ 0.03 ؋ 10 ؊9 M; for the active form, the corresponding number was too low to allow accurate measurements. The crystal structure of the active sulfoform of milk xanthine dehydrogenase complexed with TEI-6720 and determined at 2.8-Å resolution revealed the inhibitor molecule bound in a long, narrow channel leading to the molybdenum-pterin active site of the enzyme. It filled up most of the channel and the immediate environment of the cofactor, very effectively inhibiting the activity of the enzyme through the prevention of substrate binding. Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.

Inhibitors of xanthine oxidoreductase block conversion of xanthine to uric acid and are therefore potentially useful for treatment of hyperuricemia or gout. We determined the crystal structure of reduced bovine milk xanthine oxidoreductase complexed with oxipurinol at 2.0 A resolution. Clear electron density was observed between the N2 nitrogen of oxipurinol and the molybdenum atom of the molybdopterin cofactor, indicating that oxipurinol coordinated directly to molybdenum. Oxipurinol forms hydrogen bonds with glutamate 802, arginine 880, and glutamate 1261, which have previously been shown to be essential for the enzyme reaction. We discuss possible differences in the hypouricemic effect of inhibitors, including allopurinol and newly developed inhibitors, based on their mode of binding in the crystal structures.

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD ؉ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH͞XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and sitedirected mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.X anthine oxidoreductase (XOR) is a homodimer of molecular weight 290,000, and each subunit of the enzyme contains one molybdo-pterin (Mo-pt) cofactor, two distinct [2Fe-2S] centers, and one flavin adenine dinucleotide (FAD) cofactor (1, 2). The mammalian XORs catalyze the hydroxylation of hypoxanthine or xanthine at the Mo center, and reducing equivalents thus introduced into the enzymes are transferred via two [2Fe-2S] centers to FAD, where the reduction of NAD ϩ or molecular oxygen occurs (3). These enzymes are synthesized as the dehydrogenase form [xanthine dehydrogenase (XDH)] but can be readily converted to the oxidase form [xanthine oxidase (XO)] reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis (1, 2, 4-6). XDH shows a preference for NAD ϩ reduction at the FAD reaction site (although it still displays considerable reactivity with oxygen), whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to formation of superoxide anion and hydrogen peroxide (1, 7). Previous investigations have suggested that the XDH͞XO conversion is related to milk lipid secretion (8, 9) and is implicated in diseases characterized by oxygen radical-induced tissue damage such as postischemic reperfusion injury (10-13). Thus, the XDH͞XO transition has attracted much attention from both basic and clinical researchers, not only because of the mechanistic interest in the different reactivity of FAD toward NAD ϩ or oxygen subs...