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Oxygen Pressurized X-Ray Crystallography: Probing the Dioxygen Binding Site in Cofactorless Urate Oxidase and Implications for Its Catalytic Mechanism

Nathalie Colloc'h ^*, Laure Gabison , Gérald Monard , Muhannad Altarsha  , Mohamed Chiadmi , Guillaume Marassio ^*, Jana Sopkova-de Oliveira Santos ^¶, Mohamed El Hajji ^||, Bertrand Castro ^||, Jacques H. Abraini ^* and Thierry Prangé 

^* CI-NAPS, UMR 6232–UCBN–CNRS–CEA, Centre Cyceron, 14074 Caen cedex, France; LCRB, UMR 8015–Université Paris Descartes–CNRS, Faculté de Pharmacie, 75270 Paris cedex 06, France; ECBT–UMR 7565–Université Henri Poincaré, 54506 Vandoeuvre-les-Nancy, France; Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany; ^¶ Université de Caen, UPRES-EA 2126 (CERMN)–UFR Sciences Pharmaceutiques, 14000 Caen, France; and ^|| Sanofi-Aventis Recherche & Développement, 34184 Montpellier, France

Correspondence: Address reprint requests to Dr. Nathalie Colloc'h, CI-NAPS, UMR 6232–Université de Caen Basse-Normandie–CNRS–CEA, Centre Cyceron, 14074 Caen cedex, France. Tel.: 33-2-31-47-01-32; Fax: 33-2-31-47-02-22; E-mail: colloch{at}cyceron.fr.

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.