The gene coding for urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin, is inactivated in humans. Consequently, urate oxidase is used as a protein drug to overcome severe disorders induced by uric acid accumulation. The structure of the active homotetrameric enzyme reveals the existence of a small architectural domain that we call T-fold (for tunnelling-fold) domain. It assembles to form a perfect unusual dimeric alpha 8 beta 16 barrel. Urate oxidase may be the archetype of an expanding new family of tunnel-shaped proteins that now has three members; tetrahydropterin synthase, GTP cyclohydrolase I and urate oxidase. The structure of the active site of urate oxidase around the 8-azaxanthine inhibitor reveals an original mechanism of oxidation that does not require any ions or prosthetic groups.

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.

The three disulfide Ecballium eluterium trypsin inhibitor I1 (EETI 11) reduction with dithiothreitol (DTT) and reoxidation of the fuliy reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-191 bridge. The NMR study showed a very strong structural conservation as compared to the native EETI 11, suggesting that the bridges are the [9-211 and [15-271 native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-IIe 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9][10][11][12][13][14][15][16][17][18][19][20][21][15][16][17][18][19][20][21][22][23][24][25][26][27] arrangement and resulted in a well-conserved (7-28) segment.

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