Oxygen pressurized X-ray crystallography: Probing the dioxygen binding site in cofactor-less urate oxidase and implications to its catalytic mechanism
Nathalie Colloc'h 1*, Laure Gabison 2, Gerald Monard 3, Muhanad Altarsha 4, Mohamed Chiadmi 2, Guillaume Marassio 1, Jana Sopkova-de Oliveira Santos 5, Mohamed El Hajji 6, Bertrand Castro 6, Jacques H. Abraini 1 and Thierry Prangé 2
1 CI-NAPS - UMR 6232 - UCBN - CNRS
2 LCRB, UMR 8015 - Université Paris Descartes - CNRS
3 ECBT - UMR 7565 - Université Henri Poincaré
4 Max-Planck-Institut fur Kohlenforschung
5 CERMN, Université de Caen
6 Sanofi-Aventis
* To whom correspondence should be addressed. E-mail: colloch{at}cyceron.fr.
Submitted on September 17, 2007
Revised on October 8, 2007
Accepted on 12 March 2008
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Abstract |
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The localization of dioxygen sites in oxygen-binding proteins is a non-trivial experimental task and is often suggested through indirect methods, like using xenon or halide anions as oxygen probes. In the present 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 cofactor-less 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 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.
Key Words:
active site, catalase, chloride, hydroperoxide, uricase, xenon
