Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme-Substrate-Dioxygen Configuration in a Cofactor-free Oxidase

International audienceCofactor-free oxidases and oxygenases promote and control the reactivity of O-2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by incrystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5OO(H) bond at 100K, thus releasing O-2 insitu, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active sit