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Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme–Substrate–Dioxygen Configuration in a Cofactor‐free Oxidase
Author(s) -
Bui Soi,
von Stetten David,
Jambrina Pablo G.,
Prangé Thierry,
Colloc'h Nathalie,
de Sanctis Daniele,
Royant Antoine,
Rosta Edina,
Steiner Roberto A.
Publication year - 2014
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201405485
Subject(s) - chemistry , peroxide , cofactor , substrate (aquarium) , reaction intermediate , photochemistry , radical , active site , stereochemistry , reaction mechanism , superoxide , reactive intermediate , enzyme , catalysis , organic chemistry , oceanography , geology
Cofactor‐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 in crystallo 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 100 K, thus releasing O 2 in situ, 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 site.