Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.
Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.
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DOI：
10.1002/anie.201405485
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发表时间：
2014-12-08
期刊：
影响因子：
--
通讯作者：
Steiner RA
中科院分区：
文献类型：
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作者：
Bui S;von Stetten D;Jambrina PG;Prangé T;Colloc'h N;de Sanctis D;Royant A;Rosta E;Steiner RA
无辅因子的氧化酶和加氧酶利用有限的化学工具来促进和控制 O2 的反应性。它们的作用机制尚未完全了解，并且任何反应中间体的结构信息均不可用。由晶内拉曼光谱和 QM/MM 计算支持的近原子分辨率晶体学明确表明，典型的无辅因子尿酸酶通过 C5(S)-(氢)过氧化物中间体催化尿酸降解。低 X 射线剂量在 100 K 时会特异性地破坏中间 C5=OO(H) 键，从而原位释放 O2，该 O2 被捕获在底物自由基上方。结合晶体学和拉曼分析后的剂量依赖性键断裂速率表明，电离辐射启动了过氧化物的分解及其再生。过氧化可以通过底物自由基与活性位点瞬时产生的超氧化物重新结合的机制来解释。
Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 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 C5=OO(H) bond at 100 K, thus releasing O2 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.