An unprecedented dioxygen species revealed by serial femtosecond rotation crystallography in copper nitrite reductase.
An unprecedented dioxygen species revealed by serial femtosecond rotation crystallography in copper nitrite reductase.
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DOI：
10.1107/s2052252517016128
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发表时间：
2018-01-01
期刊：
影响因子：
3.9
通讯作者：
Hasnain SS
中科院分区：
文献类型：
--
作者：
Halsted TP;Yamashita K;Hirata K;Ago H;Ueno G;Tosha T;Eady RR;Antonyuk SV;Yamamoto M;Hasnain SS
利用分离的氧化酶的连续飞秒旋转结晶学观察到时间冻结结构中的O2，为CuNiRs中O2的结合模式提供了期待已久的明确证据。这提供了一个洞察木质氧化曲霉CuNiR如何作为一种氧化酶，将O2还原为H_2O_2，或作为一种超氧化物歧化酶，因为它在大约20年前被证明具有牛∼超氧化物歧化酶56%的歧化酶活性。基于同步加速器的配体结合酶的X射线结构研究是加深我们对反应机理的理解的有力工具。对于氧化还原酶来说，有必要同时研究氧化和还原的活性中心，以充分阐明反应，这一目标因潜在的X射线光还原而变得复杂。在底物存在的情况下，可以利用这一点来构建与催化相关的事件的结构电影。利用新发展的连续飞秒旋转结晶学(SF-ROX)方法，对分离的亚硝酸铜还原酶(CuNiR)的X射线无损伤结构进行了可视化。来自SACLA X射线自由电子激光器的低于10 飞秒的X射线脉冲长度使衍射数据能够在“时间冻结”状态下采集到1.6 ä分辨率。X射线脉冲的持续时间极短，确保在辐射引起的变化(包括辐射分解)开始之前捕获数据。出乎意料的是，在CuNiRs中，以一种全新的双原子配体的结合模式，确定了一种O2配体与T2Cu结合。在分离的氧化酶的时间冻结结构中观察到的O2为CuNiRs中O2的结合模式提供了期待已久的明确证据。这让我们深入了解了木糖产碱菌的铜镍是如何作为一种氧化酶，将O2还原为过氧化氢，或者作为一种超氧化物歧化酶发挥作用的，因为大约20年前，它被证明具有牛超氧化物歧化酶的56%的∼活性。
The observation of O2 in a time-frozen structure using serial femtosecond rotation crystallography of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from A. xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago. Synchrotron-based X-ray structural studies of ligand-bound enzymes are powerful tools to further our understanding of reaction mechanisms. For redox enzymes, it is necessary to study both the oxidized and reduced active sites to fully elucidate the reaction, an objective that is complicated by potential X-ray photoreduction. In the presence of the substrate, this can be exploited to construct a structural movie of the events associated with catalysis. Using the newly developed approach of serial femtosecond rotation crystallography (SF-ROX), an X-ray damage-free structure of the as-isolated copper nitrite reductase (CuNiR) was visualized. The sub-10 fs X-ray pulse length from the SACLA X-ray free-electron laser allowed diffraction data to be collected to 1.6 Å resolution in a ‘time-frozen’ state. The extremely short duration of the X-ray pulses ensures the capture of data prior to the onset of radiation-induced changes, including radiolysis. Unexpectedly, an O2 ligand was identified bound to the T2Cu in a brand-new binding mode for a diatomic ligand in CuNiRs. The observation of O2 in a time-frozen structure of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from Alcaligenes xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago.