Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.
Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.
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DOI：
10.1021/acs.biochem.5b00676
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发表时间：
2015-11
期刊：
影响因子：
2.9
通讯作者：
J. Wallen;T. C. Mallett;T. Okuno;D. Parsonage;H. Sakai;T. Tsukihara;A. Claiborne
中科院分区：
文献类型：
--
作者：
J. Wallen;T. C. Mallett;T. Okuno;D. Parsonage;H. Sakai;T. Tsukihara;A. Claiborne
在探索粪肠球菌NADH氧化酶(Nox；O2→2H2O)C42S突变体的氧反应性时，我们提供了C(4a)-过氧黄素中间体参与氧化半反应的直接证据，并描述了同源二聚体完全再氧化的限速构象或化学变化。在这项工作中，来自化脓性链球菌的NOx(SpyNox)得到了表达和结晶，并分别在2.0时和2.15时提纯了过氧化野生型[Cys44-SOH→Cys44-SO2H]和C44S突变酶结构。我们发现叠氮在溶液中与双电子还原野生型(EH2)酶和突变酶结合，但与突变蛋白的亲和力显著提高。以SpyNOxEH2形式滴定的光谱过程清楚地表明了Cys44-S(-)→Fad电荷转移相互作用的渐进位移。叠氮化物与C44S氮氧化物晶体浸泡导致了复合体的结构，在2.10？下进行了精炼。活性中心N3(-)配体位于Ser44和His11侧链的近端，Ser44侧链也发生了显著的移位。这为叠氮引起的电荷转移吸收损失提供了一个有吸引力的解释，并允许C(4a)-过氧黄素结构模型的适应。Ser44的构象和相关的螺旋元件，以及由此产生的空间调节，似乎与粪肠球菌C42s NOx氧化半反应中描述的构象变化有关。
In probing the oxygen reactivity of an Enterococcus faecalis NADH oxidase (Nox; O2 → 2H2O) C42S mutant lacking the Cys42-sulfenic acid (Cys42-SOH) redox center, we provided direct evidence of a C(4a)-peroxyflavin intermediate in the oxidative half-reaction and also described a conformational or chemical change that is rate-limiting for full reoxidation of the homodimer. In this work, the Nox from Streptococcus pyogenes (SpyNox) has been expressed and crystallized, and the overoxidized wild-type [Cys44-SOH → Cys44-sulfinic acid (Cys44-SO2H)] and C44S mutant enzyme structures have been refined at 2.0 and 2.15 Å, respectively. We show that azide binds to the two-electron reduced wild-type (EH2) enzyme and to the mutant enzyme in solution, but with a significantly higher affinity for the mutant protein. The spectral course of the titration with the SpyNox EH2 form clearly indicates progressive displacement of the Cys44-S(-) → FAD charge-transfer interaction. An azide soak with C44S Nox crystals led to the structure of the complex, as refined at 2.10 Å. The active-site N3(-) ligand is proximal to the Ser44 and His11 side chains, and a significant shift in the Ser44 side chain also appears. This provides an attractive explanation for the azide-induced loss of charge-transfer absorbance seen with the wild-type EH2 form and also permits accommodation of a C(4a)-peroxyflavin structural model. The conformation of Ser44 and the associated helical element, and the resulting steric accommodation, appear to be linked to the conformational change described in the E. faecalis C42S Nox oxidative half-reaction.