Wallen Jamie R, Mallett T Conn, Okuno Takashi, Parsonage Derek, Sakai Hiroaki, Tsukihara Tomitake, Claiborne Al
Department of Chemistry and Physics, Western Carolina University , Cullowhee, North Carolina 28723, United States.
Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157, United States.
Biochemistry. 2015 Nov 17;54(45):6815-29. doi: 10.1021/acs.biochem.5b00676. Epub 2015 Nov 6.
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.
在探究缺乏半胱氨酸42-亚磺酸（Cys42-SOH）氧化还原中心的粪肠球菌NADH氧化酶（Nox；O2 → 2H2O）C42S突变体的氧反应性时，我们提供了氧化半反应中C（4a）-过氧黄素中间体的直接证据，还描述了一种对同型二聚体完全再氧化起限速作用的构象或化学变化。在这项工作中，化脓性链球菌的Nox（SpyNox）已被表达和结晶，并且过氧化的野生型[Cys44-SOH → 半胱氨酸44-亚磺酸（Cys44-SO2H）]和C44S突变体酶结构分别在2.0和2.15 Å分辨率下得到了优化。我们表明，叠氮化物在溶液中与双电子还原的野生型（EH2）酶和突变体酶结合，但对突变体蛋白的亲和力明显更高。用SpyNox EH2形式进行滴定的光谱过程清楚地表明了半胱氨酸44-S（-）→黄素腺嘌呤二核苷酸电荷转移相互作用的逐步取代。用C44S Nox晶体进行叠氮化物浸泡得到了复合物的结构，分辨率为2.10 Å。活性位点的N3（-）配体靠近丝氨酸44和组氨酸11的侧链，丝氨酸44侧链也出现了显著位移。这为野生型EH2形式中叠氮化物诱导的电荷转移吸光度损失提供了一个有吸引力的解释，也允许容纳C（4a）-过氧黄素结构模型。丝氨酸44的构象和相关的螺旋元件以及由此产生的空间容纳似乎与粪肠球菌C42S Nox氧化半反应中描述的构象变化有关。
