Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.
Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.
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谷氨酸 350 在大肠杆菌 Ia 类核糖核苷酸还原酶长程自由基转运的构象门控中发挥重要作用
DOI：
10.1021/acs.biochem.6b01145
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发表时间：
2017-02-14
期刊：
影响因子：
2.9
通讯作者：
Stubbe J
中科院分区：
文献类型：
--
作者：
Ravichandran K;Minnihan EC;Lin Q;Yokoyama K;Taguchi AT;Shao J;Nocera DG;Stubbe J
E.大肠杆菌Ia型核糖核苷酸还原酶由两个亚基组成，形成活性α2β2复合物。α2，35 Å中的核苷二磷酸底物（NDP）由β2中必需的二铁-酪氨酰自由基（Y122·）辅因子还原而成。Y122·介导的C439在α2中的氧化通过穿过α/ β界面的途径（β2中的Y122 <$[W 48]<$Y356到7agr;2中的Y 731 <$Y730 <$C439）发生。α2β2结构的缺失妨碍了对Y356和Y 731在亚基界面上的位置的了解。β2中保守的E350与Y356的序列接近性表明其在催化和/或构象门控中的重要性。为了研究其功能，报道了在E350或E350 X（X = A，D，Q）突变体存在下wt-β2/α2和3，5-二氟酪氨酸（F2 Y）取代残基356、731或两者的突变体的pH速率曲线。对于E350，活性在pH极值下保持，表明F2 Y356和F2 Y 731的质子化和去质子化状态是活性的，并且自由基运输（RT）可以通过在低pH下的质子偶联电子转移或在高pH下的电子转移跨越界面发生。所有的RNR都是无活性的，这表明E350在界面Ys的氧化过程中可能是质子受体。为了确定E350是否在强氧化剂的构象门控中起作用，NO2 Y122·-β2和2，3，5-F3 Y122·-β2在E350和E350 X背景下与α2/CDP/ATP反应，并通过快速冷冻淬灭EPR光谱监测反应的路径自由基。只有当E350存在时才产生通路自由基，支持其在门控启动RT的构象变化中的重要作用，并掩盖其作为质子受体的作用。
E. coli Ia ribonucleotide reductase is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122• mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in 7agr;2) across the α/ β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The sequence proximity of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH rate profiles of wt-β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731 or both are reported in the presence of E350 or E350X (X = A, D, Q) mutants. With E350, activity is maintained at the pH extremes suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2 were reacted with α2/CDP/ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid-freeze quench EPR spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.
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