Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.
Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.
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DOI：
10.1021/bi101319v
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发表时间：
2011-03-01
期刊：
影响因子：
2.9
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
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作者：
Seyedsayamdost, Mohammad R.;Yee, Cyril S.;Stubbe, JoAnne
E.大肠杆菌核糖核苷酸还原酶是一种α2β2复合物，催化核苷5′-二磷酸（NDP）转化为脱氧核苷酸（dNDP）。NDP还原的活性位点位于α2，而启动自由基转移到α2（C439）中活性位点半胱氨酸的必需二铁酪氨酰自由基（Y122·）辅因子（去除35个碱基）位于β2。氧化反应可能是通过芳香族氨基酸的跳跃机制（β2中的Y122→ W 48 →Y356到α2中的Y 731 → Y 730 →C439）和可逆的质子耦合电子转移（PCET）进行的。最近，2，3，5-F3 Y（F3 Y）位点特异性地掺入β2中的Y356，3-NH 2 Y（NH 2 Y）位点特异性地掺入α2中的Y 731和Y 730。F3 Y356-β2的pH速率曲线表明，随着pH值升高，RNR的速率决定步骤可以从构象变化改变为PCET，并且通过途径中与其相邻的残基改变的F3 Y氧化驱动力是导致这种变化的原因。使用NH 2 Y 731（730）-α2/β2/CDP/ATP的研究导致检测到能够形成dNDP的NH 2 Y自由基（NH 2 Y·）中间体。在这项研究中，F3 Y356-β2/α2/CDP/ATP的反应已经通过停流（SF）吸收和快速冷冻淬灭EPR光谱进行了检查，没有发现任何自由基中间体。F3 Y356-β2/CDP/ATP与NH 2 Y 731-α2（或NH 2 Y 730-α2）在pH 6.5-9.2范围内的停流动力学也得到了验证，并揭示了NH 2 Y·形成的速率常数，这支持了pH升高时速率限制步骤的变化。
E. coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y122•) cofactor that initiates radical transfer to the active site cysteine in α2 (C439), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y122→W48→Y356 in β2 to Y731→Y730→C439 in α2) and reversible proton coupled electron transfer (PCET). Recently 2,3,5-F3Y (F3Y) was site-specifically incorporated in place of Y356 in β2, and 3-NH2Y (NH2Y) in place of Y731 and Y730 in α2. A pH rate profile with F3Y356-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F3Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH2Y731(730)-α2/β2/CDP/ATP resulted in detection of NH2Y radical (NH2Y•) intermediates capable of dNDP formation. In this study, the reaction of F3Y356-β2/α2/CDP/ATP has been examined by stopped flow (SF) absorption and rapid freeze quench EPR spectroscopy and has failed to reveal any radical intermediates. F3Y356-β2/CDP/ATP has also been examined with NH2Y731-α2 (or NH2Y730-α2) by stopped-flow kinetics from pH 6.5–9.2 and revealed rate constants for NH2Y• formation that support a change in rate limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.
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影响因子：
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作者：
DEFELIPPIS, MR;MURTHY, CP;KLAPPER, MH
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DOI：
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发表时间：
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发表时间：
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期刊：
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影响因子：
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