Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase

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• 作者：Kanchana R. Ravichandran ; Ellen C. Minnihan ; Yifeng Wei ; Daniel G. Nocera ; JoAnne Stubbe
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：November 18, 2015
• 年：2015
• 卷：137
• 期：45
• 页码：14387-14395
• 全文大小：548K
• ISSN：1520-5126

文摘

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (伪 and 尾), which form an 伪2尾2 complex that catalyzes the conversion of nucleoside 5鈥?diphosphates to deoxynucleotides (dNDPs). 尾2 contains the essential tyrosyl radical (Y₁₂₂^{鈥?/sup>) that generates a thiyl radical (C₄₃₉鈥?/sup>) in 伪2 where dNDPs are made. This oxidation occurs over 35 脜 through a pathway of amino acid radical intermediates (Y₁₂₂ 鈫?[W₄₈] 鈫?Y₃₅₆ in 尾2 to Y₇₃₁ 鈫?Y₇₃₀ 鈫?C₄₃₉ in 伪2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of 尾2 (F₃Y鈥?/sup>-尾2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s鈥?). Rapid freeze鈥搎uench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F₃Y鈥?/sup>-尾2, 伪2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y₃₅₆鈥?/sup> and 0.5 equiv of dCDP, both at 30 s鈥?. In the absence of an external reducing system, Y₃₅₆鈥?/sup> reduction occurs concomitant with F₃Y reoxidation (0.4 s鈥?) and subsequent to oxidation of all 伪2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s鈥?) is observed prior to steady-state turnover (1.7 s鈥?). The [Y₃₅₆鈥?/sup>] does not change, consistent with rate-limiting F₃Y reoxidation. The data support a mechanism where Y₁₂₂鈥?/sup> is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active 伪2尾2 complex to complete the catalytic cycle.}