A Chemically Competent Thiosulfuranyl Radical on the Escherichia coli Class III Ribonucleotide Reductase

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• 作者：Yifeng Wei ; Guinevere Mathies ; Kenichi Yokoyama ; Jiahao Chen ; Robert G. Griffin ; JoAnne Stubbe
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：June 25, 2014
• 年：2014
• 卷：136
• 期：25
• 页码：9001-9013
• 全文大小：618K
• ISSN：1520-5126

文摘

The class III ribonucleotide reductases (RNRs) are glycyl radical (G鈥? enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and II RNRs, where reducing equivalents for the reaction are delivered by a redoxin (thioredoxin, glutaredoxin, or NrdH) via a pair of conserved active site cysteines, the class III RNRs examined to date use formate as the reductant. Here, we report that reaction of the Escherichia coli class III RNR with CTP (substrate) and ATP (allosteric effector) in the absence of formate leads to loss of the G鈥?concomitant with stoichiometric formation of a new radical species and a 鈥渢rapped鈥?cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G鈥?and reduction of the cytidine derivative, proposed to be 3鈥?keto-deoxycytidine, to dCTP and a small amount of cytosine. The structure of the new radical has been identified by 9.5 and 140 GHz EPR spectroscopy on isotopically labeled varieties of the protein to be a thiosulfuranyl radical [RSSR₂]鈥? composed of a cysteine thiyl radical stabilized by an interaction with a methionine residue. The presence of a stable radical species on the reaction pathway rationalizes the previously reported [³H]-(k_cat/K_M) isotope effect of 2.3 with [³H]-formate, requiring formate to exchange between the active site and solution during nucleotide reduction. Analogies with the disulfide anion radical proposed to provide the reducing equivalent to the 3鈥?keto-deoxycytidine intermediate by the class I and II RNRs provide further evidence for the involvement of thiyl radicals in the reductive half-reaction catalyzed by all RNRs.