A Chemically Competent Thiosulfuranyl Radical on the Escherichia coli Class III Ribonucleotide Reductase
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Wei-2014-Chemically competent.pdf
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Author(s)
Wei, Yifeng
Mathies, Guinevere
Yokoyama, Kenichi
Chen, Jiahao
Stubbe, JoAnne
Griffin, Robert Guy
Date Issued
May 2014
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Citation
Wei, Yifeng, Guinevere Mathies, Kenichi Yokoyama, Jiahao Chen, Robert G. Griffin, and JoAnne Stubbe. “A Chemically Competent Thiosulfuranyl Radical on the Escherichia Coli Class III Ribonucleotide Reductase.” Journal of the American Chemical Society 136, no. 25 (June 25, 2014): 9001–9013. © 2014 American Chemical Society
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Final published version
Abstract
The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) 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• concomitant with stoichiometric formation of a new radical species and a “trapped” cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G• and reduction of the cytidine derivative, proposed to be 3′-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 [RSSR[subscript 2]]•, 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 [[superscript 3]H]-(k[subscript cat]/K[subscript M]) isotope effect of 2.3 with [[superscript 3]H]-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′-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.
MIT Department
Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
Francis Bitter Magnet Laboratory (Massachusetts Institute of Technology)
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DOI of Published Version
https://doi.org/10.1021/ja5030194
