Kinetics of hydrogen atom abstraction from substrate by an active site thiyl radical in ribonucleotide reductase

Lisa Olshansky, Arturo A. Pizano, Yifeng Wei, Joanne Stubbe, Daniel G. Nocera

Research output: Contribution to journalArticlepeer-review


Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans 35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2,or C439S-α2, as well as with 3-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3-C-H bond of substrate by the transiently formed C439·thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s-1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.

Original languageEnglish (US)
Pages (from-to)16210-16216
Number of pages7
JournalJournal of the American Chemical Society
Issue number46
StatePublished - Nov 19 2014
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


Dive into the research topics of 'Kinetics of hydrogen atom abstraction from substrate by an active site thiyl radical in ribonucleotide reductase'. Together they form a unique fingerprint.

Cite this