Use of 2,3,5-F3Y-α2 and 3-NH2Y-α2 to study proton-coupled electron transfer in escherichia coli ribonucleotide reductase

Mohammad R. Seyedsayamdost, Cyril S. Yee, Joanne Stubbe

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Escherichia coli ribonucleotide reductase is an β2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in β2, and the essential diferric-tyrosyl radical (Y122) cofactor that initiates transfer of the radical to the active site cysteine in β2 (C439), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y 122 → W48 → Y356 in β2 to Y 731 → Y730 → C439 in β2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F 3Y (F3Y) was site-specifically incorporated in place of Y356 in β2 and 3-NH2Y (NH2Y) in place of Y731 and Y730 in β2. A pH-rate profile with F 3Y356-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)-β2, β2, CDP, and ATP resulted in detection of NH2Y radical (NH2Y) intermediates capable of dNDP formation. In this study, the reaction of F 3Y356-β2, β2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F3Y356-β2, CDP, and ATP has also been examined with NH2Y731-β2 (or NH2Y 730-β2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH2Y formation that support a change in the 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.

Original languageEnglish (US)
Pages (from-to)1403-1411
Number of pages9
Issue number8
StatePublished - Mar 1 2011
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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