Use of 2,3,5-F[subscript 3]Y-beta 2 and 3-NH[subscript 2]Y-alpha 2 To Study Proton-Coupled Electron Transfer in Escherichia coli Ribonucleotide Reductase
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Author(s)
Seyedsayamdost, Mohammad R.
Yee, Cyril S.
Stubbe, JoAnne
Date Issued
December 2010
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Seyedsayamdost, Mohammad R., Cyril S. Yee, and JoAnne Stubbe. “Use of 2,3,5-F[subscript 3]Y-beta 2 and 3-NH[subscript 2]Y-alpha 2 To Study Proton-Coupled Electron Transfer in Escherichia coli Ribonucleotide Reductase.” Biochemistry 50.8 (2010): 1403–1411.
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Author's final manuscript
Abstract
Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y[subscript 122][superscript •]) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C[subscript 439]), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y[subscript 122] → W[subscript 48] → Y[subscript 356] in β2 to Y[subscript 731] → Y[subscript 730] → C[subscript 439] in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F[subscript 3]Y (F[subscript 3]Y) was site-specifically incorporated in place of Y[subscript 356] in β2 and 3-NH[subscript 2]Y (NH[subscript 2]Y) in place of Y[subscript 731] and Y[subscript 730] in α2. A pH−rate profile with F[subscript 3]Y[subscript 356-]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 F[subscript 3]Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH[subscript 2]Y[subscript 731(730)-]α2, β2, CDP, and ATP resulted in detection of NH[subscript 2]Y radical (NH[subscript 2]Y•) intermediates capable of dNDP formation. In this study, the reaction of F[subscript 3]Y[subscript 356-]β2, α2, 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 F[subscript 3]Y[subscript 356-]β2, CDP, and ATP has also been examined with NH[subscript 2]Y[subscript 731-]α2 (or NH[subscript 2]Y[subscript 730-]α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH[subscript 2]Y• 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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/bi101319v
