Title:
Identification of redox-linked structural changes in ribonucleotide reductase (RNR) by way of reaction-induced FTIR spectroscopy

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Watson, Ryan A.
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Barry, Bridgette A.
Oyelere, Adegboyega K.
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Abstract
Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient 2β2 complex forms when a ribonucleotide substrate binds to the 2 subunit. A tyrosyl radical (Y122Oo)-differic cofactor in β2 initiates substrate reduction in 2 via long distance, proton-coupled electron transfer (PCET). Reaction-induced FT-IR spectroscopy was used to describe the 2β2 structural landscapes associated with catalysis and inhibition by anti-cancer chemotherapeutics. Mixing of 2 and β2 subunits to form the active complex yielded amide I (C=O) and II (CN/NH) bands indicative of structural rearrangements associated with catalysis. Radical trap, AzUDP, demonstrates backbone rearrangement associated with a redox-induced shift associated with conformational changes associated with PCET. Isotope labeling of β2 tyrosines was performed to assign specific contributions to each subunit. FTIR spectroscopy allows for the detection of single amino acid changes that take place as a result of RNR inhibition due to radical quenching or trapping. The FTIR spectra highlight the distinction between substrates and effectors during active turnover, and the conformational impact of inhibition during active turnover of the QC.
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2018-07-31
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Dissertation
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