Redox-Linked Changes to the Hydrogen-Bonding Network

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleotides, a critical step in DNA replication and repair in all organisms. The class Ia RNRs, which are found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. β2 contains an essential diferric-tyrosyl radical (Y122O•) cofactor required to initiate reduction of NDPs in the α2 subunit. Here, we investigate the Y122O• reduction mechanism in E. coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We test the hypothesis that Y122OH redox reactions cause structural changes at the diferric cluster. The reduction of Y122O• is studied using reaction-induced FT-IR spectroscopy and [13C]-aspartate labeled β2. These spectra, Y122O•–Y122OH, provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration (υas) of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in hydrogen bonding between Y122OH and D84, which may regulate proton transfer reactions on the HU-mediated inactivation pathway in isolated β2.