Ribonueleotide reductase (RNR) catalyzes. conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found hi aerobic bacteria and all eukaryotes, are a complex of two subunits: alpha 2 and beta 2. The beta 2 subunit contains an essential diferrictyrosyl radical (Y122O(center dot)) cofactor that is needed to initiate reduction of NDPs in the alpha 2 subunit. In this work, we investigated the Y122O(center dot) reduction mechanism in Escherichia coli beta 2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O(center dot) was studied using reaction-induced FT-IR spectroscopy and [C-13]aspartate-labeled beta 2. These Y122O(center dot) minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated beta 2.