Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of alpha 2 and beta 2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(center dot))-diferric cofactor is located in beta 2. Although Y122O(center dot) is extremely stable in free beta 2, Y122O(center dot) is highly reactive in the quaternary substrate-alpha 2 beta 2 complex and serves as a radical initiator in catalytic PCET between beta 2 and alpha 2. In this report, we investigate the structural interactions that control the reactivity of Y122O(center dot) in a model system, isolated E. coli beta 2. Y122O(center dot) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific C-13-labeling of the tyrosine C-1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 angstrom translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIB = 16.7 +/- 1.0 at 25 degrees C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(center dot) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(center dot)-diferric cofactor (SIB = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(center dot) and the iron cluster.