Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient alpha 2 beta 2 complex forms when a ribonucleotide substrate, such as CDP, binds to the alpha 2 subunit. A tyrosyl radical (Y122O center dot)-diferric cofactor in beta 2 initiates substrate reduction in alpha 2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the alpha 2 beta 2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli alpha 2 and beta 2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, C-13 alpha 2 beta 2 and alpha 2(13)C beta 2, were used to define subunit-specific structural changes. Mixing of alpha 2 and beta 2 under turnover conditions yielded amide I (C=O) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to beta strands and disordered structures. Significantly, HU-mediated reduction of Y122O center dot was associated with structural changes in alpha 2, as well as beta 2. To define the spectral contributions of Y122O center dot/Y122OH in the quaternary complex, H-2(4) labeling of beta 2 tyrosines and HU editing were performed. The bands of Y122O center dot, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated beta 2, were observed in the alpha 2 beta 2 complex. These spectra also provide evidence for a conformational rearrangement at an additional beta 2 tyrosine(s), Y-x, in the alpha 2 beta 2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.