Redox-Dependent Structural Coupling between the α2 and β2 Subunits in E. coli Ribonucleotide Reductase

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.