文摘
Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient 伪2尾2 complex forms when a ribonucleotide substrate, such as CDP, binds to the 伪2 subunit. A tyrosyl radical (Y122O鈥?-diferric cofactor in 尾2 initiates substrate reduction in 伪2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the 伪2尾2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli 伪2 and 尾2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, 13C伪2尾2 and 伪213C尾2, were used to define subunit-specific structural changes. Mixing of 伪2 and 尾2 under turnover conditions yielded amide I (C鈺怬) 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 尾 strands and disordered structures. Significantly, HU-mediated reduction of Y122O鈥?was associated with structural changes in 伪2, as well as 尾2. To define the spectral contributions of Y122O鈥?Y122OH in the quaternary complex, 2H4 labeling of 尾2 tyrosines and HU editing were performed. The bands of Y122O鈥? Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated 尾2, were observed in the 伪2尾2 complex. These spectra also provide evidence for a conformational rearrangement at an additional 尾2 tyrosine(s), Yx, in the 伪2尾2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.