Redox-Dependent Structural Coupling between the 伪2 and 尾2 Subunits in E. coli Ribonucleotide Reductase

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• 作者：Adam R. Offenbacher ; R. Atlee Watson ; Cynthia V. Pagba ; Bridgette A. Barry
• 刊名：Journal of Physical Chemistry B
• 出版年：2014
• 出版时间：March 20, 2014
• 年：2014
• 卷：118
• 期：11
• 页码：2993-3004
• 全文大小：623K
• 年卷期：v.118,no.11(March 20, 2014)
• ISSN：1520-5207

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), Y_x, 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.