Use of 2,3,5-F3Y-尾2 and 3-NH2Y-伪2 To Study Proton-Coupled Electron Transfer in Escherichia coli Ribonucleotide Reductase

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• 作者：Mohammad R. Seyedsayamdost ; Cyril S. Yee ; JoAnne Stubbe
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：March 1, 2011
• 年：2011
• 卷：50
• 期：8
• 页码：1403-1411
• 全文大小：968K
• 年卷期：v.50,no.8(March 1, 2011)
• ISSN：1520-4995

文摘

Escherichia coli ribonucleotide reductase is an 伪2尾2 complex that catalyzes the conversion of nucleoside 5鈥?diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in 伪2, and the essential diferric-tyrosyl radical (Y<sub>122sub><sup>鈥?/sup>) cofactor that initiates transfer of the radical to the active site cysteine in 伪2 (C<sub>439sub>), 35 脜 removed, is in 尾2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y<sub>122sub> 鈫?W<sub>48sub> 鈫?Y<sub>356sub> in 尾2 to Y<sub>731sub> 鈫?Y<sub>730sub> 鈫?C<sub>439sub> in 伪2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F<sub>3sub>Y (F<sub>3sub>Y) was site-specifically incorporated in place of Y<sub>356sub> in 尾2 and 3-NH<sub>2sub>Y (NH<sub>2sub>Y) in place of Y<sub>731sub> and Y<sub>730sub> in 伪2. A pH鈭抮ate profile with F<sub>3sub>Y<sub>356sub>-尾2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F<sub>3sub>Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH<sub>2sub>Y<sub>731(730)sub>-伪2, 尾2, CDP, and ATP resulted in detection of NH<sub>2sub>Y radical (NH<sub>2sub>Y<sup>鈥?/sup>) intermediates capable of dNDP formation. In this study, the reaction of F<sub>3sub>Y<sub>356sub>-尾2, 伪2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F<sub>3sub>Y<sub>356sub>-尾2, CDP, and ATP has also been examined with NH<sub>2sub>Y<sub>731sub>-伪2 (or NH<sub>2sub>Y<sub>730sub>-伪2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH<sub>2sub>Y<sup>鈥?/sup> formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.