Redox-Linked Conformational Control of Proton-Coupled Electron Transfer: Y122 in the Ribonucleotide Reductase 尾2 Subunit

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• 作者：Adam R. Offenbacher ; Lori A. Burns ; C. David Sherrill ; Bridgette A. Barry
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：July 18, 2013
• 年：2013
• 卷：117
• 期：28
• 页码：8457-8468
• 全文大小：590K
• 年卷期：v.117,no.28(July 18, 2013)
• ISSN：1520-5207

文摘

Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of 伪2 and 尾2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O^{鈥?/sup>)-diferric cofactor is located in 尾2. Although Y122O鈥?/sup> is extremely stable in free 尾2, Y122O鈥?/sup> is highly reactive in the quaternary substrate-伪2尾2 complex and serves as a radical initiator in catalytic PCET between 尾2 and 伪2. In this report, we investigate the structural interactions that control the reactivity of Y122O鈥?/sup> in a model system, isolated E. coli 尾2. Y122O鈥?/sup> was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific 13C-labeling of the tyrosine C₁ carbon assigned a component of these bands to the Y122鈥揟123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine鈥搕hreonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 脜 translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 卤 1.0 at 25 掳C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O鈥?/sup> is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O鈥?/sup>-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O鈥?/sup> and the iron cluster.}