FADH-和306Trp•在大肠杆菌光解酶中电荷重组中协同电子质子转移的证据,Journal of the American Chemical Society

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FADH-和306Trp•在大肠杆菌光解酶中电荷重组中协同电子质子转移的证据
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2011-05-25 , DOI: 10.1021/ja2001488
Agnieszka A. Zieba ₁ , Caroline Richardson ₂ , Carlos Lucero ₁ , Senghane D. Dieng ₁ , Yvonne M. Gindt ₂ , Johannes P. M. Schelvis ₁

Affiliation

质子耦合电子转移 (PCET) 是蛋白质电子转移和酶催化中非常重要的机制，芳香族氨基酸在该过程中的参与备受关注。DNA 修复酶光解酶提供了一个自然系统，允许使用中性自由基色氨酸 (Trp(•)) 研究 PCET。在大肠杆菌光解酶中，黄素腺嘌呤二核苷酸 (FAD) 辅因子以其中性自由基半醌形式 (FADH(•)) 的光还原导致 FADH(-) 和 (306)Trp(•) 的形成。这两种中间体之间的电荷重组需要 (306)Trp(•) 吸收质子。在 5.5 到 10.0 的 pH 值范围内，电荷复合的速率常数已被测量为温度的函数，并使用经典 Marcus 和半经典 Hopfield 电子转移理论对数据进行分析。与电荷重组过程相关的重组能显示出 pH 依赖性范围从 pH ≤ 7 时的 2.3 eV 到 pH(D) 10.0 时的 1.2 eV。这些发现表明电荷重组反应至少涉及两种机制。数据的整体分析支持这样一个假设，即电荷重组期间的 PCET 可以遵循两种不同的机制，在 pH 值 6.5 附近有明显的转换。在较低的 pH 值下，协同电子质子转移 (CEPT) 是有利的机制，重组能为 2.1-2.3 eV。在较高的 pH 值下，顺序机制变得占主导地位，限速电子转移，然后是质子摄取，其重组能为 1.0-1.3 eV。观察到的“逆” pH < 8 时的氘同位素效应可以通过溶剂同位素效应来解释，该效应影响反应的自由能变化并掩盖 CEPT 机制预期的正常的、与质量相关的动力学同位素效应。据我们所知，这是第一次在蛋白质中观察到 PCET 机制的转换。

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Evidence for Concerted Electron Proton Transfer in Charge Recombination between FADH–and306Trp•inEscherichia coliPhotolyase

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp(•)). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH(•)) results in the formation of FADH(-) and (306)Trp(•). Charge recombination between these two intermediates requires the uptake of a proton by (306)Trp(•). The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH ≤ 7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1-2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0-1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.

更新日期：2011-05-25

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