Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, New Jersey 07043, USA.
J Am Chem Soc. 2011 May 25;133(20):7824-36. doi: 10.1021/ja2001488. Epub 2011 May 2.
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.
质子偶联电子转移（PCET）是蛋白质电子转移和酶催化中的一种重要机制，而芳香族氨基酸在这个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，可以使用中性自由基色氨酸（Trp(•)）来研究 PCET。在大肠杆菌光解酶中，黄素腺嘌呤二核苷酸（FAD）辅因子的光还原会导致 FADH(-)和（306）Trp(•)的形成。这两种中间体之间的电荷复合需要（306）Trp(•)吸收一个质子。已经在 pH 范围为 5.5 到 10.0 之间测量了电荷复合速率常数作为温度的函数，并使用经典 Marcus 和半经典 Hopfield 电子转移理论对数据进行了分析。与电荷复合过程相关的重组能显示出从 pH≤7 时的 2.3 eV 到 pH(D)10.0 时的 1.2 eV 的 pH 依赖性。这些发现表明至少有两种机制参与了电荷复合反应。数据的全局分析支持这样的假设，即在电荷复合过程中，PCET 可以遵循两种不同的机制，在 pH 约 6.5 处存在明显的转换。在较低的 pH 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 下，顺序机制变得占主导地位，限速电子转移后是质子吸收，重组能为 1.0-1.3 eV。在 pH<8 时观察到的“反向”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，并掩盖了预期的 CEPT 机制的正常、与质量相关的动力学同位素效应。据我们所知，这是首次在蛋白质中观察到 PCET 机制的转换。
