†Chemistry Department, Emory University, Atlanta, Georgia 30322, United States.
‡Department of Biochemistry, University of Georgia, Athens, Georgia 30602, United States.
J Am Chem Soc. 2015 Apr 8;137(13):4558-66. doi: 10.1021/jacs.5b01791. Epub 2015 Mar 30.
The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.
质子和电子的运动是所有化学燃料（如 H2）合成的共同特征。氢化酶能够催化质子的可逆还原，这需要质子和电子在两者之间进行传输和反应，但迄今为止，其详细的作用机制仍难以捉摸。在这里，我们使用光触发化学势跃变方法来快速启动 [NiFe]氢化酶的质子还原活性。将光化学引发方法与纳秒瞬态红外和可见吸收光谱相结合，可直接观察到界面电子转移和活性位点化学。通过 pH 值和同位素取代来调节分子内质子传输，揭示了在催化过程中存在独特的协同和分步质子耦合电子转移机制。在两个连续的质子相关还原过程中观察到的异质性表明，高度工程化的蛋白质环境可以调节催化作用，并暗示了三个新的反应中间体；Nia-I、Nia-D 和 Nia-SR(-)。该结果建立了对 [NiFe]氢化酶催化作用的基本机制理解，这对酶促质子耦合电子转移和仿生催化剂设计具有重要意义。
