[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe−4S] electron relay covalently attached to the H₂ binding site and a diiron cluster ligated by CO, CN^–, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe−4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe−4S]_H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

中文翻译：

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时间分辨红外光谱揭示了 [FeFe] 氢化酶催化循环中第一个电子转移步骤的 pH 依赖性

[FeFe] 氢化酶是用于氢转化的高活性催化剂。它们的活性位点有两个组成部分：共价连接到 H ₂结合位点的 [4Fe-4S] 电子中继和由 CO、CN ^–和 2-azapropane-1,3-dithiolate (ADT) 配体连接的二铁簇。[4Fe-4S] 位点的还原被提议与其半胱氨酸配体之一的质子化相结合。在这里，我们对来自莱茵衣藻( Cr HydA1)的 [FeFe] 氢化酶使用时间分辨红外 (TRIR) 光谱，其中含有丙烷-1,3-二硫醇盐 (PDT) 配体而不是天然 ADT 配体。PDT 修饰不影响电子转移到 [4Fe-4S] _H的步骤但会阻止酶在催化循环中进一步进行。我们表明，第一个电子转移步骤的速率与 pH 值无关，支持简单的电子转移而不是质子耦合事件。这些结果对我们理解[FeFe]氢化酶的催化机制具有重要意义，并突出了TRIR的实用性。