Max-Planck-Institut für Chemische Energiekonversion , Stiftstrasse 34-36, 45470 Mülheim/Ruhr, Germany.
J Am Chem Soc. 2017 Feb 1;139(4):1440-1443. doi: 10.1021/jacs.6b12636. Epub 2017 Jan 17.
The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] ⇔ [4Fe-4S] and [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)] thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + 2e ⇄ H. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H of the H-cluster actually consists of two species: H = [4Fe-4S] - [Fe(I)Fe(II)] and HH = [4Fe-4S] - [Fe(I)Fe(I)] (H) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H/HH) has a pK ≈ 7.2.
[FeFe]氢化酶的活性位点是 H 簇，由一个 [4Fe-4S]簇通过桥连半胱氨酸与一个带有 CO 和 CN 配体以及桥连氮杂二硫代羧酸配体（ADT）的 [2Fe] 配合物相连，其中胺部分作为质子穿梭体在蛋白质和 H 簇之间传递质子。在催化循环中，两个亚簇改变氧化态：[4Fe-4S] ⇔ [4Fe-4S]和 [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)]，从而能够储存催化反应 2H + 2e ⇄ H 所需的两个电子。我们使用不同 pH 值下的莱茵衣藻（Chlamydomonas reinhardtii）[FeFe]氢化酶（CrHydA1）的 FTIR 光谱电化学方法，解析了催化循环中的氧化还原和质子化事件，并确定了它们的内在热力学参数。我们表明，H 簇的单还原态 H 实际上由两种物质组成：H = [4Fe-4S] - [Fe(I)Fe(II)]和 HH = [4Fe-4S] - [Fe(I)Fe(I)]（H），它们通过质子耦合电子重排相关。催化循环中的两个氧化还原事件发生在 [4Fe-4S] 亚簇上，其中点电位相似（-375 与-418 mV）；质子化事件（H/HH）的 pK 值约为 7.2。
