Martini Maria Alessandra, Bikbaev Konstantin, Pang Yunjie, Lorent Christian, Wiemann Charlotte, Breuer Nina, Zebger Ingo, DeBeer Serena, Span Ingrid, Bjornsson Ragnar, Birrell James A, Rodríguez-Maciá Patricia
Department of Inorganic Spectroscopy, Max Planck Institute for Chemical Energy Conversion Stiftstraße 34-36 45470 Mülheim an der Ruhr Germany
Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nürnberg Bioinorganic Chemistry Erlangen Germany.
Chem Sci. 2023 Feb 8;14(11):2826-2838. doi: 10.1039/d2sc06098a. eCollection 2023 Mar 15.
[FeFe] hydrogenases are highly efficient metalloenyzmes for hydrogen conversion. Their active site cofactor (the H-cluster) is composed of a canonical [4Fe-4S] cluster ([4Fe-4S]) linked to a unique organometallic di-iron subcluster ([2Fe]). In [2Fe] the two Fe ions are coordinated by a bridging 2-azapropane-1,3-dithiolate (ADT) ligand, three CO and two CN ligands, leaving an open coordination site on one Fe where substrates (H and H) as well as inhibitors ( O, CO, HS) may bind. Here, we investigate two new active site states that accumulate in [FeFe] hydrogenase variants where the cysteine (Cys) in the proton transfer pathway is mutated to alanine (Ala). Our experimental data, including atomic resolution crystal structures and supported by calculations, suggest that in these two states a third CN ligand is bound to the apical position of [2Fe]. These states can be generated both by "cannibalization" of CN from damaged [2Fe] subclusters as well as by addition of exogenous CN. This is the first detailed spectroscopic and computational characterisation of the interaction of exogenous CN with [FeFe] hydrogenases. Similar CN-bound states can also be generated in wild-type hydrogenases, but do not form as readily as with the Cys to Ala variants. These results highlight how the interaction between the first amino acid in the proton transfer pathway and the active site tunes ligand binding to the open coordination site and affects the electronic structure of the H-cluster.
[铁铁]氢化酶是用于氢转化的高效金属酶。它们的活性位点辅因子（H-簇）由一个与独特的有机金属二铁亚簇（[2Fe]）相连的典型[4Fe-4S]簇（[4Fe-4S]）组成。在[2Fe]中，两个铁离子由一个桥连的2-氮杂丙烷-1,3-二硫醇盐（ADT）配体、三个CO和两个CN配体配位，在一个铁上留下一个开放的配位位点，底物（H⁺和H⁻）以及抑制剂（O₂、CO、HS⁻）可能会结合于此。在此，我们研究了在[铁铁]氢化酶变体中积累的两种新的活性位点状态，其中质子转移途径中的半胱氨酸（Cys）被突变为丙氨酸（Ala）。我们的实验数据，包括原子分辨率晶体结构，并得到计算结果的支持，表明在这两种状态下，第三个CN配体与[2Fe]的顶端位置结合。这些状态既可以通过从受损的[2Fe]亚簇中“夺取”CN产生，也可以通过添加外源CN产生。这是首次对外源CN与[铁铁]氢化酶相互作用进行详细的光谱和计算表征。类似的CN结合状态也可以在野生型氢化酶中产生，但形成的速度不如半胱氨酸到丙氨酸变体那样快。这些结果突出了质子转移途径中的第一个氨基酸与活性位点之间的相互作用如何调节配体与开放配位位点的结合，并影响H-簇的电子结构。
