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Hydrogenases catalyze the reversible heterolytic formation of H2 from protons and electrons. According to the type of metals in the active site, these enzymes are classified into three groups:[NiFe],[FeFe], and [Fe] hydrogenases.[1, 2] All of these enzymes are of high interest in biotechnology, aiming at the generation and conversion of H2 as renewable energy carrier.[1, 3] Since [FeFe] hydrogenases are highly efficient in hydrogen production in vivo, elucidation of their catalytic mechanism is of particular relevance for developing artificial hydrogen production systems.[4, 5] The active site of [FeFe] hydrogenase contains the socalled H-cluster (Figure 1) consisting of a di-iron center [2Fe], which is covalently attached via a cysteine thiol bridge to a cubane-like [4Fe-4S] subcluster.[1, 6–8] In the [2Fe] subcluster, both Fe ions are coordinated by CO and CN ligands, keeping the iron centers in low oxidation and spin states.[8–11] The iron atoms in the [2Fe] subcluster are bridged by an azadithiolate group (adt,(SCH2) 2NH).[8, 12] The iron distal to the [4Fe4S] subcluster (Fed) has an open coordination site, which is most likely the H2 binding site (Figure 1).[1, 13] It has been proposed that the adt-amine group, which is in a perfect position with respect to the open coordination site, is involved in the proton transfer to and from the catalytic site.[8, 12] Until now, two redox states of the H-cluster were identified that are believed to take part in the catalytic cycle: that is, the active “oxidized” state Hox which is paramagnetic and characterized by a mixed-valence (FeIFeII) binuclear part, and the active “reduced” state Hred, which adopts the (FeIFeI) configuration.[1 …