Scheme 1.
In vitro assembly of the H-cluster. Our present data (in combination also with earlier reports, refs [32, 33]) suggest at least three consecutive steps during the cofactor assembly. Fusion of the [4Fe4S]H cluster and the [2Fe]adt complex results in an early adduct, here assigned to the Hred′-CO state, followed by H-cluster oxidation to form Hox-CO, and final CO release yielding the active Hox state of the cofactor, which is the entry species into the catalytic H2-conversion cycle of [FeFe]-hydrogenases. Oxidation states of the [4Fe–4S]H and [2Fe]H components are indicated in red