[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy

[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe–4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH 2 S) 2 )·[FeFe]-hydrogenase apo-protein binding only the [4Fe–4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe] adt ). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe] adt . The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred′-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster. Graphic abstract