The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions.

[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research due to their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the "H-cluster". The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a pre-catalyst to the apo-hydrogenase, but the details of this process are not well understood. Herein we utilize Gas-phase Electrophoretic MacroMolecule Analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase, and that the formation of this complex is dependent on the cofactor content on HydF. Moreover, FTIR, EPR and UV/Vis spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the pre-catalyst in a reversible fashion, and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in final stages of H-cluster assembly, i.e. in binding and delivering the pre-catalyst.