The [4Fe4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies.

[FeFe]-hydrogenases are key enzymes for bio(photo)production of hydrogen and several efforts are underway to understand how their complex active site (the H-cluster) is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins (HydE, HydF and HydG) are required for its biosynthesis. HydF, is a maturase protein which drives the last step of the H-cluster assembly process and it has been proposed to play a double function acting as a scaffold for the building of the cluster and at the same time as a carrier for the delivery of the active metal center to HydA. HydF maturase is an iron sulphur protein containing a [4Fe-4S] cluster. Its dual role is associated with the ability of HydF to bind and dissociate a FeS center to be transferred to HydA. We solved the X-ray structure of the apo-HydF from Thermotoga neapolitana, and described the domain containing the consensus sequence (CxHx46-53HCxxC), with the three highly conserved cysteines that represent the putative FeS cluster binding site possibly along with a histidine residue as fourth metal ligand. To explore the role of these residues on the biosynthesis/transfer of the H-cluster precursor, we analyzed by EPR and HYSCORE spectroscopies the HydF holoproteins from T. neapolitana and Clostridium acetobutylicum. To investigate the [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF, we used electron paramagnetic spectroscopy, EPR and HYSCORE spectroscopy, combined with site directed mutagenesis of the recombinant proteins overexpressed in E. coli.