Probing the solvent accessibility of the [4Fe-4S] cluster of the hydrogenase maturation protein HydF from Thermotoga neapolitana by HYSCORE and 3p-ESEEM.

The catalytic site of [FeFe]-hydrogenase, the “H-cluster”, composed by a [4Fe-4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN- and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE and HydF. HydF is a complex enzyme, which is thought to act as scaffold and carrier for the [2Fe] subunit of the H-cluster. This maturase protein contains itself a [4Fe-4S] cluster binding site, with three conserved cysteine residues and a non-cysteinyl fourth ligand. In this work we have exploited 3p-ESEEM and HYSCORE spectroscopies to get insight into the structure and the chemical environment of the [4Fe-4S] cluster of HydF from the hyperthermophilic organism Thermotoga neapolitana. The nature of the fourth ligand and the solvent accessibility of the active site comprising the [4Fe-4S] cluster is discussed on the basis of the spectroscopic results obtained upon H/D exchange. We propose that the non-cysteinyl ligated Fe atom of the [4Fe-4S] cluster is the site where the [2Fe] subcluster precursor is anchored and finally processed to be delivered to the hydrogenase (HydA).