Activation of HydAΔEFG Requires a Preformed 4Fe-4S Cluster

The H-cluster is a complex bridged metal assembly at the active site of FeFe-hydrogenases that consists of a 4Fe-4S subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active FeFe-hydrogenase have previously been identified and shown to be required for the heterologous expression of active FeFe-hydrogenase Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA(DeltaEFG) (the FeFe-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a 4Fe-4S cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(DeltaEFG)) and S(2-) (1.8 +/- 0.5 S(2-) atoms per HydA(DeltaEFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = (1)/(2) EPR signal (g = 2.04 and 1.91) characteristic of a reduced 4Fe-4S(+) cluster. Mossbauer spectroscopic characterization of (57)Fe-enriched HydA(DeltaEFG) provided further evidence of the presence of a redox active 4Fe-4S(2+/+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a 4Fe-4S cluster in HydA(DeltaEFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(DeltaEFG) can be activated by the specific maturases only when a 4Fe-4S cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.