Activation of HydA^ΔEFG Requires a Preformed [4Fe-4S] Cluster

Abstract

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.

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

“…Detailed spectroscopic and structural studies of HydA ΔEFG have shown that it contains a [4Fe-4S] cluster and is poised to accept a 2Fe subcluster prior to activation by the accessory proteins (9,10). We have also shown that HydF purified from E. coli in which all three accessory proteins were coexpressed (HydF EG ) was competent in HydA ΔEFG activation, indicating that HydF acts as a scaffold or carrier protein during activation of HydA (20).…”

“…Assembly of a catalytically competent H cluster requires the actions of three hydrogenase-specific accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine (SAM) enzymes and the third of which (HydF) is a GTPase (7,8). These accessory proteins are directed at synthesis of the 2Fe subcluster of the H cluster, which is subsequently transferred to the hydrogenase structural protein (HydA) containing a preformed [4Fe-4S] cluster (9,10) to produce the active hydrogenase. The detailed stepwise mechanism of H-cluster assembly, as well as the specific roles of and interactions between the three accessory proteins in this assembly process, remains largely unknown.…”

Biochemical Analysis of the Interactions between the Proteins Involved in the [FeFe]-Hydrogenase Maturation Process

“…According to several recent in vitro studies, this process occurs in a multistep pathway, in which the [4Fe-4S] unit is synthesized by the Isc/Suf FeS general cell machinery, whereas the biosynthesis and insertion of the 2Fe subcluster, together with its ligands, is driven by HydE, HydG, and HydF maturases (5)(6)(7)(8)17). Current data indicate that the addition of the CO/CN Ϫ / dithiolate ligands to the 2Fe subcluster is accomplished through the action of the HydE/HydG radical SAM chemistry coupled to the presence of HydF, which would have a double involvement as scaffold and carrier to build and insert the modified 2Fe subcluster into a hydrogenase containing a preformed [4Fe-4S] unit (17)(18)(19)(20)(21).…”

In Vivo EPR Characterization of Semi‐Synthetic [FeFe] Hydrogenases

“…Already prior to the formation of [2Fe] pdt ‐HydA1, the [4Fe4S] cluster present in apo‐HydA1 is potentially detectable under our experimental conditions 8, 27. As seen in Figure 2 (spectrum a), the EPR spectrum of apo‐HydA1‐expressing cells featured a number of signals, but we could only observe minor differences compared to control samples of BL21(DE3) cells lacking the HydA1 plasmid (Supporting Information, Figure S2).…”

“…Consequently, we assign the signal to the formation of [2Fe] pdt ‐HydA1, residing in the paramagnetic H ox ‐like state previously described as [4Fe‐4S] 2+ ‐[Fe I Fe II ] pdt 14, 16. To further support the assignment of the observed EPR signal we generated purified [2Fe] pdt ‐HydA1 ([2Fe] pdt ‐HydA1 pur ) through a modified literature protocol 8, 13, 27. The purified semi‐synthetic protein was oxidized by thionine to generate the H ox ‐like state, and the EPR spectrum of [2Fe] pdt ‐HydA1 pur showed the expected rhombic signal (Figure 2, spectrum d) 14.…”