Activation of HydA(Delta EFG) Requires a Preformed [4Fe-4S] Cluster

作者：Mulder David W; Ortillo Danilo O; Gardenghi David J; Naumov Anatoli V; Ruebush Shane S; Szilagyi Robert K; Huynh BoiHanh; Broderick Joan B; Peters John W^*

来源：Biochemistry, 2009, 48(26): 6240-6248.

DOI：10.1021/bi9000563

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 subduster 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 subduster. The spectroscopic and biochemical characterization of HydA(Delta EFG) (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(Delta EFG)) and S2- (1.8 +/- 0.5 S2- atoms per HydA(Delta EFG)) 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 Fe-57-enriched HydA(Delta EFG) 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(Delta EFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(Delta EFG) 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 subduster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.

• 出版日期2009-7-7