The role of the maturase HydG in [FeFe]-hydrogenase active site synthesis and assembly.
ABSTRACT [FeFe]-hydrogenases catalyze the protons/hydrogen interconversion through a unique di-iron active site consisting of three CO and two CN ligands, and a non-protein SCH(2)XCH(2)S (X=N or O) dithiolate bridge. Site assembly requires two "Radical-S-adenosylmethionine (SAM or AdoMet)" iron-sulfur enzymes, HydE and HydG, and one GTPase, HydF. The sequence homology between HydG and ThiH, a Radical-SAM enzyme which cleaves tyrosine into p-cresol and dehydroglycine, and the finding of a similar cleavage reaction catalyzed by HydG suggests a mechanism for hydrogenase maturation. Here we propose that HydG is specifically involved in the synthesis of the dithiolate ligand, with two tyrosine-derived dehydroglycines as precursors along with an [FeS] cluster of HydG functioning both as electron shuttle and source of the sulfur atoms.
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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.Biochemistry 05/2009; 48(26):6240-8. · 3.42 Impact Factor
Article: The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN- ligated iron cofactor.[show abstract] [hide abstract]
ABSTRACT: Biosynthesis of the [FeFe] hydrogenases active site (H-cluster) requires three maturation factors whose respective roles are not understood yet. The clostridial maturation enzymes (CaHydE, CaHydF and CaHydG) were homologously overexpressed in their native host Clostridium acetobutylicum. CaHydF was able to activate Chlamydomonas reinhardtii [FeFe] hydrogenase apoprotein (CrHydA1(apo)) to almost 100% compared to the native specific hydrogen evolution activity. Based on electron paramagnetic resonance spectroscopy and Fourier-transform infrared spectroscopy data the existence of a [4Fe4S] cluster and a CO and CN(-) ligand coordinated di-iron cluster is suggested. This study contains the first experimental evidence that the bi-nuclear part of the H-cluster is assembled in HydF.FEBS letters 12/2009; 584(3):638-42. · 3.54 Impact Factor
Article: Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.[show abstract] [hide abstract]
ABSTRACT: [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.PLoS ONE 01/2011; 6(5):e20346. · 4.09 Impact Factor