Activation of HydA(DeltaEFG) 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.
Mulder
David W
DW
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715, USA.
Ortillo
Danilo O
DO
Gardenghi
David J
DJ
Naumov
Anatoli V
AV
Ruebush
Shane S
SS
Szilagyi
Robert K
RK
Huynh
BoiHanh
B
Broderick
Joan B
JB
Peters
John W
JW
D015502:Absorptiometry, Photon; D000818:Animals; D001051:Apoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D055162:Biocatalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002712:Chlorides; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005290:Ferric Compounds / Q000737:chemistry; D005583:Fourier Analysis; D006864:Hydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D013440:Sulfides / Q000737:chemistry
D001051:Apoenzymes; D002712:Chlorides; D005290:Ferric Compounds; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D013440:Sulfides; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase; C024555:ferric chloride; C033479:sodium sulfide