title: The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases.
abstract: Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3.
authors.affiliation: Department I der Fakultät für Biologie, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638 Münich, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Blokesch
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
authors.forename: Berthold F
authors.initials: BF
authors.lastname: Matzanke
authors.forename: Nikola M
authors.initials: NM
authors.lastname: Drapal
authors.forename: Alexander
authors.initials: A
authors.lastname: Jacobi
authors.forename: August
authors.initials: A
authors.lastname: Böck
mesh_terms: D020134:Catalytic Domain; D003486:Cyanides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D046911:Macromolecular Substances; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015204:Spectroscopy, Mossbauer
chemical_list: D003486:Cyanides; D029968:Escherichia coli Proteins; C491587:HypC protein, E coli; C108875:HypD protein, Bacteria; D046911:Macromolecular Substances; D011506:Proteins; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase