title: The auxiliary protein HypX provides oxygen tolerance to the soluble [NiFe]-hydrogenase of ralstonia eutropha H16 by way of a cyanide ligand to nickel.
abstract: The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD+ -reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H2 oxidation with NAD+ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.
authors.affiliation: Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands.
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Thorsten
authors.initials: T
authors.lastname: Buhrke
authors.forename: Eddy
authors.initials: E
authors.lastname: van der Linden
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D002474:Cell-Free System; D020118:Cupriavidus necator / Q000201:enzymology*; D003486:Cyanides / Q000737:chemistry*; D004583:Electrons; D017353:Gene Deletion; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry*; D008024:Ligands; D008956:Models, Chemical; D009154:Mutation; D009532:Nickel / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; C101839:HypX protein, Rhizobium leguminosarum; D008024:Ligands; D009532:Nickel; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen