title: The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen.
abstract: Infrared spectra of (15)N-enriched preparations of the soluble cytoplasmic NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)(2)(CN)Ni(micro-RS)(2)Fe(CN)(3)(CO) centre (R=Cys) and that H(2) activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.
authors.affiliation: Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
authors.forename: Eddy
authors.initials: E
authors.lastname: Van der Linden
authors.forename: Tanja
authors.initials: T
authors.lastname: Burgdorf
authors.forename: Michael
authors.initials: M
authors.lastname: Bernhard
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
mesh_terms: D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D003486:Cyanides / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D008024:Ligands; D009243:NAD / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D003486:Cyanides; D008024:Ligands; D009243:NAD; D009532:Nickel; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen