title: Selective release and function of one of the two FMN groups in the cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha.
abstract: The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity--the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.
authors.affiliation: Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, The Netherlands.
authors.forename: Eddy
authors.initials: E
authors.lastname: van der Linden
authors.forename: Bart W
authors.initials: BW
authors.lastname: Faber
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Tanja
authors.initials: T
authors.lastname: Burgdorf
authors.forename: Michael
authors.initials: M
authors.lastname: Bernhard
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D002850:Chromatography, Gel; D020118:Cupriavidus necator / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D005292:Ferricyanides / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
chemical_list: D005292:Ferricyanides; D009243:NAD; D005486:Flavin Mononucleotide; D006859:Hydrogen; D005949:Glucose Oxidase; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D005947:Glucose; C028033:potassium ferricyanide