title: Use of a reverse micelle system for study of oligomeric structure of NAD+-reducing hydrogenase from Ralstonia eutropha H16.
abstract: Inclusion of an oligomeric enzyme, NAD+-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD+/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.
authors.affiliation: Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia. ttikhonova@inbi.ras.ru
authors.forename: T V
authors.initials: TV
authors.lastname: Tikhonova
authors.forename: S A
authors.initials: SA
authors.lastname: Kurkin
authors.forename: N L
authors.initials: NL
authors.lastname: Klyachko
authors.forename: V O
authors.initials: VO
authors.lastname: Popov
mesh_terms: D002384:Catalysis; D020118:Cupriavidus necator / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000235:genetics*; D007123:Immunoelectrophoresis, Two-Dimensional; D008823:Micelles; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D008823:Micelles; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase; D009245:NADH Dehydrogenase