title: A reversible electron-bifurcating ferredoxin- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Moorella thermoacetica.
abstract: Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO(2). Depending on the growth substrate, this Gram-positive bacterium can either form H(2) or consume it. Despite the importance of H(2) in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H(2) formation and H(2) uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD(+) with H(2) at 55 °C at pH 7.5 at a specific rate of about 100 μmol min(-1) mg protein(-1) and the reverse reaction, the coupled reduction of protons to H(2) with reduced ferredoxin and NADH, at a specific rate of about 10 μmol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) Fd(red)(2-) + NADH + 3H(+). When ferredoxin from Clostridium pasteurianum, NAD(+), and the enzyme were incubated at pH 7.0 under 100% H(2) in the gas phase (E(0)' = -414 mV), more than 95% of the ferredoxin (E(0)' = -400 mV) was reduced, which indicated that ferredoxin reduction with H(2) is driven by the exergonic reduction of NAD(+) (E(0)' = -320 mV) with H(2). In the absence of NAD(+), ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site.
authors: Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors: Shuning
authors: S
authors: Wang
authors: Haiyan
authors: H
authors: Huang
authors: Jörg
authors: J
authors: Kahnt
authors: Rudolf K
authors: RK
authors: Thauer
mesh_terms: D055162:Biocatalysis; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D058845:Moorella / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D024363:Transcription Initiation Site
chemical_list: D005288:Ferredoxins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D009243:NAD; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase