A Reversible Electron-Bifurcating Ferredoxin- and NAD-Dependent [FeFe]-Hydrogenase (HydABC) in Moorella thermoacetica

Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO2. 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 degrees C at pH 7.5 at a specific rate of about 100 mu mol 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 mu mol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) reversible arrow 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.