Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes ( fdhA/hytA-E ) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP ⁺ with H ₂ or formate and the reversible formation of H ₂ and CO ₂ from formate. We propose the complex to have two functions in vivo , namely, to normally catalyze CO ₂ reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H ₂ formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO ( E _0′ = −520 mV).

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex fromClostridium kluyvericouples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex inC. kluyvericapable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺with NADH in a reversible reaction: Fd_red²⁻+ NADH + 2 NADP⁺+ H⁺= Fd_ox+ NAD⁺+ 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation ofC. kluyveriis discussed.

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO ₂ molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd _red ²⁻ ) molecules. In the reductive part, 2 CO ₂ molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO ₂ are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO ₂ reduction to acetic acid are generated by the reduction of 2 NADP ⁺ molecules with 1 NADH and 1 Fd _red ²⁻ catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP ⁺ oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli , purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD ⁺ and Fd with 2 H ₂ molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H ₂ -CO ₂ -grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H ₂ -CO ₂ -grown cells additionally contain high specific activities of an NADP ⁺ -dependent hydrogenase that catalyzes the reduction of NADP ⁺ with H ₂ . This activity is hardly detectable in glucose-grown cells.

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 H₂ or consume it. Despite the importance of H₂ 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 H₂ formation and H₂ uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD⁺ with H₂ 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 H₂ with reduced ferredoxin and NADH, at a specific rate of about 10 μmol min⁻¹ mg protein⁻¹ in the stoichiometry Fd_ox + NAD⁺ + 2H₂ ⇋ Fd_red²⁻ + NADH + 3H⁺. When ferredoxin from Clostridium pasteurianum, NAD⁺, and the enzyme were incubated at pH 7.0 under 100% H₂ in the gas phase (E₀′ = −414 mV), more than 95% of the ferredoxin (E₀′ = −400 mV) was reduced, which indicated that ferredoxin reduction with H₂ is driven by the exergonic reduction of NAD⁺ (E₀′ = −320 mV) with H₂. 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.

Moorella thermoaceticacan grow with H₂and CO₂, forming acetic acid from 2 CO₂via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that theM. thermoaceticagene that putatively encodes this enzyme,metF, is part of a transcription unit also containing the geneshdrCBA,mvhD, andmetV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of themetFgene inEscherichia colirevealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction inM. thermoaceticais NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.

NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD(+). Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg(187) is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH(•)/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed.