In methanogenic archaea growing on H 2 and CO 2 the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO 2 with H 2 to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H 2 to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB-dependent reduction of ferredoxin with H 2 . Per mole CoM-S-S-CoB added, 1 mol of ferredoxin (Fd) was reduced, indicating an electron bifurcation coupling mechanism: This stoichiometry of coupling is consistent with an ATP gain per mole methane from 4 H 2 and CO 2 of near 0.5 deduced from an H 2 -threshold concentration of 8 Pa and a growth yield of up to 3 g/mol methane.

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples 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 in C. kluyveri capable 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:The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H 2 (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO 2 . Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27).During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate Ϫ , one H ϩ , and two H 2 (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P i (⌬G o Ј ϭ ϩ32 kJ/ mol) has remained unclear for many years.We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E o Ј ϭ Ϫ10 mV) with NADH (E o Ј ϭ Ϫ320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd ox ) (E o Ј ϭ Ϫ420 mV) with NADH (E o Ј ϭ Ϫ320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12):The reduced ferredoxin (Fd red 2Ϫ ) is assumed to be used for rereduction of NAD ϩ via a membrane-associated, protontranslocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membraneassociated F 1 F 0 ATP synthetase (reaction 4):The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP ϩ -and an NAD ϩ -specific ␤-hydroxybutyrylCoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8,9,13).The presence of a reduced fe...

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bMoorella 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. F lavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Via this novel mechanism, e.g., the endergonic reduction of ferredoxin (Fd) (two [4Fe4S] clusters, each with an E o = of ϽϪ400 mV) with NADH (E o = ϭ Ϫ320 mV) is coupled to the exergonic reduction of crotonyl coenzyme A (CoA) to butyryl-CoA (E o = ϭ Ϫ10 mV) with NADH (E o = ϭ Ϫ320 mV) in butyric acid-forming clostridia (reaction 1). The coupled reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron transfer flavoprotein complex (Bcd/EtfAB) containing only flavin adenine dinucleotides (FADs) as prosthetic groups (28, 37). The mechanism of coupling was proposed previously to proceed similarly to that of ubiquinone-based electron bifurcation in the cytochrome bc 1 complex of the respiratory system (9, 29, 46). One of the main differences between flavin-and ubiquinone-based electron bifurcations appears to be that flavin-based electron bifurcation is associated with a cytoplasmic enzyme complex and operates at redox potentials around that of free flavins (Ϫ200 mV), whereas ubiquinone-based electron bifurcation is associated with a membrane enzyme complex and operates at around the redox potential of ubiquinone (ϩ110 mV).Another example of flavin-based electron bifurcation is the coupling of ferredoxin reduction with H 2 (E o = ϭ Ϫ414 mV) to the reduction of the heterodisulfide CoM-S-S-CoB (E o = ϭ Ϫ140 mV) with H 2 in methanogenic archaea growing on H 2 and CO 2 (reaction 2) (⌬G o = calculated by using an E o = of Ϫ400 mV for ferredoxin from Clostridium pasteurianum [65] Reaction 3 was demonstrated previously by Schut and Adams only in the direction of H 2 formation (61). In this direction, the enzyme catalyzing the reaction is actually confurcating rather than bifurcating. However, the flavin mononucleotide (FMN)-depen-

An open and closed case: The structure of a binary complex of C176A [Fe]‐hydrogenase with methylenetetrahydromethanopterin was solved at 2.15 Å resolution in an open conformation. A closed form of the complex was modeled on the basis of the experimentally determined structure. In this model, the iron‐site trans to the acyl carbon is located next to the C14a and therefore considered as H2 binding site.

Growth of Methanobacterium thermoautotrophicum on H2 and CO2 as sole energy and carbon sources was found to be dependent on Ni, Co, and Mo. At low concentrations of Ni (less than 100 nM), Co (less than 10 nM) and Mo (less than 10 nM) the amount of cells formed was roughly proportional to the amount of transition metal added to the medium; for the formation of 1 g cells (dry weight) approximately 150 nmol NiCl2, 20 nmol CoCl2 and 20 nmol Na2MoO4 were required. A dependence of growth on Cu, Mn, Zn, Ca, Al, and B could not be demonstrated. Conditions are described under which the bacterium grew exponentially with a doubling time of 1.8 h up to a cell density of 2 g cells (dry weight)/l.

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An open and closed case: The structure of a binary complex of C176A [Fe]‐hydrogenase with methylenetetrahydromethanopterin was solved at 2.15 Å resolution in an open conformation. A closed form of the complex was modeled on the basis of the experimentally determined structure. In this model, the iron‐site trans to the acyl carbon is located next to the C14a and therefore considered as H2 binding site.