c 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 ironsulfur 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). Five years ago it was discovered that in butyric acid-forming clostridia, the exergonic reduction of crotonyl coenzyme A (crotonyl-CoA; E 0 = ϭ Ϫ10 mV) with NADH (E 0 = ϭ Ϫ320 mV) is coupled with the endergonic reduction of ferredoxin (Fd) (E 0 = ϭ Ϫ400 mV) with NADH (reaction 1) catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron transfer flavoprotein complex Bcd/EtfAB (1, 2).The available evidence indicates that electron bifurcation is flavin based: a protein-bound flavin is reduced by NADH to the hydroquinone, which is subsequently reoxidized by crotonylCoA to the semiquinone radical that has a redox potential sufficiently negative to reduce ferredoxin (3). The proposed mechanism is analogous to the mechanisms of ubiquinonebased electron bifurcation in the bc1 complex of the aerobic respiratory chain and plastoquinone-based electron bifurcation in the b6f-complex in oxygenic photosynthesis (4-6). The main differences between the electron bifurcation mechanisms are that flavin-based electron bifurcation is associated with cytoplasmic proteins and operates at more negative redox potentials (Ϫ300 mV Ϯ 200 mV), whereas ubiquinone/plastoquinone-based electron bifurcation is associated with membranes and operates at more positive redox potentials (ϩ100 mV Ϯ 200 mV) (3,(7)(8)(9).Since t...

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...

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-