Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.; Upadhyay, Vikrant; Koelzer, S.; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgañg

doi:10.1074/jbc.m113.521013

Cited by 107 publications

(291 citation statements)

References 50 publications

Supporting

Mentioning

270

Contrasting

Order By: Relevance

“…The proposal is substantiated by the finding that in anaerobic bacteria the complexes of EtfAB with butyryl-CoA dehydrogenase (43,70), with caffeyl-CoA reductase (71) and with lactate dehydrogenase (72) catalyze electron-bifurcating reactions, in which the exergonic reduction of crotonylCoA, caffeyl-CoA and pyruvate, respectively, with NADH are coupled with the endergonic reduction of ferredoxin with NADH. In these complexes EtfAB is assumed to be the site of electron bifurcation (38,70). (12) MetFV alone catalyzes only the reduction of methylene-H 4 F with reduced viologen dyes (62), and EtfAB alone only catalyzes the reduction of viologen dyes with NADH (70).…”

Section: H 2 Activationmentioning

confidence: 80%

“…In these complexes EtfAB is assumed to be the site of electron bifurcation (38,70). (12) MetFV alone catalyzes only the reduction of methylene-H 4 F with reduced viologen dyes (62), and EtfAB alone only catalyzes the reduction of viologen dyes with NADH (70). Both activities are found in cell extracts of C. autoethanogenum but a ferredoxindependent reduction of methylene-H 4 F with NADH (reaction 12) or NADPH has not yet been demonstrated, maybe because MetFV and EtfAB only form a loose complex which dissociates in the cell extracts.…”

Section: H 2 Activationmentioning

confidence: 99%

See 1 more Smart Citation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

View full text Add to dashboard Cite

Most acetogens can reduce CO 2 with H 2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO 2 and H 2 . How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H 2 /CO 2 -grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD ؉ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H 2 /CO 2 -grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H 2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO 2 and H 2 . IMPORTANCEEthanol formation from syngas (H 2 , CO, and CO 2 ) and from H 2 and CO 2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H 2 and CO 2 , considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H 2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

show abstract

Section: H 2 Activationmentioning

confidence: 80%

Section: H 2 Activationmentioning

confidence: 99%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

View full text Add to dashboard Cite

show abstract

“…In dissimilatory sulfate reduction, a function in coupling of cytoplasmic redox reactions to membrane-associated redox reactions has been proposed (16,69). The HdrABC complex appears to be an electron-bifurcating module used over and over again in different contexts, as is the EtfAB complex (70), which is used in many anaerobes as an electronbifurcating module in crotonyl-CoA reduction (71), caffeyl-CoA reduction (72), and lactate oxidation (73). This hypothesis still remains to be experimentally verified.…”

Section: Discussionmentioning

confidence: 99%

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

et al. 2014

View full text Add to dashboard Cite

c Moorella thermoacetica can grow with H 2 and CO 2 , forming acetic acid from 2 CO 2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. 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 the metF gene in Escherichia coli revealed 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 in M. thermoacetica is 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.

show abstract

“…Finally, the bacteria were shifted to a medium with 50 mM either 4-hydroxy [ 10 mM CoA, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM NADH, and 175 mM sodium formate. The following proteins were added: 4-hydroxybutyrate CoA-transferase (1.0 U/ml), 4HBD (0.13 U/ml), electron-transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium tetanomorphum (4.5 U/ml, ferricenium assay) (6,43,44), ferredoxin from C. tetanomorphum (1 mg/ml) (6), formate dehydrogenase from Candida boidinii (3.13 U/ml; Sigma-Aldrich), and hydrogenase from Clostridium pasteurianum (4.5 U/ml) (45). The reaction was carried out under anoxic conditions for 18 h at room temperature.…”

Section: Mutationmentioning

confidence: 99%

“…Activation to the CoA thioester and dehydration affords crotonyl-CoA, which disproportionates to butyrate and acetate. Energy is conserved via substrate level phosphorylation via acetylphosphate and via electron bifurcation at the electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (5,6). The reduced ferredoxin obtained thereby recycles NADH, mediated by NAD-ferredoxin oxidoreductase, also called Rnf, which generates an electrochemical Na ϩ gradient for ATP synthesis (7,8).…”

mentioning

confidence: 99%

Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

Zhang

Friedrich

Pierik

et al. 2015

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

2؉ cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B 12 , S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose. 4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible ⌬-isomerization of vinylacetyl-CoA to crotonyl-CoA (Fig. 1). The enzyme was discovered in the fermentation of 4-aminobutyrate (␥-aminobutyrate [GABA]) to ammonia, acetate, and butyrate (Fig. 2) by Clostridium aminobutyricum (1, 2), attributed to cluster XI of the clostridia (3, 4). In this pathway, 4-aminobutyrate is transaminated with 2-oxoglutarate to yield glutamate and succinic semialdehyde (4-oxobutanoate), which is reduced to 4-hydroxybutyrate. Activation to the CoA thioester and dehydration affords crotonyl-CoA, which disproportionates to butyrate and acetate. Energy is conserved via substrate level phosphorylation via acetylphosphate and via electron bifurcation at the electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (5, 6). The reduced ferredoxin obtained thereby recycles NADH, mediated by NAD-ferredoxin oxidoreductase, also called Rnf, which generates an electrochemical Na ϩ gradient for ATP synthesis (7,8). With one additional dehydrogenase, this pathway is used by Clostridium kluyveri to reduce succinyl-CoA to butyrate (Fig. 2) (9). Autotrophic CO 2 -fixing Crenarchaeota synthesize succinyl-CoA by reductive carboxylations of acetyl-CoA. The pathway depicted in Fig. 2 recycles the carrier molecule acetyl-CoA and forms a second one for biosynthesis (10, 11). The key enzyme of all these conversions is 4HBD, which links lipid and carbohydrate metabolisms. In this work, we study the enzyme in more detail and propose a plausible mechanism of action.4HBD from C. aminobutyricum is a homotetrameric enzyme (4ϫ 56 kDa) containing one 2ϩ...

show abstract

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Cited by 107 publications

References 50 publications

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

Contact Info

Product

Resources

About

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Cited by 107 publications

References 50 publications

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

Contact Info

Product

Resources

About

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation