The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

Hess, Verena; Schuchmann, Kai; Müller, Volker

doi:10.1074/jbc.m113.510255

Cited by 141 publications

(167 citation statements)

References 33 publications

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“…The two organisms have different membrane-associated enzyme complexes required for conserving energy during growth with H 2 and CO 2 , as revealed from genome sequences (20,75). A. woodii involves a membrane-associated sodium ion-translocating reduced ferredoxin:NAD oxidoreductase (RnfA to -G) (75,76), which is lacking in M. thermoacetica. On the other hand, it is predicted that M. thermoacetica contains a membrane-associated, energy-converting [NiFe]-hydrogenase (EchABCE to -I) in complex with formate dehydrogenase (FdhA2) (20) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

et al. 2014

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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.

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Section: Discussionmentioning

confidence: 99%

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

et al. 2014

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“…In A. woodii, 2 NADH, 1 ferredoxin, and 1 H 2 (equivalent to 0.5 NADH and 0.5 ferredoxin) are necessary for the reduction of 2 CO 2 to acetate (6,8,24). The endergonic reduction of ferredoxin from NADH is catalyzed by the membrane-bound Rnf complex energized by a sodium ion gradient generated by ATP hydrolysis by the ATP synthase (7,25). This reverse electron flow consumes ATP and thus the conversion of 1,2-PD to propionate with CO 2 , as the electron acceptor would result in a theoretical ATP gain of 0.75 mol ATP per mol of 1,2-PD.…”

Section: Discussionmentioning

confidence: 99%

Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

Schuchmann¹,

Schmidt²,

López³

et al. 2014

J. Bacteriol.

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dAcetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO 2 in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.A cetogenic bacteria are a diverse group of anaerobic bacteria able to reduce two molecules of CO 2 to acetate by the WoodLjungdahl pathway (WLP) (1-4). Electrons may derive from molecular hydrogen (autotrophic growth) or from organic donors (heterotrophic growth), such as hexoses, pentoses, formate, lactate, alcohols, and methyl group donors (1). This not only provides the cell with organic material for biomass formation, but the pathway is also coupled to energy conservation for ATP supply (2, 5). The energy-conserving reactions remained an enigma for a long time, but recent discoveries in the model acetogen Acetobacterium woodii provided insights into the energy metabolism of this group of anaerobic bacteria (6, 7). In A. woodii, the reactions for the oxidation of the substrate can in general be regarded as isolated modules separate from the reactions of the WLP for the reoxidation of the electron carriers by the reduction of CO 2 . One has to emphasize that all enzymes of the WLP are soluble and located in the cytoplasm (6). With molecular hydrogen as the electron donor, only one enzyme is necessary for its oxidation, providing the reducing equivalents as reduced ferredoxin (Fd) and NADH in a 1:1 stoichiometry (8). Oxidation of organic substrates, such as hexoses, also yields reduced ferredoxin and NADH that are reoxidized in the WLP. The WLP in A. woodii does not use both electron carriers in equal amounts; therefore, a membranebound Fd:NAD oxidoreductase (presumably the Rnf complex) provides NADH from reduced ferredoxin, thereby translocating sodium ions across the cytoplasmic membrane that are used for subsequent ATP synthesis by a membrane-bound, sodium iondependent ATP synthase (7, 9, 10). The reaction is reversible, and the enzyme can drive Fd reduction at the expense of the electrochemical sodium ion potential.Besides CO 2 , acetogenic bacteria can use different alternative electron acceptors, e.g., nitrate (Moorella thermoacetica [11]) or phenylacrylates (A. woodii [12]). These acceptors have a more positive redox potential than the CO 2 -acetate pair,...

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“…Then, 1.8 mM NAD, 0.2 mM CoA, and 10 mM acetaldehyde were added to the assay mixture. Formate dehydrogenase (hydrogen-dependent CO 2 reductase [HDCR]), Rnf, and CO dehydrogenase (CODH) activities were measured as described previously (22,23).…”

Section: Methodsmentioning

confidence: 99%

Ethylene Glycol Metabolism in the Acetogen Acetobacterium woodii

2016

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The acetogenic bacterium Acetobacterium woodii is able to grow by the oxidation of diols, such as 1,2-propanediol, 2,3-butanediol, or ethylene glycol. Recent analyses demonstrated fundamentally different ways for oxidation of 1,2-propanediol and 2,3-butanediol. Here, we analyzed the metabolism of ethylene glycol. Our data demonstrate that ethylene glycol is dehydrated to acetaldehyde, which is then disproportionated to ethanol and acetyl coenzyme A (acetyl-CoA). The latter is further converted to acetate, and this pathway is coupled to ATP formation by substrate-level phosphorylation. Apparently, the product ethanol is in part further oxidized and the reducing equivalents are recycled by reduction of CO 2 to acetate in the Wood-Ljungdahl pathway. Biochemical data as well as the results of protein synthesis analysis are consistent with the hypothesis that the propane diol dehydratase (PduCDE) and CoA-dependent propionaldehyde dehydrogenase (PduP) proteins, encoded by the pdu gene cluster, also catalyze ethylene glycol dehydration to acetaldehyde and its CoA-dependent oxidation to acetyl-CoA. Moreover, genes encoding bacterial microcompartments as part of the pdu gene cluster are also expressed during growth on ethylene glycol, arguing for a dual function of the Pdu microcompartment system. IMPORTANCEAcetogenic bacteria are characterized by their ability to use CO 2 as a terminal electron acceptor by a specific pathway, the WoodLjungdahl pathway, enabling in most acetogens chemolithoautotrophic growth with H 2 and CO 2 . However, acetogens are very versatile and can use a wide variety of different substrates for growth. Here we report on the elucidation of the pathway for utilization of ethylene glycol by the model acetogen Acetobacterium woodii. This diol is degraded by dehydration to acetaldehyde followed by a disproportionation to acetate and ethanol. We present evidence that this pathway is catalyzed by the same enzyme system recently described for the utilization of 1,2-propanediol. The enzymes for ethylene glycol utilization seem to be encapsulated in protein compartments, known as bacterial microcompartments. In the absence of other energetically more favorable electron acceptors, CO 2 is the only electron acceptor used for energy conservation in anoxic ecosystems. Only two groups of organisms are able to utilize CO 2 as a terminal electron acceptor, namely, methanogenic archaea and acetogenic bacteria. Acetogenic bacteria are a specialized group of strictly anaerobic bacteria that, in most cases, can use molecular hydrogen as an electron donor for CO 2 reduction and can thus grow autotrophically with H 2 and CO 2 . Therefore, they reduce two molecules of CO 2 to acetyl coenzyme A (acetyl-CoA) via the reductive acetyl-CoA pathway (the WoodLjungdahl pathway [WLP]) (1-3). This pathway of CO 2 fixation also provides energy for growth by a chemiosmotic mechanism: an electrochemical ion gradient is established in the course of CO 2 reduction (4-6) and can be used by an ATP synthase to drive the phosphory...

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The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

Cited by 141 publications

References 33 publications

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

Ethylene Glycol Metabolism in the Acetogen Acetobacterium woodii

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