Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by Syntrophomonas wolfei

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H₂ generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.Butyrate is fermented to methane and CO₂ by syntrophic communities in which a methanogenic partner organism maintains a low hydrogen partial pressure to allow the oxidation of butyrate to acetate (19, 20, 29). Only under such conditions can butyrate-oxidizing bacteria such as Syntrophomonas wolfei gain energy from the latter reaction in a range of approximately −20 kJ per mol of butyrate, which is just sufficient to support microbial growth (29). It was postulated that S. wolfei has to invest some of the ATP that is formed in the acetate kinase reaction during the β-oxidation of butyrate into an ATP-driven reversed electron transport in order to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH (34).Experimental evidence for the involvement of a proton gradient and of ATPase activity in this process was obtained with intact cell suspensions (36), and it was hypothesized that menaquinone-7 could play an essential role in this reaction (36). This would imply that membrane-bound enzymes similar to complex I of the aerobic respiratory chain, i.e., NADH dehydrogenase (NDH), operate in reverse to reduce NAD⁺ with butyrate electrons.Another option for a reversed electron transport during butyrate oxidation and hydrogen formation in S. wolfei could be a reversal of the so-called Buckel-Thauer reaction. In this reaction that was described for ethanol-acetate fermentation by Clostridium kluyveri, electrons from NADH are disproportionated to reduce both crotonyl-CoA and ferredoxin simultaneously. The reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron-transfer flavoprotein (Bcd/EtfAB) complex (13, 18). Very recently, another “bifurcating” electron pathway has been described for an NADH- and ferredoxin-coaccepting di-iron hydrogenase complex in Thermotoga maritima (30). Here, electrons from NADH and from ferredoxin are combined to produce hydrogen, and the genome sequence of S. wolfei has been shown to contain candidate genes for such a three-component hydrogenase complex (30). Nonetheless, the energetic situation of syntrophic butyrate oxidation is basically different from that of ethanol or glucose degradation: electrons arise at comparably positive redox potentials, i.e., at −125 mV/−10 mV (12, 28) and −250 mV, and there is no oxidation step involved that could be coupled directly with ferredoxin reduction.In the present study, we report that butyrate oxidation by S. wolfei cell suspensions can be inhibited by trifluoperazine (TPZ), an antitubercular agent that has been shown to inhibit type II NADH:menaquinone oxidoreductase NDH-2 in Mycobacterium tuberculosis (40), and that a TPZ-sensitive NADH:acceptor oxidoreductase activity can be measured in cell extracts of S. wolfei cells. This enzyme system and a butyryl-CoA dehydrogenase were enriched by anion-exchange chromatography, and the obtained proteins were identified by peptide mass fingerprinting.