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

Abstract

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 H 2 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. Butyrate is fermented to methane and CO 2 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 butyrateoxidizing bacteria such as Syntrophomonas wolfei gain energy from the latter reaction in a range of approximately Ϫ20 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 ␤-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 ϩ 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 ferredoxincoaccepting 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 sho...

Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

“…A link between a low [NAD ϩ ]/[NADH] ratio and the stimulation of ␤-oxidation was observed previously (8,9). Furthermore, participation of an NADH:acceptor oxidoreductase as a physiological partner of a butyryl-CoA dehydrogenase was recently proposed in Syntrophomonas wolfei, although the enzyme was not identified (54).…”

Biogas process parameters—energetics and kinetics of secondary fermentations in methanogenic biomass degradation

“…Since the redox potential of the H + /H 2 pair and the CO 2 /formate pair at pH 7.0 are nearly identical (E 0 ' = −414 vs. −430 mV) and most methanogenic partners can use both hydrogen and formate as electron donors, it has remained a matter of discussion whether actually hydrogen or formate is the electron carrier between secondary fermenters and their methanogenic partners (Bryant et al 1967;McInerney et al 1979). Recent evidence obtained in biochemical, genomic, and proteomic studies of secondary fermenters indicates that formate may be at least as important as hydrogen in interspecies electron transfer (Müller et al 2009;Müller et al 2010;Schmidt et al 2013;Worm et al 2011). We therefore included measurement of formate pool sizes in the present study.…”

“…In only few of these studies, hydrogen partial pressures could be measured with reliable methods (e.g., (Pauss et al 1990)), and measurement of formate at micromolar concentrations was not possible at all so far. However, recent studies on the biochemistry of syntrophic oxidations (Müller et al 2009;Müller et al 2010;Schmidt et al 2013;Worm et al 2011) have shown that formate may be at least as important as hydrogen as an electron carrier between syntrophic partners. For a more exact assessment of the energetic situation of the various trophic groups within the biogas-forming community, we measured in the present study pool sizes of fatty acids including formate and of hydrogen and carbon monoxide at high sensitivity in digesting sludge from four different digesters operating at different rates with different feeds and efficiencies.…”

Effect of illumination on the hydrogen-production capability of anaerobic activated sludge

“…The volume of biogas was measured by gas measurement collector, while the biogas content, volatile fatty acids (VFAs) and ethanol were determined by gas chromatography [19]. The dehydrogenase activity of the activated sludge was detected using iodonitrotetrazolium chloride (INT) [20,21].…”