Evidence of reversed electron transport in syntrophic butyrate or benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii

Abstract

Evidence of reversed electron transport in syntrophic butyrateor benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii Received: 2 March 1994 / Accepted: 20 April 1994 Abstract Syntrophomonas wolfei and Syntrophus buswellii were grown with butyrate or benzoate in a defined binary coculture with Methanospirillum hungatei. Both strains also grew independent of the partner bacteria with crotonate as substrate. Localization of enzymes involved in butyrate oxidation by S. wolfei revealed that ATP synthase, hydrogenase, and butyryl-CoA dehydrogenase were at least partially membrane-associated whereas 3-hydroxybutyryl-CoA dehydrogenase and crotonase were entirely cytoplasmic. Inhibition experiments with copper chloride indicated that hydrogenase faced the outer surface of the cytoplasmic membrane. Suspensions of butyrate-or benzoate-grown cells of either strain accumulated hydrogen during oxidation of butyrate or benzoate to a low concentration that was thermodynamically in equilibrium with calculated reaction energetics. The protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and the proton-translocating ATPase inhibitor N,N'dicyclohexylcarbodiimide (DCCD) both specifically inhibited hydrogen formation from butyrate or benzoate at low concentrations, whereas hydrogen formation from crotonate was not affected. A menaquinone was extracted from cells of S. wolfei and S. buswellii grown syntrophically in a binary methanogenic culture. The results indicate that a proton-potential-driven process is involved in hydrogen release from butyrate or benzoate oxidation. Degradation of both compounds becomes feasible only at low hydrogen partial pressure (10-4-10 -5 atm; Schink 1992), which can be maintained by hydrogen-oxidizing anaerobes such as methanogenic bacteria (Zehnder 1978;Dolfing 1988). The pathways of butyrate and benzoate degradation in these bacteria have been at least tentatively elucidated (see Schink 1992). The energetically most difficult electron transfer steps are the oxidations of the saturated acid esters butyryl-CoA or glutaryl-CoA to the respective unsaturated compounds. The electrons released in the butyryl-CoA dehydrogenase reaction (E 0, = -125 mV; Gustafson et al. 1986) and glutaryl-CoA dehydrogenase, for which a similar redox potential is assumed, are used to reduce protons to molecular hydrogen (E 0" = -414 mV). Even at 10 .4 atm hydrogen, the redox potential of the couple 2 H+/H2 is still -295 mV and is much lower than that of the electron donor. It has been hypothesized, therefore, that part of the ATP gained by substrate level phosphorylation during butyrate oxidation has to be spent in a reversed electron transport step to shift these electrons to a lower redox potential (Thauer and Morris 1984). Similar problems arise with oxidation of saturated intermediates in syntrophic benzoate degradation, and involvement of reversed electron transport in this oxidation also has been postulated (Schink 1992). However, experimental evidence of such energy-driven processes in syntrophic ...

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

“…An early hypothesis how such a reversed electron transport could operate in S. wolfei assumed the involvement of a menaquinone (29,36). This concept seemed plausible also for glycolate-oxidizing syntrophs (8,29).…”

“…Axenic cultures of S. wolfei were grown with 20 mM sodium crotonate (36). In addition, the medium contained resazurine (0.4 mg liter…”

“…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.…”

Energetics and biochemistry of fermentative benzoate degradation by Syntrophus gentianae

“…Further energy has to be invested in reversed electron transport steps to allow release of electrons from oxidation reactions of comparably high redox potential [e.g., the pimelyl-CoA dehydrogenase reaction (compound 6 to compound 7) and the glutaryl-CoA dehydrogenase reaction (compound 10 to compound 11)], to be released as molecular hydrogen at hydrogen pressures in the range of 10 Pa, as observed in such cultures (Schöcke and Schink 1997;Schink 1997). In analogy to the butyryl-CoA dehydrogenase reaction in syntrophic butyrate oxidation, these reversed electron transport reactions can be assumed to consume two-thirds of an ATP unit each (Wallrabenstein and Schink 1994). With this, the overall energy balance of benzoate degradation by S. gentianae comes to a net energy gain of one-third of an ATP unit per reaction run provided that the dearomatizing benzoyl-CoA reduction does not require any energy input, as hypothesized above.…”

Initial steps in the fermentation of 3-hydroxybenzoate by Sporotomaculum hydroxybenzoicum

“…Acetate and butyrate were measured according to Galushko et al (1999). Hydrogen concentrations were determined by gas chromatography (Wallrabenstein and Schink 1994). Corrinoids were determined by HPLC analysis in cell-free extracts containing 144-172 mg protein according to Kengen et al (1988).…”