Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri

Abstract

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)-and ferredoxin-dependent formation of H 2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E 0 ‫؍‬ ؊410 mV) with NADH (E 0 ‫؍‬ ؊320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E 0 ‫؍‬ ؊10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH ؉ 1 oxidized ferredoxin ؉ 1 crotonyl-CoA ‫؍‬ 2 NAD ؉ ؉ 1 ferredoxin reduced by two electrons ؉ 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.Clostridium kluyveri is a strictly anaerobic gram-positive endospore-forming bacterium (2). Among the clostridia this organism is unique in fermenting ethanol and acetate to butyrate, caproate, and H 2 (38, 49) and in deriving a large portion (30%) of its cell carbon from CO 2 (52). Both its energy metabolism and its pathways of biosynthesis have therefore been the subject of many investigations (26, 39a). In particular, understanding the energy metabolism of C. kluyveri remains a challenge for microbiologists, and one of the pertinent questions is how this organism generates H 2 (36, 39a, 48).

A Bacterial Electron-bifurcating Hydrogenase

“…Electron bifurcation was first demonstrated for the Etf/butyryl-CoA dehydrogenase complex of Clostridium kluyveri (20) in which the energetic downhill flux two-times one electron from NADH ϩ H ϩ to crotonyl-CoA drives the endergonic uphill transport of two-times one electron from NADH ϩ H ϩ to ferredoxin. The reduced ferredoxin is then used to produce hydrogen with a ferredoxin-dependent hydrogenase or to fuel the Rnf complex (21).…”

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

“…This process was originally described for an electron transferring flavoprotein containing two FAD cofactors. It coupled the exergonic electron transfer from NADH to crotonyl-CoA to the endergonic one from NADH to ferredoxin (26,27). The flavin cofactors were considered essential for this bifurcation of electrons.…”

Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla

“…The last step in E. rectale's butyrate production pathway is catalyzed by the butyrylCoA dehydrogenase/electron transfer f lavoprotein (Bcd/Etf) complex (EUBREC 0735-0737; EC 1.3.99.2), and offers a recently discovered additional pathway for energy conservation, via a bifurcation of electrons from NADH to crotonylCoA and ferredoxin (20). Reduced ferredoxin, in turn, can be reoxidized via hydrogenases, or via the membranebound oxidoreductase, Rnf, which generates sodium-motive force (Fig.…”