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 H2 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 (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -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.

FIG. 1.

SDS-PAGE of the butyryl-CoA dehydrogenase/Etf complex at different purification stages. Each lane contained 15 μg of protein. Lane 1, cell extract (3,500 U; specific activity, 1.6 U/mg); lane 2, 150,000-×-g supernatant (1,600 U; specific activity, 1 U/mg) (40% of the activity was lost in the pellet [see Materials and Methods]); lane 3, DEAE-Sepharose (1,600 U; specific activity, 3.8 U/mg); lane 4, 50% (NH₄)₂SO₄ pellet (500 U; specific activity, 2 U/mg) (despite the decrease in specific activity, this precipitation step was employed since it was the only step found to remove an abundant 60-kDa chaperone [see Materials and Methods]); lane 5, Superdex 200 (450 U; specific activity, 7 U/mg); lane 6, molecular mass standards (PageRuler; Fermentas). The specific activities were determined at 25°C by the NADH oxidation assay.

FIG. 2.

H₂ formation from NADH catalyzed by purified butyryl-CoA dehydrogenase/Etf complex from C. kluyveri in the presence of hydrogenase, ferredoxin, and crotonyl-CoA. (A) Amount of H₂ formed as a function of the amount of crotonyl-CoA added in the presence of excess amounts of NADH. (B) Amount of H₂ formed as a function of the amount of NADH added in the presence of excess amounts of crotonyl-CoA. Each 0.5-ml assay mixture at 37°C contained 100 mM Tris-HCl (pH 7.5), 20 mM 2-mercaptoethanol, NADH at a concentration of 2.5 mM (A) or at the concentration indicated (B), crotonyl-CoA at a concentration of 5 mM (B) or at the concentration indicated (A), 10 μM FAD, 20 μM ferredoxin, 0.4 U hydrogenase from C. pasteurianum, and 0.2 mg purified complex. The reaction was started by addition of crotonyl-CoA. For panel A the NADH was regenerated by galactose (20 mM) and galactose dehydrogenase (0.5 U).

FIG. 3.

Proposed mechanism of endergonic ferredoxin reduction with NADH coupled to exergonic crotonyl-CoA reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex (Bcd/EtfAB complex) from C. kluyveri.