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J Bacteriol. Feb 2008; 190(3): 843–850.
Published online Nov 9, 2007. doi:  10.1128/JB.01417-07
PMCID: PMC2223550

Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri[down-pointing small open triangle]

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.

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 H2 (38, 49) and in deriving a large portion (30%) of its cell carbon from CO2 (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 H2 (36, 39a, 48).

During growth of C. kluyveri on ethanol and acetate approximately 6 ethanol and 3 acetate are converted to 3 butyrate, 1 caproate, 1 H+, and 2 H2, and 1 mol of ATP is synthesized from ADP and phosphate per 2 H2 formed by substrate-level phosphorylation (49).

equation M1
(1)

A detailed analysis revealed that in the oxidative part of the fermentation, the dehydrogenation of ethanol to acetyl coenzyme A (acetyl-CoA), only pyridine nucleotide-dependent steps are involved.

equation M2
(2)

equation M3
(3)

Whereas the ethanol dehydrogenase in cell extracts of C. kluyveri is NAD+ specific (reaction 2), the aldehyde dehydrogenase (CoA acetylating) can use NAD+ and NADP+ (reaction 3), although NAD+ is used with higher catalytic efficiency (kcat/Km) (14, 15). Taking into account the fact that in C. kluyveri the intracellular concentration of NAD+ is more than 10 times higher than the intracellular concentration of NADP+ (48), it is most likely that in reaction 3 in vivo only NAD+ is reduced. Based on these findings, it was concluded that in C. kluyveri the H2 generated in the fermentation must be derived from NADH (reaction 4), despite the fact that this is an endergonic reaction (48).

equation M4
(4)

H2 formation from NADH is thermodynamically unfavorable also under physiological conditions. The NADH/NAD+ ratio in C. kluyveri has been determined to be 0.3 (48), indicating that the redox potential (E0′) of the NAD+/NADH couple is only −300 mV. The redox potential of the H+/H2 couple is −410 mV or more negative, as deduced from the finding that in cultures at pH 7.0 the H2 partial pressure builds up to 105 Pa or higher.

Cell extracts of C. kluyveri were found to catalyze reaction 4, and the activity was shown to be associated with the soluble cell fraction. H2 formation was ferredoxin (Fd) dependent and required the presence of acetyl-CoA. Formyl-CoA, monofluoroacetyl-CoA, propionyl-CoA, and butyryl-CoA could not substitute for acetyl-CoA. The findings were interpreted to indicate that C. kluyveri contains a cytoplasmic NADH:ferredoxin oxidoreductase allosterically regulated by acetyl-CoA and a ferredoxin-dependent hydrogenase catalyzing reactions 5 and 6, respectively (20-23, 50):

equation M5
(5)

equation M6
(6)

where Fdox is oxidized ferredoxin and Fdred2− is ferredoxin reduced by two electrons.

C. kluyveri, like other clostridia, contains high concentrations of a cytoplasmic 6-kDa ferredoxin (39) with two [4Fe4S] clusters (E0′ [Fdox/Fd] = −340 mV; E0′ [Fd/Fdred2−] = −410 mV) (41, 46) and of a cytoplasmic ferredoxin-dependent hydrogenase of the [FeFe]-hydrogenase type (21, 39a, 50).

To be able to observe H2 formation from NADH in cell extracts, both an NADH-regenerating system (galactose plus NAD+-specific galactose dehydrogenase) and an acetyl-CoA-regenerating system (acetyl-phosphate, CoA, and phosphotransacetylase) had to be added, since the cell extracts rapidly catalyzed the reduction of acetyl-CoA to ethanol and butyryl-CoA with NADH (21, 50). However, all attempts to purify the acetyl-CoA-dependent NADH:ferredoxin oxidoreductase failed. After the first chromatographic separation step most or all the activity was always lost even under strictly anaerobic conditions. The protein catalyzing reaction 5 thus remained unknown.

We report here that cell extracts of C. kluyveri catalyze ferredoxin reduction with NADH not only in the presence of acetyl-CoA but also in the presence of crotonyl-CoA, indicating that a reduction product formed from acetyl-CoA rather than acetyl-CoA itself is required for ferredoxin reduction with NADH. The crotonyl-CoA-dependent activity was found to be associated with the butyryl-CoA dehydrogenase/Etf complex, which was purified and characterized.

MATERIALS AND METHODS

Biochemicals.

Crotonyl-CoA (epsilon260 = 22.6 mM−1 cm−1) (44) was synthesized from crotonic anhydride and CoA (40). Butyryl-CoA, acetyl-CoA, hexanoyl-CoA, acetyl-phosphate, ferrocenium hexafluorophosphate, triphenyltetrazolium chloride (TTC), and phosphotransacetylase were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Propionyl-phosphate was synthesized from propionic anhydride (24). d-(+)-Galactose was obtained from Merck (Darmstadt, Germany). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and β-galactose dehydrogenase were obtained from Roche (Mannheim, Germany). 4-Hydroxybutyryl-CoA:acetate CoA transferase was purified from Clostridium aminobutyricum (35). Ferredoxin from Clostridium pasteurianum (epsilon390 = 30 mM−1 cm−1) (16) was either purified from cell extracts of C. pasteurianum by chromatography on Q-Sepharose, Phenyl-Sepharose, and Superdex 75 (42) or obtained from Sigma-Aldrich Chemie GmbH. Essentially identical results were obtained with both ferredoxins.

Hydrogenase from C. pasteurianum was purified from a cell extract of C. pasteurianum by employing a heat treatment step, followed by chromatography on Q-Sepharose (2.6 by 15 cm), hydroxyapatite (1.6 by 8 cm), Phenyl-Sepharose (1.6 by 10 cm), Superdex 200 (2.6 by 60 cm), and Mono Q (1 by 10 cm) columns (4). Hydrogenase activity was determined at pH 7.5 and 37°C by measuring the H2 formation from ferredoxin reduced by dithionite (51). During purification the specific activity increased from 0.6 to 325 U/mg (1 U = 1 μmol H2 formed per min). The purified hydrogenase showed only one protein band at 60 kDa after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Growth of clostridia.

C. kluyveri DSM 555 was grown at 37°C on acetate and ethanol in a 50-liter plastic fermentor (51). C. pasteurianum DSM 525 was cultivated in a 10-liter glass fermentor on a glucose-NH4+ medium (22). C. aminobutyricum DSM 2643 was grown on a modified 4-aminobutyrate-yeast extract medium (11). Cells were harvested at mid-exponential growth phase and stored at −80°C until they were used.

Cell lysate preparation.

Frozen cells of C. kluyveri (3 g, wet weight) were suspended in 6 ml of 10 mM Tris-HCl (pH 7.5) containing 10 mM 2-mercaptoethanol and were subsequently lysed by incubating the cell suspension with lysozyme (100 U/mg cells) at 37°C for 30 min. Cell debris was removed by centrifugation at 40,000 × g for 30 min and 4°C to obtain cell lysate containing 45 mg protein/ml. Where indicated, low-molecular-mass compounds (<1,000 Da) were removed from the cell lysate by gel filtration on a Sephadex G-25 column (2.6 by 10 cm; GE Healthcare, Freiburg, Germany). All steps were performed under strictly anoxic conditions.

The protein content was determined by the Bradford assay with bovine serum albumin as the standard (3).

Enzyme purification.

Frozen cells of C. kluyveri (15 g, wet weight) were suspended in 20 ml of 50 mM morpholineethanesulfonic acid (MOPS)-KOH (pH 7.5) containing 2 mM dithiothreitol (DTT) and 5 μM flavin adenine dinucleotide (FAD) and were subsequently disrupted by passing the suspension three times through a French press cell at 120 MPa and 4°C. Cell debris was removed by centrifugation at 30,000 × g for 30 min and 4°C to obtain 30 ml of cell extract containing 75 mg protein/ml with 1.6 U butyryl-CoA dehydrogenase activity (NADH oxidation assay)/mg protein. The cell extract was centrifuged at 150,000 × g for 2 h and 4°C to remove the membrane fraction. (In this step approximately 40% of the butyryl-CoA dehydrogenase activity was lost to the sediment; this was accepted since when centrifugation at 150,000 × g was used, other proteins that in later steps were difficult to separate from the butyryl-CoA dehydrogenase/Etf complex sedimented.) The supernatant (25 ml), which contained 1,600 U butyryl-CoA dehydrogenase activity (NADH oxidation assay) and 1,700 mg protein, was applied to a DEAE-Sepharose column (2.6 by 15 cm) equilibrated with 50 mM MOPS-KOH (pH 7.5) containing 2 mM DTT and 5 μM FAD. Protein was eluted with a stepwise NaCl gradient (100 ml each of 0.15, 0.3, 0.6, and 0.9 M NaCl in the same buffer) at a flow rate of 4 ml min−1. The butyryl-CoA dehydrogenase activity eluted at 0.3 M NaCl. Combined active fractions (1,600 U and 430 mg protein) were concentrated by ultrafiltration (10-kDa-cutoff ultrafiltration membranes; Millipore) to obtain 20 ml and brought to 50% ammonium sulfate, with which only part of the butyryl-CoA dehydrogenase activity was precipitated (approximately 500 U and 260 mg protein). 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. The precipitate was dissolved in 100 ml of 50 mM MOPS-KOH (pH 7.5) containing 2 mM DTT, 5 μM FAD, and 0.1 M NaCl, concentrated, and applied to a Superdex 200 gel filtration column (2.6 by 60 cm) equilibrated with the same buffer. The enzyme (450 U) eluted after 160 ml (maximum peak). The protein (66 mg) was concentrated by ultrafiltration to obtain a concentration of 20 mg protein/ml, and the concentrate was then stored at either 4 or −80°C. Using this procedure, the butyryl-CoA dehydrogenase/Etf complex was purified almost 10-fold to a specific activity of approximately 7 U/mg (NADH oxidation assay) with a 10 to 15% yield.

All chromatographic steps were performed in an anaerobic chamber (Coy, Ann Arbor, MI) filled with 95% N2-5% H2 and containing a palladium catalyst for O2 reduction with H2. All chromatographic material was obtained from GE Healthcare.

Assays of butyryl-CoA dehydrogenase/Etf complex activities.

Assays to determine the reduction of ferredoxin via H2 formation, the oxidation of NADH, the reduction of TTC (E0′ = −80 mV; n = 2) (19), and the reduction of ferrocenium (E0′ = 380 mV; n = 1) (32) were performed under strictly anoxic conditions.

(i) H2 formation assay.

Ferredoxin reduction catalyzed by the butyryl-CoA dehydrogenase/Etf complex was assayed by coupling this reaction with the hydrogenase reaction (reaction 6). Using 0.5- or 1-ml mixtures, the assays were performed at 37°C in 6.5-ml serum bottles closed with a rubber stopper and filled with N2 at a pressure of 1.2 × 105 Pa. (For the compositions of the assay mixtures, see Tables Tables11 and and2.)2.) The gas phase and the liquid phase were equilibrated by continuous shaking. After the reaction was started, 0.2-ml gas samples were withdrawn at 1-min intervals and assayed for H2 by gas chromatography. One unit was defined as the formation of 1 μmol H2 per min.

TABLE 1.
H2 formation from NADH or NADPH in cell lysates of C. kluyveri
TABLE 2.
Crotonyl-CoA-dependent NADH oxidation and ferredoxin reduction catalyzed by purified butyryl-CoA dehydrogenase/Etf complex from C. kluyveri

(ii) NADH oxidation assay.

NADH oxidation catalyzed by the butyryl-CoA dehydrogenase/Etf complex was assayed spectrophotometrically (epsilon340 = 6.2 mM−1 cm−1) at 25°C. Each 1-ml assay mixture contained 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.1 mM NADH, 0.1 mM crotonyl-CoA, 5 μM FAD, and enzyme. Where indicated, 20 μM ferredoxin, 0.4 U hydrogenase from C. pasteurianum, and/or methyl viologen was added. The reaction was started with crotonyl-CoA. One unit was defined as the oxidation of 1 μmol NADH per min.

(iii) TTC reduction assay.

TTC reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex at 25°C was assayed spectrophotometrically (epsilon546 = 9.1 mM−1 cm−1) (23). Each 1-ml assay mixture contained 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.2 mM NADH, 0.4 mM TTC, 0.1 mM crotonyl-CoA, 5 μM FAD, and enzyme. Where indicated, 20 μM ferredoxin and/or 0.4 U hydrogenase from C. pasteurianum was added. The reaction was started with crotonyl-CoA. One unit was defined as the reduction of 1 μmol TTC per min.

(iv) Ferrocenium reduction assay.

Ferrocenium reduction with butyryl-CoA catalyzed by the butyryl-CoA dehydrogenase/Etf complex was assayed spectrophotometrically (epsilon300 = 4.3 mM−1 cm−1) (25). Each 1-ml assay mixture contained 50 mM Tris-HCl (pH 7.5), 0.4 mM ferrocenium, 0.05 mM butyryl-CoA (or hexanoyl-CoA), 5 μM FAD, and enzyme. The reaction was started with the enzyme. One unit was defined as the reduction of 2 μmol ferrocenium per min.

Butyryl-CoA determination.

Samples of the assay mixtures described above were withdrawn at 1-min intervals and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry. At time zero a peak at 836 Da (mass of crotonyl-CoA) was found, which was converted to a peak at 838 Da (mass of butyryl-CoA) upon incubation.

H2 determination.

H2 was measured using a gas chromatograph equipped with a thermal conductivity detector (Carlo Erba GC series 6000). Gases were separated using a molecular sieve (0.5-nm) column (3 mm by 1 m). The injector and detector temperature was 105°C, and the oven temperature was 110°C. The carrier gas was N2 at a flow rate of 30 ml min−1. The peak heights were correlated to a standard curve.

RESULTS

Herrmann et al. (12) put forward the hypothesis that in clostridial fermentations the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) (9) by NADH (E0′ = −320 mV) could be coupled with the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH. These authors proposed that the acetyl-CoA-dependent reduction of ferredoxin with NADH (reaction 5) catalyzed by cell lysates of C. kluyveri and C. pasteurianum (21, 22, 50) could be explained by the fact that cell extracts of these organisms catalyze the reduction of acetyl-CoA to butyryl-CoA. Therefore, we first tested whether cell extracts of C. kluyveri catalyze the formation of H2 from NADH in the presence of crotonyl-CoA. After this was verified, we showed using purified butyryl-CoA dehydrogenase/Etf complex from C. kluyveri that indeed this complex catalyzes crotonyl-CoA-dependent reduction of ferredoxin with NADH.

Crotonyl-CoA-dependent H2 formation from NADH in cell lysates.

It was confirmed that cell lysates of C. kluyveri did not catalyze the formation of H2 from NADH even when the H2 partial pressure was only a few pascals (H+/H2; E0′ > −300 mV) and when the NADH/NAD+ ratio was kept above 10 (E0′ < −350 mV) via an NADH-regenerating system (galactose and galactose dehydrogenase) (Table (Table1).1). However, hydrogen was formed from NADH when acetyl-phosphate and CoA or propionyl-phosphate plus acetate and CoA were added, from which acetyl-CoA was generated either via reaction 7 or via reactions 8 and 9.

equation M7
(7)

equation M8
(8)

equation M9
(9)

Cell lysates of C. kluyveri contain phosphotransacetylase catalyzing reactions 7 and 8 and a CoA transferase catalyzing reaction 9. The finding that for H2 formation from NADH both acetyl-phosphate and CoA or propionyl-phosphate, CoA, and acetate are required (Table (Table1)1) thus confirms the previous reports that H2 formation from NADH in cell lysates of C. kluyveri is acetyl-CoA dependent (21, 50).

To test the idea that H2 formation from NADH could be coupled to crotonyl-CoA reduction to butyryl-CoA, we exploited the finding that CoA transferase from C. kluyveri not only catalyzes reaction 9 but also the transfer of CoA from propionyl-CoA to vinylacetate (reaction 10) (but not to crotonate) (43) and the finding that cell extracts rapidly catalyze the isomerization of vinylacetyl-CoA to crotonyl-CoA (reaction 11) using an enzyme that has not been characterized yet. The isomerase activity can be separated from crotonase (3-hydroxybutyryl-CoA dehydratase) activity present in cell extracts by anion-exchange chromatography (37). As shown below, purified butyryl-CoA dehydrogenase/Etf complex can catalyze the isomerization of vinylacetyl-CoA to crotonyl-CoA (reaction 11).

equation M10
(10)

equation M11
(11)

When propionyl-phosphate, CoA, and vinylacetate were added to cell lysates, H2 was formed from NADH. H2 was also formed when instead of the crotonyl-CoA-regenerating system (reactions 8, 10, and 11) chemically synthesized crotonyl-CoA was added (Table (Table11).

Cell lysates of C. kluyveri did not catalyze the formation of H2 from NADPH either in the absence or in the presence of acetyl-CoA or crotonyl-CoA (Table (Table1).1). Acetyl-CoA was reduced by NADPH to 3-hydroxybutyryl-CoA but not to butyryl-CoA, consistent with the finding that C. kluyveri contains an NAD+- and NADP+-dependent 3-hydroxybutyryl-CoA dehydrogenase (27) but only an NAD+-specific butyryl-CoA dehydrogenase (see below).

It has been reported that cell lysates of C. kluyveri catalyze the ferredoxin-dependent formation of H2 from NADPH when NAD+ is present (51). This finding was confirmed (Table (Table11).

Purification of the butyryl-CoA dehydrogenase/Etf complex.

High concentrations of a complex composed of three subunits having apparent molecular masses of 41, 36, and 28 kDa were present in cell extracts of C. kluyveri, as revealed by SDS-PAGE (Fig. (Fig.1,1, lane 1). This complex was purified only sevenfold to apparently homogeneity (Fig. (Fig.1,1, lane 5). During purification the specific activity increased from 1 U/mg (140,000-×-g supernatant) to 7 U/mg (NADH oxidation assay). Purification with a 10 to 15% activity yield was achieved by anion-exchange chromatography on DEAE-Sepharose, precipitation by ammonium sulfate (50%), and gel filtration with Superdex 200 (Fig. (Fig.11).

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 ...

Purification of the butyryl-CoA dehydrogenase/Etf complex had to be performed in the presence of FAD and under strictly anoxic conditions. In the absence of added FAD the activity was rapidly lost. Flavin mononucleotide (FMN) could not substitute for FAD in stabilizing the enzyme activity. However, even in the presence of FAD and under anoxic conditions the butyryl-CoA dehydrogenase/Etf complex lost 50% of its activity within 2 to 3 days at 4°C.

During purification the butyryl-CoA dehydrogenase/Etf complex partially dissociated into its components, as shown by the finding that fractions containing mainly the 41-kDa subunit and other fractions containing mainly the 36- or 28-kDa subunit were obtained. In the NADH oxidation assay only the complete complex appeared to be active.

Molecular properties.

The molecular mass of the purified complex was determined by gel filtration with Superdex 200 to be 320 kDa, which best fits a complex consisting of four 41-kDa subunits, two 36-kDa subunits, and two 28-kDa subunits (total molecular mass, 292 kDa). Photometric scans of SDS-PAGE gels after they were stained with Coomassie brilliant blue yielded a stoichiometry of 1.8:1:1 (not shown). A stoichiometry of 2:1:1 has been reported for the propionyl-CoA dehydrogenase/Etf complex from Clostridium propionicum (13).

Partial amino acid sequencing revealed that the 41-kDa subunit is encoded by the bcd (CKL_0455) gene, the 36-kDa subunit is encoded by the etfA (CKL_0457) gene, and the 28-kDa subunit is encoded by the etfB (CKL_0456) gene. In this respect it is noteworthy that the genome of C. kluyveri harbors a second set of bcd (CKL_3515 and CKL_0633), etfA, and etfB (CLK_3516 and CLK_3517) genes (39a) which have been implicated to have a function in caproate formation from ethanol and butyrate. However, based on partial amino acid sequencing of the proteins present in the cell extracts (Fig. (Fig.1,1, lane 1), we obtained no evidence that this second gene cluster is expressed to a measurable extent.

Catalytic properties.

The purified complex catalyzed the ferredoxin-dependent oxidation of NADH with crotonyl-CoA (14 U/mg) at 37°C (Table (Table2),2), the crotonyl-CoA-dependent reduction of TTC with NADH (12 U/mg) at 25°C (Table (Table2),2), the crotonyl-CoA-dependent oxidation of reduced methyl viologen (19 U/mg; 1 U = 2 μmol reduced methyl viologen oxidized per min) at 25°C, the crotonyl-CoA-dependent reduction of ferredoxin with NADH (5 U/mg) at 37°C (Table (Table2),2), and the isomerization of vinylacetyl-CoA to crotonyl-CoA (in the absence of NADH) (0.5 U/mg) at 25°C. It also catalyzed the oxidation of butyryl-CoA and caproyl-CoA (hexanoyl-CoA) with ferrocenium (an artificial one-electron acceptor) at almost the same specific activity (4 U/mg) at 25°C, indicating that the complex is involved in both butyrate formation and caproate formation.

Both the absolute and relative values of the specific activities varied from preparation to preparation. This was not only because the enzyme complex was very labile but also because there were inherent difficulties with the activity measurements. Some of the reactions started with a considerable lag period, and the rates of the reactions were not always proportional to the amounts of enzyme added.

Ferredoxin-dependent NADH oxidation with crotonyl-CoA.

In the absence of ferredoxin and hydrogenase, the purified butyryl-CoA dehydrogenase/Etf complex catalyzed the oxidation of NADH with crotonyl-CoA with a specific activity of only 0.3 to 0.4 U/mg protein. The specific activity increased approximately 20-fold to 7 U/mg when ferredoxin and hydrogenase were added to the assay mixture, indicating that ferredoxin was required as an electron acceptor for full activity. In the absence of crotonyl-CoA NADH was not oxidized (Table (Table2).2). Vinylacetyl-CoA could substitute for crotonyl-CoA, indicating that butyryl-CoA dehydrogenase catalyzes the isomerization of vinylacetyl-CoA to crotonyl-CoA. This was substantiated by the finding that the enzyme complex catalyzed the formation of crotonyl-CoA from vinylacetyl-CoA in the absence of NADH and ferredoxin (results not shown).

Crotonyl-CoA-dependent reduction of TTC with NADH.

The purified enzyme complex also catalyzed crotonyl-CoA-dependent oxidation of NADH with TTC (a two-electron acceptor). Reduction of TTC was not dependent on the presence of ferredoxin (Table (Table22).

Crotonyl-CoA-dependent reduction of ferredoxin with NADH.

The butyryl-CoA dehydrogenase/Etf complex catalyzed the reduction of ferredoxin with NADH (measured by H2 formation) only in the presence of crotonyl-CoA (5 U/mg) or vinylacetyl-CoA (4 U/mg). In the absence of these compounds the specific activity was essentially zero (Table (Table22).

Addition of methyl viologen (1 mM) partially inhibited NADH oxidation (Table (Table2)2) but completely quenched H2 formation (Table (Table2),2), indicating that the one-electron-accepting herbicide (E0′ = −420 mV) uncoupled crotonyl-CoA reduction and ferredoxin reduction.

All the assay mixtures contained FAD. When the flavin was omitted, the measured rates were lower (Table (Table2).2). The FAD dependence was more pronounced when older and less active enzyme preparations were tested. FMN could not substitute for FAD for stimulating the activity.

The butyryl-CoA dehydrogenase did not catalyze the oxidation of NADPH or the reduction of ferredoxin with NADPH under any of the conditions tested with NADH (not shown).

Stoichiometry of the reaction.

To obtain insight into the stoichiometry of the butyryl-CoA dehydrogenase/Etf complex-catalyzed reaction, the concentrations of crotonyl-CoA (Fig. (Fig.2A)2A) and NADH (Fig. (Fig.2B)2B) were varied using constant concentrations of ferredoxin and hydrogenase. When the NADH concentration was varied (Fig. (Fig.2B),2B), the assay mixtures contained excess crotonyl-CoA, and when the crotonyl-CoA concentration was varied (Fig. (Fig.2A),2A), they contained excess NADH. The reaction was started by addition of crotonyl-CoA and was allowed to proceed to completion. From the slope in Fig. Fig.2B2B it was calculated that in the presence of excess crotonyl-CoA 2.2 mol NADH was required for the formation of 1 mol H2, and from the slope in Fig. Fig.2A2A it was calculated that in the presence of excess NADH 1.4 mol crotonyl-CoA was required for the formation of 1 mol H2. The observed stoichiometry (2.2 NADH + 1.4 crotonyl-CoA = 2.2 NAD+ + 1.4 butyryl-CoA + 1 H2) was independent of whether NADH or an NADH-regenerating system was employed in the assays. Since the purified butyryl-CoA dehydrogenase/Etf complex catalyzed the reduction of crotonyl-CoA with NADH at low rates also in the absence of ferredoxin (Table (Table2),2), the results indicate that under fully coupled conditions probably 1 mol ferredoxin is reduced per 2 mol NADH and 1 mol crotonyl-CoA (reaction 12), considering that the hydrogenase catalyzes reaction 6. The finding that the butyryl-CoA dehydrogenase/Etf complex catalyzed the oxidation of NADH in the presence of ferredoxin and hydrogenase at a rate that was nearly three times the rate of H2 formation (14 U/mg versus 5 U/mg at 37°C) (Table (Table2)2) is also consistent with this stoichiometry, which predicts that under fully coupled conditions the specific activity of NADH oxidation should be twice that of H2 formation.

equation M12
(12)

The apparent Km values for all three substrates were found to be approximately 10 μM (NADH oxidation assay) (not shown).

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 ...

Specific activity.

In our best preparations, the specific activity of the purified butyryl-CoA dehydrogenase/Etf complex in the presence of ferredoxin and hydrogenase was only 5 U/mg protein at 37°C (H2 formation assay) (Table (Table2),2), probably reflecting the enzyme's complex function (namely, to couple the endergonic reduction of ferredoxin with NADH to the exergonic reduction of crotonyl-CoA with NADH). The relative low specific activity is compensated for in the cells by a very high enzyme concentration. Over 10% of the soluble proteins in C. kluyveri were found to be the butyryl-CoA dehydrogenase/Etf complex (Fig. (Fig.1).1). Cell suspensions of C. kluyveri catalyze the formation of butyrate and caproate from ethanol and acetate at a specific rate of 0.3 U/mg protein (37°C) (31). The specific activity and intracellular concentration of the butyryl-CoA dehydrogenase/Etf complex are high enough to account for this specific rate.

DISCUSSION

How does the butyryl-CoA dehydrogenase/Etf complex couple the reduction of ferredoxin with NADH to the reduction of crotonyl-CoA? Most probably, FAD is involved since each of the three different subunits of the complex contains an FAD molecule and one of the FAD molecules in Etf is only loosely bound (6, 13, 34). It is known that in some flavoproteins the flavin nucleotide can be reduced by one electron to a stable semiquinone flavin radical (FADH or FMNH), which can then be reduced by a second electron to the fully reduced flavin nucleotide (FADH2 or FMNH2). For example, in flavodoxin from Acidaminococcus fermentans the first FMN reduction step has a redox potential (E1) of about −60 mV and the redox potential of the second step (E2) is about −430 mV (10, 33). Similar properties have been described for the electron-transferring proteins (54). The midpoint potentials for free flavins in water have been determined to be −172 mV (E1) and −238 mV (E2) (7). Thus, the redox potentials of flavins are modulated significantly by the proteins with which they are associated. Therefore, all one has to assume is that in the butyryl-CoA dehydrogenase/Etf complex the electron flow from NADH via FADH2 is directed so that the electron of FADH2 with the more negative redox potential is used to reduce ferredoxin (E0′ = −410 mV) (41, 46) and the electron with the more positive potential is used to reduce crotonyl-CoA (E0′ = −10 mV) (9). This is illustrated in Fig. Fig.33.

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.

The driving force for the proposed mechanism is the oxidation of the flavin semiquinone radical by crotonyl-CoA. Oxidation-driven reduction reactions are not without precedent. In the bc1 complex of the respiratory chain the oxidation of reduced ubiquinone by cytochrome c drives the reduction of cytochrome b (5, 17, 30), in ribonucleotide reductases the oxidation of a cysteine thiol to a thiyl radical drives the reduction of ribonucleotides to deoxyribonucleotides (28, 29, 45), and in methyl-CoM reductase the oxidation of the thiol group of CoM or CoB to the thiyl radical drives the rereduction of the nickel tetrapyrrole from the Ni(II) state to the Ni(I) state (18, 47). In all these cases an endergonic one-electron reduction step is coupled to an exergonic one-electron oxidation step involving radical intermediates which have to be stabilized in the enzyme. However, only in the case of the butyryl-CoA dehydrogenase/Etf complex is the reduced product released from the enzyme and thus the free energy of the oxidation reaction is conserved in an isolatable compound, namely, reduced ferredoxin. In the case of the bc1 complex the energy is conserved in an electrochemical proton potential, and in the case of ribonucleotide reductase and methyl-CoM reductase it is used to lower the activation energy of the two enzyme-catalyzed reactions.

Just recently, a novel enzyme that catalyzes the reductive carboxylation of crotonyl-CoA to ethyl-malonyl-CoA (8) (reaction 13) was found in Rhodobacter sphaeroides.

equation M13
(13)

This enzyme was named crotonyl-CoA carboxylase/reductase. It does not show sequence similarity to butyryl-CoA dehydrogenases (1, 53). In the absence of CO2 the enzyme catalyzes the reduction of crotonyl-CoA with NADPH to butyryl-CoA. Thus, the crotonyl-CoA reductase/carboxylase also catalyzes two energetically coupled reactions, an exergonic reduction reaction and an endergonic carboxylation reaction. Although the mechanism of coupling is quite different from that described above for the butyryl-CoA dehydrogenase/Etf complex from C. kluyveri, it is another example of how the free energy change associated with the reduction of crotonyl-CoA with NAD(P)H can be exploited to drive an endergonic reaction, for which ATP or an ATP equivalent would otherwise be required.

Acknowledgments

This work was supported by the Max Planck Society, the Deutsch Forschungsgemeinschaft, and the Fonds der Chemischen Industrie.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 November 2007.

Dedicated to Karl Decker, Emeritus Professor of Biochemistry, University Freiburg, Freiburg, Germany.

REFERENCES

1. Alber, B. E., R. Spanheimer, C. Ebenau-Jehle, and G. Fuchs. 2006. Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides. Mol. Microbiol. 61297-309. [PubMed]
2. Barker, H. A., and S. M. Taha. 1942. Clostridium kluyverii, an organism concerned in the formation of caproic acid from ethyl alcohol. J. Bacteriol. 43347-363. [PMC free article] [PubMed]
3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72248-254. [PubMed]
4. Chen, J. S., and L. E. Mortenson. 1974. Purification and properties of hydrogenase from Clostridium pasteurianum W5. Biochim. Biophys. Acta 371283-298. [PubMed]
5. Darrouzet, E., J. W. Cooley, and F. Daldal. 2004. The cytochrome bc1 complex and its homologue the b6 f complex: similarities and differences. Photosynth. Res. 7925-44. [PubMed]
6. Djordjevic, S., C. P. Pace, M. T. Stankovich, and J. J. Kim. 1995. Three-dimensional structure of butyryl-CoA dehydrogenase from Megasphaera elsdenii. Biochemistry 342163-2171. [PubMed]
7. Draper, R. D., and L. L. Ingraham. 1968. A potentiometric study of the flavin semiquinone equilibrium. Arch. Biochem. Biophys. 125802-808. [PubMed]
8. Erb, T. J., I. A. Berg, V. Brecht, M. Müller, G. Fuchs, and B. E. Alber. 2007. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc. Natl. Acad. Sci. USA 10410631-10636. [PMC free article] [PubMed]
9. Fink, C. W., M. T. Stankovich, and S. Soltysik. 1986. Oxidation-reduction potentials of butyryl-CoA dehydrogenase. Biochemistry 256637-6643. [PubMed]
10. Hans, M., E. Bill, I. Cirpus, A. J. Pierik, M. Hetzel, D. Alber, and W. Buckel. 2002. Adenosine triphosphate-induced electron transfer in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Biochemistry 415873-5882. [PubMed]
11. Hardman, J. K. 1962. γ-Hydroxybutyrate dehydrogenase from Clostridium aminobutyricum. Methods Enzymol. 5778-783.
12. Herrmann, G., E. Jayamani, G. Mai, and W. Buckel. 2008. Energy conservation via electron-transferring flavoprotein in anaerobic bacteria. J. Bacteriol. 190784-791. [PMC free article] [PubMed]
13. Hetzel, M., M. Brock, T. Selmer, A. J. Pierik, B. T. Golding, and W. Buckel. 2003. Acryloyl-CoA reductase from Clostridium propionicum: an enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein. Eur. J. Biochem. 270902-910. [PubMed]
14. Hillmer, P., and G. Gottschalk. 1972. Particulate nature of enzymes involved in the fermentation of ethanol and acetate by Clostridium kluyveri. FEBS Lett. 21351-354. [PubMed]
15. Hillmer, P., and G. Gottschalk. 1974. Solubilization and partial characterization of particulate dehydrogenases from Clostridium kluyveri. Biochim. Biophys. Acta 33412-23.
16. Hong, J. S., and J. C. Rabinowitz. 1970. Molar extinction coefficient and iron and sulfide content of clostridial ferredoxin. J. Biol. Chem. 2454982-4987. [PubMed]
17. Hunte, C., H. Palsdottir, and B. L. Trumpower. 2003. Protonmotive pathways and mechanisms in the cytochrome bc1 complex. FEBS Lett. 54539-46. [PubMed]
18. Jaun, B., and R. K. Thauer. 2007. Methyl-coenzyme M reductase and its nickel corphin coenzyme F430 in methanogenic archaea, p. 323-356. In A. Sigel, H. Sigel, and R. K. O. Sigel (ed.), Nickel and its surprising impact in nature, vol. 2. Wiley & Sons Ltd., Chichester, United Kingdom.
19. Jerchel, D., and W. Mohle. 1944. The determination of reduction potential of tetrazolium compounds. Ber. Dtsch. Chem. Ges. B 77591-601.
20. Jungermann, K., H. Kirchniawy, N. Katz, and R. K. Thauer. 1974. NADH, a physiological electron donor in clostridial nitrogen fixation. FEBS Lett. 43203-206. [PubMed]
21. Jungermann, K., E. Rupprecht, C. Ohrloff, R. Thauer, and K. Decker. 1971. Regulation of the reduced nicotinamide adenine dinucleotide-ferredoxin reductase system in Clostridium kluyveri. J. Biol. Chem. 246960-963. [PubMed]
22. Jungermann, K., R. K. Thauer, G. Leimenstoll, and K. Decker. 1973. Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic clostridia. Biochim. Biophys. Acta Bioenerg. 305268-280. [PubMed]
23. Katz, N. 1972. Die Reduktion von Triphenyltetrazoliumchlorid. Eine spezifische Indikatorreaktion zur optischen Messung der NADH-Ferredoxin Oxidoreduktase und weiterer Ferredoxin-Reduktasen. (The reduction of triphenyl-tetrazolium chloride. A specific indicator reaction for optical assay of NADH-ferredoxin oxidoreductase and other ferredoxin reductases.) Diploma thesis. Albert-Ludwigs University, Freiburg, Germany.
24. Kornberg, A., S. R. Kornberg, and E. S. Simms. 1956. Metaphosphate synthesis by an enzyme from Escherichia coli. Biochim. Biophys. Acta 20215-227. [PubMed]
25. Lehman, T. C., and C. Thorpe. 1990. Alternate electron-acceptors for medium-chain acyl-CoA dehydrogenase—use of ferricenium salts. Biochemistry 2910594-10602. [PubMed]
26. Li, F., C. H. Hagemeier, H. Seedorf, G. Gottschalk, and R. K. Thauer. 2007. Re-citrate synthase from Clostridium kluyveri is phylogenetically related to homocitrate synthase and isopropylmalate synthase rather than to Si-citrate synthase. J. Bacteriol. 1894299-4304. [PMC free article] [PubMed]
27. Madan, V. K., P. Hillmer, and G. Gottschalk. 1973. Purification and properties of NADP-dependent l(+)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri. Eur. J. Biochem. 3251-56. [PubMed]
28. Mulliez, E., D. Padovani, M. Atta, C. Alcouffe, and M. Fontecave. 2001. Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center. Biochemistry 403730-3736. [PubMed]
29. Nordlund, P., and P. Reichard. 2006. Ribonucleotide reductases. Annu. Rev. Biochem. 75681-706. [PubMed]
30. Osyczka, A., C. C. Moser, and P. L. Dutton. 2005. Fixing the Q cycle. Trends Biochem. Sci. 30176-182. [PubMed]
31. Pfeiff, B. 1991. Untersuchungen zur Kopplung von H2-Bildung und Fettsäuresynthese in Clostridium kluyveri. (Investigations of the coupling of H2 formation and butyric acid/caproic acid synthesis in Clostridium kluyveri.) Diploma thesis. Philipps University, Marburg, Germany.
32. Pladziewicz, J. R., M. S. Brenner, D. A. Rodeberg, and M. D. Likar. 1985. Kinetic study of the oxidation of spinach plastocyanin by ferrocenium ion derivatives. Inorg. Chem. 241450-1453.
33. Sancho, J. 2006. Flavodoxins: sequence, folding, binding, function and beyond. Cell. Mol. Life Sci. 63855-864. [PubMed]
34. Sato, K., Y. Nishina, and K. Shiga. 2003. Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum. J. Biochem. (Tokyo) 134719-729. [PubMed]
35. Scherf, U., and W. Buckel. 1991. Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum. Appl. Environ. Microbiol. 572699-2702. [PMC free article] [PubMed]
36. Scherf, U., B. Söhling, G. Gottschalk, D. Linder, and W. Buckel. 1994. Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ32-isomerase. Arch. Microbiol. 161239-245. [PubMed]
37. Schleicher, E., and H. Simon. 1976. On the mechanism and some properties of vinylacetyl-CoA delta-isomerase of Clostridium kluyveri. Hoppe Seyler's Z. Physiol. Chem. 357535-541. [PubMed]
38. Schoberth, S., and G. Gottschalk. 1969. Considerations on the energy metabolism of Clostridium kluyveri. Arch. Mikrobiol. 65318-328. [PubMed]
39. Schönheit, P., C. Wascher, and R. K. Thauer. 1978. A rapid procedure for the purification of ferredoxin from clostridia using polyethyleneimine. FEBS Lett. 89219-222. [PubMed]
39a. Seedorf, H., W. F. Fricke, B. Veith, H. Brüggemann, H. Liesegang, A. Strittmatter, M. Miethke, W. Buckel, J. Hinderberger, F. Li, C. Hagemeier, R. K. Thauer, and G. Gottschalk. The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc. Natl. Acad. Sci. USA, in press. [PMC free article] [PubMed]
40. Simon, E. J., and D. Shemin. 1953. The preparation of S-succinyl coenzyme A. J. Am. Chem. Soc. 752520.
41. Smith, E. T., J. M. Tomich, T. Iwamoto, J. H. Richards, Y. Mao, and B. A. Feinberg. 1991. A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties. Biochemistry 3011669-11676. [PubMed]
42. Soboh, B., D. Linder, and R. Hedderich. 2004. A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis. Microbiology 1502451-2463. [PubMed]
43. Stadtman, E. R. 1953. The coenzyme A transphorase system in Clostridium kluyveri. J. Biol. Chem. 203501-512. [PubMed]
44. Stadtman, E. R. 1957. Preparation and assay of acyl coenzyme A and other thiol esters: use of hydroxylamine. Methods Enzymol. 3931-941.
45. Stubbe, J., and W. A. van Der Donk. 1998. Protein radicals in enzyme catalysis. Chem. Rev. 98705-762. [PubMed]
46. Thamer, W., I. Cirpus, M. Hans, A. J. Pierik, T. Selmer, E. Bill, D. Linder, and W. Buckel. 2003. A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Arch. Microbiol. 179197-204. [PubMed]
47. Thauer, R. K. 1998. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture. Microbiology 1442377-2406. [PubMed]
48. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Microbiol. Mol. Biol. Rev. 41100-180. [PMC free article] [PubMed]
49. Thauer, R. K., K. Jungermann, H. Henninger, J. Wenning, and K. Decker. 1968. The energy metabolism of Clostridium kluyveri. Eur. J. Biochem. 4173-180. [PubMed]
50. Thauer, R. K., K. Jungermann, E. Rupprecht, and K. Decker. 1969. Hydrogen formation from NADH in cell-free extracts of Clostridium kluyveri: acetyl coenzyme A requirement and ferredoxin dependence. FEBS Lett. 4108-112. [PubMed]
51. Thauer, R. K., E. Rupprecht, C. Ohrloff, K. Jungermann, and K. Decker. 1971. Regulation of the reduced nicotinamide adenine dinucleotide phosphate-ferredoxin reductase system in Clostridium kluyveri. J. Biol. Chem. 246954-959. [PubMed]
52. Tomlinson, N., and H. A. Barker. 1954. Carbon dioxide and acetate utilization by Clostridium kluyveri. I. Influence of nutritional conditions on utilization patterns. J. Biol. Chem. 209585-595. [PubMed]
53. Wallace, K. K., Z.-Y. Bao, H. Dai, R. Digate, G. Schuler, M. K. Speedie, and K. A. Reynolds. 1995. Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. Eur. J. Biochem. 233954-962. [PubMed]
54. Yang, K. Y., and R. P. Swenson. 2007. Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: role of alphaSer254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1. Biochemistry 462289-2297. [PubMed]

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