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PubMed1983

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.

Cell Membrane↗
PubMed1979

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

Anaerobiosis↗
PubMed1996

Effect of fumaric acid, its dimethylester, and topical antipsoriatic drugs on epidermal differentiation in the mouse tail model.

Fumaric acid, fumaric acid dimethylester, and the dithranol derivative C4-lactone were studied in the mouse tail test to evaluate their effects on epidermal cell differentiation compared with other topical antipsoriatic drugs, such as betamethasone, calcipotriol, and dithranol. Mouse tails were treated for 2 weeks and longitudinal histological sections prepared of the tail skin. The length of the orthokeratotic regions (stratum granulosum) was measured on 10 sequential scales per tail and expressed as percentage of the full length of the scale. In addition, epidermal thickness was measured and the efficacy of the various compounds evaluated. In comparison to 2% salicylic acid ointment, all tested compounds except fumaric acid significantly (p < or = 0.05) increased the proportion of the orthokeratotic region. C4-lactone and calcipotriol were less effective than dithranol, fumaric acid dimethylester only moderately influenced cell differentiation, and betamethasone showed the least potent effect. Dithranol was the most potent substance inducing orthokeratosis without increasing epidermal thickness.

Administration, Topical↗
PubMed1990

[Fumaric acid therapy in psoriasis; a double-blind, placebo-controlled study].

Thirty-nine patients with psoriasis (12 females, 27 males) entered a randomised, double-blind, placebo-controlled study on the efficacy of fumaric acid therapy in an outpatient setting. During 16 weeks the patients were treated with tablets containing a combination of dimethylfumarate and different salts of monoethylfumarate, with octylhydrogen fumarate or with placebo tablets. All patients were treated with identical indifferent topical therapy and followed an elimination diet (avoidance of spices, wine and nuts). Thirty-four patients completed the study. Five patients dropped out because of side effects or aggravation of the skin lesions. The patients treated with the combination of monoethyl- and dimethylfumarate showed a significantly better therapeutic response compared with those who were treated with placebo or octylhydrogen fumarate. Side effects of the fumarate containing tablets were flushing, diarrhoea, a reversible elevation of transaminases, lymphocytopenia and eosinophilia. One patient developed a disturbance of the kidney function which normalised after discontinuation of the therapy.

Adult↗
PubMed1981

[Kinetics of fumarate hydratase reaction catalyzed by free cells of Escherichia coli].

The kinetics of the fumarate hydratase (fumarase) reaction catalyzed by the cells of E. coli strain 85 at high concentrations of the substrate (potassium fumarate) were studied. An automatic procedure for determination of the reaction product--malonic acid--including the use of commercial malate dehydrogenase from porcine heart was developed. The fumarate activity of bacterial cells was studied at different concentrations of the substrate and at different pH values with intact and disrupted cells of E. coli 85 used as the enzyme source. The rate of the fumarase reaction in the E. coli cells was shown to depend on the diffusion and transport processes of the reagent transfer across the cell wall and the cytoplasmic membrane of bacterial cells. The pH optimum of the reaction in free E. coli cells (8-9) and the rate of malonic acid synthesis from potassium fumarate under optimal conditions, which varies within the concentration range of (6--13) x 10(-5) mkmole per mg of protein depending on the quality of cell, were determined.

Escherichia coli↗
PubMed1985

A sodium-stimulated membrane-bound fumarate reductase system in Bacteroides amylophilus.

Membrane vesicles derived from whole cells of the strictly anaerobic rumen bacterium Bacteroides amylophilus exhibited fumarate reductase activity with NADH, FADH2, FMNH2, or reduced viologens as electron donors. The fumarate reductase system is most likely localized on the cytoplasmic side of the plasma membrane. Cytochromes and menaquinone were not detectable. The NADH-dependent activity was inactivated by oxygen, an endogenous protease, and by irradiation at 254 nm. The electron transport inhibitor HpHOQnO and Zn2+ were identified as strong inhibitors of the fumarate reductase reaction. Two types of functional SH-groups might be operative in this system as probed by ClHgSO3H. The oxidation of NADH by fumarate was stimulated by low concentrations of Na+. Concentrations of Na+ in the range of 4 to 30 mM had a pronounced influence on growth rate and cell yield of B. amylophilus. In the presence of 1 mM NaCl growth was observed only after a lag-period of 15 h.

Bacteroides↗
PubMed1989

Interactions of oxaloacetate with Escherichia coli fumarate reductase.

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV).

Escherichia coli↗
PubMed1980

The orientation of the substrate sites of formate dehydrogenase and fumarate reductase in the membrane of Vibrio succinogenes.

Formate dehydrogenase and fumarate reductase are involved in the electron transport phosphorylation system of Vibrio succinogenes. The orientation of the active sites of these enzymes in the cytoplasmic membrane of the bacterium was investigated with the aim of elucidating the mechanism of energy transduction. This was done by measuring the accessiblities of the enzymes to substrates, dyes and inhibitors both in cells and in cell-derived particles obtained with the French press. 1. After treatment of the cells with lysozyme and EDTA, followed by fractionation, both enzymes were found exclusively in the membranous fractions, while the periplasmic as well as the cytoplasmic fractions were devoid of both of the enzymic activities. 2. The sites of dye interaction of fumarate reductase were inaccessible to non-permeant dyes in cells, but were fully accessible in French-press particles. The Km for succinate as measured with the permeant methylene blue as acceptor was increased ten fold on lysis of the cells. The Km measured in the particles was similar to that of lysed cells and was not altered by lysis. 3. The rates of formate oxidation in the presence of non-permeant dyes and the Km for formate were unaffected by cell lysis. On lysis of French-press particles, formate oxidation with both permeant and non-permeant acceptors was increased about three fold. The extent of stimulation was not altered by inhibition of the enzymic activities. 4. Succinate oxidation by particles was fully inhibited by 4-chloromercuriphenyl sulfonate whereas that of cells was fully resistant. Formate dehydrogenase in cells was inhibited by 4-diazophenyl sulfonate when added together with formate. This compound also inhibited the enzyme in the particles when given in the absence of a lytic agent or after its addition. However, most of the enzyme remained active if the inhibitor was added before the lytic agent. 5. Fumarate and succinate were actively taken up by the cells from the medium, while formate did not even penetrate through the membrane of the bacteria. 6. It is concluded that the substrate and the dye-reactive sites of formate dehydrogenase face the outside, while those of fumarate reductase face the inside of the cytoplasmic membrane of cells of V. succinogenes. About 70% of the French-press particles were inverted with respect to the substrate and dye sites of the enzymes. The membrane is impermeable to formate and does not contain a specific transporter. The electrogenic liberation of protons on the outisde and the uptake of protons from the cytoplasm of the bacteria, which accompany electron transport, can be explained on the basis of the orientation of the substrate sites of the enzymes without net transport of protons across the membrane.

Aldehyde Oxidoreductases↗
PubMed1985

In vivo detection of a three iron cluster in fumarate reductase from Escherichia coli.

Escherichia coli with plasmid amplified expression of fumarate reductase was grown anaerobically on a medium containing fumarate and glycerol and investigated by electron paramagnetic resonance spectroscopy. Anaerobically harvested cells exhibit an EPR signal characteristic of a reduced [2Fe-2S] cluster. Anaerobic addition of fumarate results in diminution of the reduced [2Fe-2S] signal and the appearance of the EPR signal associated with the oxidized 3Fe cluster. The results provide the first evidence for a trinuclear iron-sulfur cluster that exists in vivo, and suggest that the 3Fe cluster in purified fumarate reductase samples is not an artifact of the isolation procedure. The significance of this observation is discussed in relation to the physiological relevance of trinuclear iron-sulfur clusters.

Circular Dichroism↗
PubMed1986

Isolation of fumarate reductase from Desulfovibrio multispirans, a sulfate reducing bacterium.

Fumarate reductase was isolated and purified 100-fold to homogeneity from Desulfovibrio multispirans, a new species of sulfate-reducing bacteria. The enzyme contained 1 mol of non-covalently bound FAD and four subunits with Mr 45,000, 32,000, 30,000 and 27,000. EPR spectroscopy showed the existence of two iron-sulfur clusters. The absorption spectrum showed a broad region of high absorbance from 450 nm to 300 nm with a protein peak at 278 nm. The ratio of A278:A400 was 2.60. The specific activity was 110 mumoles H2/mg of protein. The Km for fumarate was 2.5 mM. The activation energy was 8.7 kcal/mol. Electron transport from H2 to fumarate in intact cells was inhibited by 2-heptyl-4-hydroxy-quinoline-N-oxide, a quinone inhibitor, indicating the participation of quinone (probably menaquinone) in fumarate reduction.

Desulfovibrio↗
PubMed1986

Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi.

Subcellular fractions obtained from Trypanosoma cruzi epimastigotes broken by freezing and thawing were assayed for fumarate reductase activity with reduced methyl viologen as electron donor and fumarate as electron acceptor under anaerobic conditions. Two distinct activities were detected: one in the mitochondrial membranes, 115 mU(mg protein)-1, accounting for 96% of the total and the other in the cytosol, 3 mU(mg protein)-1, accounting for 3% of the total. The activity of membrane-bound fumarate reductase correlated statistically with either the activity or the amount of mitochondrial markers such as succinate and NADH dehydrogenases, cytochromes b + c558, cytochrome a611 and 5,7-diene sterols in the obtained subcellular fractions (580 X g, 12 000 X g, and 105 000 X g sediments and supernatant). Mitochondrial fumarate reductase was inhibited by succinate, malonate, cyanide, and 2-thenoyltrifluoroacetone (TTFA); whereas the soluble enzyme was inhibited by succinate and not by TTFA. The 12 000 X g sediment (mitochondrial membranes) showed after dithionite addition, absorption maxima at 611, 560 and 530 nm accounting for the presence of cytochrome b560, c558 and a611. A CO-binding cytochrome o was also detected. A scheme of the T. cruzi mitochondrial respiratory chain is presented.

Animals↗
PubMed1994

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Amino Acid Sequence↗
PubMed1987

The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli.

Escherichia coli, which can utilize O2, nitrate, fumarate, or trimethylamine N-oxide (Me3NO) as terminal electron acceptor, preferentially utilizes the one with the highest redox potential. Thus O2 prevents induction of nitrate, fumarate, and Me3NO reductases, and nitrate curtails the induction of fumarate and Me3NO reductases. Under anaerobic conditions the narL gene product, in the presence of nitrate, is known to activate transcription of the narC operon, which encodes nitrate reductase. This study shows that the same product plays a role in the repression by nitrate of the operons (frd and tor) that encode fumarate and Me3NO reductases. In contrast, the anaerobic repression of ethanol dehydrogenase by nitrate does not require the narL product. Expression of narL does not require the fnr gene product, a pleiotropic activator that is required for full expression of narC, frd, and tor.

Bacterial Proteins↗
PubMed1984

Location of the catalytic site of the respiratory fumarate reductase of Escherichia coli.

The location of the catalytic site of the membrane-bound respiratory fumarate reductase of Escherichia coli was investigated using mutants and inhibitors of dicarboxylic acid transport. Comparison of apparent Km and Vmax values for fumarate in intact cells and in inverted membrane vesicles showed that externally added fumarate was required to be transported across the cytoplasmic membrane prior to reduction. The catalytic site of fumarate reductase must therefore be located on the cytoplasmic face of the membrane.

Aspartic Acid↗
PubMed1987

Nucleotide sequence and comparative analysis of the frd operon encoding the fumarate reductase of Proteus vulgaris. Extensive sequence divergence of the membrane anchors and absence of an frd-linked ampC cephalosporinase gene.

The fumarate reductase of Escherichia coli is a bioenergetically important membrane-bound flavoenzyme consisting of four subunits. A and B comprise a membrane-extrinsic catalytic domain whereas C and D are hydrophobic polypeptides which link the catalytic centres to the electron-transport chain. The nucleotide sequence of the frd operon encoding the fumarate reductase of the distantly related bacterium, Proteus vulgaris has been determined and used to predict the primary structures of the respective subunits. Extensive amino acid sequence identity (greater than 80%) was found between the fumarate reductase A and B subunits of P. vulgaris and E. coli. In contrast, the primary structures of the P. vulgaris and E. coli C and D proteins are much less closely related (about 60% homology) although the overall hydrophobicity of their three membrane-spanning segments has been conserved. In most enteric bacteria, the frd operon is followed by genes, ampR and/or ampC, required for the genetic regulation and biosynthesis of a cephalosporinase. The corresponding region of the P. vulgaris genome is occupied by an operon (orf A'BCD) containing at least four genes which are clearly unrelated to the ampC system. Intriguingly the primary structures of the OrfA and OrfD proteins suggest that, like fumarate reductase, they may be components of a membrane-bound enzyme complex involved in energy metabolism.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes.

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum.

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.

Anaerobiosis↗
PubMed1983

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Adenosine Triphosphatases↗