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PubMed1994

The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria.

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.

Binding Sites↗
PubMed2005

Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture.

A system for growing Geobacter sulfurreducens under anaerobic conditions in chemostats was developed in order to study the physiology of this organism under conditions that might more closely approximate those found in the subsurface than batch cultures. Geobacter sulfurreducens could be cultured under acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was threefold higher with fumarate as the electron acceptor than with Fe(III), despite the lower mid-point potential of the fumarate/succinate redox couple. When growth was limited by availability of fumarate, high steady-state concentrations were detected, suggesting that fumarate is unlikely to be an important electron acceptor in sedimentary environments. The half-saturation constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that the growth of Geobacter species is likely to be acetate limited in most subsurface sediments, but that when millimolar quantities of acetate are added to the subsurface in order to promote the growth of Geobacter for bioremediation applications, this should be enough to overcome any acetate limitations. When the availability of electron acceptors, rather than acetate, limited growth, G. sulfurreducens was less efficient in incorporating acetate into biomass but had higher respiration rates, a desirable physiological characteristic when adding acetate to stimulate the activity of Geobacter species during in situ uranium bioremediation. These results demonstrate that the ability to study the growth of G. sulfurreducens under steady-state conditions can provide insights into its physiological characteristics that have relevance for its activity in a diversity of sedimentary environments.

Acetates↗
PubMed1999

Characterization of the respiratory chain of Helicobacter pylori.

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.

Electron Transport↗
PubMed2001

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Amino Acid Sequence↗
PubMed1972

Requirement of succinate for the growth of Vibrio succinogenes.

Vibrio succinogenes required relatively small amounts of succinate for growth when formate plus nitrate was supplied as the energy source. The requirement for succinate was not apparent when formate plus fumarate was the energy source because fumarate is reduced to succinate. l-Asparagine, fumarate, and malate replaced succinate, and it appears likely that they do so by being converted to succinate. Formate plus l-aspartate or l-asparagine served as energy sources for growth. The stoichiometry of the reduction of aspartate with H(2) by resting cells suggests an aspartase reaction followed by reduction of fumarate to succinate. Oxalacetate or pyruvate plus bicarbonate did not substitute for succinate, nor did many other compounds that were tested. (14)C-succinate was mainly incorporated into the alcohol-soluble fraction of cells, although there was significant incorporation into the hot trichloroacetic acid-soluble and -insoluble fractions.

Asparagine↗
PubMed1973

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis.

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.

Aspartic Acid↗
PubMed1988

Regulation of carbon and electron flow in Propionispira arboris: relationship of catabolic enzyme levels to carbon substrates fermented during propionate formation via the methylmalonyl coenzyme A pathway.

A detailed study of the glucose fermentation pathway and the modulation of catabolic oxidoreductase activities by energy sources (i.e., glucose versus lactate or fumarate) in Propionispira arboris was performed. 14C radiotracer data show the CO2 produced from pyruvate oxidation comes exclusively from the C-3 and C-4 positions of glucose. Significant specific activities of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were detected, which substantiates the utilization of the Embden-Meyerhoff-Parnas path for glucose metabolism. The methylmalonyl coenzyme A pathway for pyruvate reduction to propionate was established by detection of significant activities (greater than 16 nmol/min per mg of protein) of methylmalonyl coenzyme A transcarboxylase, malate dehydrogenase, and fumarate reductase in cell-free extracts and by 13C nuclear magnetic resonance spectroscopic demonstration of randomization of label from [2-13C]pyruvate into positions 2 and 3 of propionate. The specific activity of pyruvate-ferredoxin oxidoreductase, malate dehydrogenase, fumarate reductase, and transcarboxylase varied significantly in cells grown on different energy sources. D-Lactate dehydrogenase (non-NADH linked) was present in cells of P. arboris grown on lactate but not in cells grown on glucose or fumarate. These results indicate that growth substrates regulate synthesis of enzymes specific for the methylmalonyl coenzyme A path and initial substrate transformation.

Acyl Coenzyme A↗
PubMed1962

Cytochromelinked fermentation in Bacteroides ruminicola.

White, D. C. (Rockefeller Institute, New York, N.Y.), M. P. Bryant, and D. R. Caldwell. Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol. 84:822-828. 1962-Previous studies showed that Bacteroides ruminicola, an anaerobic, saccharolytic, ruminal bacterium, ferments glucose with the production of succinic, acetic, and formic acids, requires a large amount of CO(2), and most strains require heme for growth. Difference spectra of cell suspensions of both heme-requiring strain 23, B. ruminicola subsp. ruminicola, and heme-independent strain GA33, B. ruminicola subsp. brevis, showed the presence of a cytochrome (absorption maxima at 560 mmu, near 530 mmu, and 428 mmu) similar to cytochrome b. This cytochrome and flavoprotein (trough at 450 mmu) in the cells, reduced by endogenous metabolism, were oxidized on addition of air, CO(2), oxalacetate, malate, or fumarate but no oxidation occurred in the presence of succinate, malonate, lactate, pyruvate, aspartate, citrate, NO(3) (-), SO(4) (=), 2-n-heptyl or hydroxyquinoline-N-oxide (HOQNO), amytal or azide. The oxidation of these cellular pigments by fumarate was not inhibited by CN(-), CO, malonate, succinate, amytal, or HOQNO. Glucose and reduced diphosphopyridine nucleotide (DPNH), but not succinate, reduced the pigments in frozen-thawed cells previously exposed to air for 4 hr at room temperature. The results suggest that this cytochrome and flavoprotein form an electron transport system for fumarate reduction to succinate by DPNH generated by glycolysis, and that succinate is produced via CO(2) condensation with pyruvate or phosphoenolpyruvate and with oxalacetate, malate, and fumarate as intermediates. A pigment similar to cytochrome o (absorption maxima at 570, 555, and 416 mmu) was observed when reduced cells were treated with CO and compared to reduced cells, but there was no detectable cytochrome oxidase activity. The function of this pigment is obscure. No peroxidase or catalase activity was detected in either strain. Pyridine hemochromogens of both strains indicate one major heme, a protoheme-like pigment, with absorption in the alpha region maximum at 556 mmu. As B. ruminicola is one of the most numerous of rumen bacteria and ferments a wide variety of carbohydrates of importance in ruminant rations, cytochrome must be of importance in electron transport in rumen contents, a highly anaerobic environment.

Animals↗
PubMed1975

Anaerobic transport in Escherichia coli membrane vesicles.

Anaerobic lactose and/or amino acid transport by membrane vesicles prepared from Escherichia coli ML 308-225 can be coupled to at least four electron transfer systems: alpha-glycerol-P-dehydrogenase:nitrate reductase, formate dehydrogenase:nitrate reductase, alpha-glycerol-P dehydrogenase:fumarate reductase, and formate dehydrogenase:fumarate reductase. Vesicles contain one or more of these electron transfer systems depending on the growth conditions of the parent cells. alpha-Glycerol-P dehydrogenase and fumarate reductase are present only in vesicles prepared from cells grown in the presence of glycerol or fumarate, respectively. Formate dehydrogenase and nitrate reductase activities, on the other hand, are present in vesicles from cells grown on a variety of media. alpha-Glycerol-P and formate are able to drive aerobic transport in vesicles prepared from anaerobically grown cells, indicating coupling between aerobic and anaerobic electron transfer systems.

Aerobiosis↗
PubMedDate not supplied

[Unsaturated organic acids as terminal electron acceptors for reductase chains of anaerobic bacteria].

This paper summarizes the current knowledge of unsaturated organic acids in their role as terminal electron acceptors of anaerobic bacteria. The mechanisms and enzyme systems involved in the reduction of fumarate by Escherichia coli, Wolinella succinogenes, and some species of the genus Shewanella are considered. Particular attention is given to reduction of the double bond of the unnatural compound methacrylate by the sigma-proteobacterium Geobacter sulfurreducens Am-1. Soluble periplasmic flavocytochromes c, found in bacteria of the genera Shewanella and Geobacter, are involved in the hydration of fumarate (in Shewanella species) and methacrylate (in G. sulfurreducens Am-1). In E. coli and W. succinogenes, fumarate is reduced in cytosol by membrane-bound fumarate reductases. The prospects for research into organic acid reduction at double bonds in bacteria are discussed.

Bacteria, Anaerobic↗
PubMed1986

The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments.

A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.

Animals↗
PubMed1982

Effect of the carbon oxidation level of the energy source on heat of reaction in biosynthesis of a Pseudomonas strain.

The heats of reaction for cell biosynthesis of a strain of Pseudomonas aeruginosa grown on ammonium-nitrogen and fumarate or butyrate as carbon-energy sources have been determined by microcalorimetric experiments, CO2 and molar growth yield determinations. The different oxidation level of the fumarate-carbon (C1+) and butyrate-carbon (C1-) caused a difference on the sign of the heat of reaction for cellular biosynthesis: positive for fumarate (delta H less than 0) and negative for butyrate (delta H greater than 0). It has been possible to state the stoichiometry of the biosynthetic reaction for fumarate only and not for butyrate because of the large pigment production under the experimental conditions adopted: pure oxygen as gas phase at 30 degrees C.

Butyrates↗
PubMed1981

Relationships between pyruvate decarboxylation and branched-chain volatile acid synthesis in Ascaris mitochondria.

The rate of 14CO2 evolution from 1-[14C]pyruvate by intact Ascaris mitochondria was very slow, but increased with increasing concentrations of pyruvate. At all concentrations of pyruvate, in an aerobic environment, pyruvate decarboxylation was stimulated greatly by the addition of fumarate, malate, or succinate. However, under anaerobic conditions, only malate and fumarate stimulated pyruvate decarboxylation; succinate had no effect. This implies that the aerobic metabolism of succinate, presumably to other dicarboxylic acids, may be required for the stimulation. Incubation of sonicated mitochondria with pyruvate plus fumarate, under rate-limiting concentrations of NAD+, resulted in approximately equal quantities of pyruvate utilized and succinate formed, suggesting that pyruvate oxidation and fumarate reduction may be linked. Branched-chain, volatile fatty acids were not formed during incubations with either malate or succinate, or succinate plus acetate. However, incubations of intact Ascaris mitochondria with pyruvate plus succinate yielded 2-methylbutyrate and 2-methylvalerate, whereas incubations with pyruvate plus propionate yielded almost exclusively 2-methylvalerate. Oxygen dramatically inhibited the synthesis of the branched-chain acids from succinate plus pyruvate, attesting to the apparent anaerobic nature of Ascaris mitochondrial metabolism. Significantly, the addition of glucose plus ADP stimulated the formation of all volatile fatty acids. Therefore, the synthesis of branched-chain acids may be related directly to increased energy generation. Alternatively, they may function in the regulatory role of maintaining the mitochondrial redox balance.

Adenosine Diphosphate↗
PubMed1995

Characterization of the cDNA and the gene encoding murine adenylosuccinate lyase.

Adenylosuccinate lyase catalyzes two similar reactions in the de novo purine biosynthetic pathway; the cleavage of succinylaminoimidazole carboxamide ribotide to aminoimidazole carboxamide ribotide and fumarate and the cleavage of adenylosuccinate to adenylate and fumarate. Adenylosuccinate lyase is also a participant in the purine nucleotide cycle which plays an important role in maintaining the AMP levels in muscle. In order to understand the structure/function and evolutionary relationships of the members of the fumarate gene family and to evaluate the possible existence of tissue specific isoforms of adenylosuccinate lyase, we have isolated and characterized the murine cDNA and gene encoding adenylosuccinate lyase. The cDNA has 94% and 87% identity to the human sequence at the amino acid and nucleotide levels respectively. The gene is about 27 kb and contains 13 exons. Comparison of the exon/intron structure of this gene with the argininosuccinate lyase gene did not suggest gene duplication or exon shuffling as a mechanism of evolution in the fumarate gene family.

Adenylosuccinate Lyase↗
PubMed1981

Molecular cloning of the fnr gene of Escherichia coli K12.

Mutations in the fnr gene of Escherichia coli have pleiotrophic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (lambda fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The lambda fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated lambda fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA : DNA hybridization.

Anaerobiosis↗
PubMedDate not supplied

Genetic change may be caused by interference with protein-protein interactions.

Several aprotic polar solvents were shown to induce mitotic aneuploidy in yeast: diethyl ketone, gamma-valerolactone, pyridine, pivalinic acid nitrile, phenylacetonitrile and fumaric acid dinitrile. Only fumaric acid dinitrile also strongly induced other types of genetic effects including mitotic crossing-over, mitotic gene conversion and point mutation. The other substances only induced aneuploidy and this only over a very narrow dose range. The treatment protocol used suggested that these chemicals acted via interference with tubulin assembly and disassembly causing a malfunctioning of spindle fiber microtubules. This hypothesis was tested using twice recycled porcine brain tubulin. Diethyl ketone, gamma-valerolactone, pyridine and phenylacetonitrile inhibited GTP-promoted assembly of porcine brain tubulin in vitro in the concentration range needed for the induction of mitotic aneuploidy in yeast. Pivalinic acid nitrile accelerated tubulin aggregation whereas fumaric acid dinitrile had no effect even at concentrations 18 times higher than the lowest tested concentration effective in yeast. The in vitro experiments with porcine brain tubulin further suggest that genetic change can result from interference with specific protein-protein interactions. Fumaric acid dinitrile was the only exception since it did induce aneuploidy but had no effects on the assembly of porcine brain tubulin. This could be caused either by interference with protein-protein interactions other than between molecules during assembly and disassembly of microtubules or species-specific differences in susceptibility between yeast spindle and porcine brain tubulin.

Aneuploidy↗
PubMed1988

Rhodoquinone requirement of the Hymenolepis diminuta mitochondrial electron transport system.

The occurrence of rhodoquinone as a mitochondrial membrane component was demonstrated in adult Hymenolepis diminuta. Chromatographic separation of pentane extracts, from lyophilized mitochondrial membranes, coupled with spectral analyses of separated material demonstrated the presence of rhodoquinone. The presence of ubiquinone was not apparent. Rhodoquinone content of membranes was about 1.2 micrograms (mg protein)-1. The rhodoquinone requirement of the H. diminuta electron transport system was demonstrated both in terms of the less active NADH oxidase and the physiologically required, NADH-dependent fumarate reductase employing lyophilized mitochondrial membranes as the source of activities. Pentane extraction of membranes virtually abolished the oxidase and fumarate reductase systems. Supplementation of pentane-treated membranes with H. diminuta rhodoquinone restored oxidase and fumarate reductase activities to levels simulating those of lyophilized membranes. Ubiquinone did not substitute for rhodoquinone. The rhodoquinone-reconstituted membranes displayed rotenone sensitivity. These findings represent the first direct demonstration of the rhodoquinone requirement of helminth electron transport-coupled oxidase and fumarate reductase.

Animals↗
PubMed1996

Rhodoquinone is synthesized de novo by Fasciola hepatica.

Most adult parasitic helminths have an anaerobic energy metabolism in which fumarate is reduced to succinate by fumarate reductase. Rhodoquinone (RQ) is an essential component of the electron transport associated with this fumarate reduction, whereas ubiquinone (UQ) is used in the aerobic energy metabolism of parasites. Not known yet, however, is the RQ and UQ composition during the entire life cycle nor the origin of RQ in parasitic helminths. This report demonstrates the essential function of RQ in anaerobic energy metabolism during the entire life cycle of Fasciola hepatica, as the amount of RQ present reflected the importance of fumarate reduction in various stages. We also studied the origin of RQ, as earlier studies on the protozoan Euglena gracilis suggested that RQ is synthesized from UQ. Therefore, in parasitic helminths RQ might be synthesized by modification of UQ obtained from the host. However, we demonstrated that in F. hepatica adults RQ was not produced by modification of UQ obtained from the host but that RQ was synthesized de novo, as (i) the chain-length of the quinones of F. hepatica adults was not related to the chain length of the quinone of the host, (ii) despite many attempts we could never detect any in vitro conversion of UQ9 into RQ9 or into UQ10, neither by intact adult flukes nor by homogenates of F. hepatica adults and (iii) F. hepatica adults used mevalonate as precursor for the synthesis of RQ. We also showed that the rate of quinone synthesis in F. hepatica adults was comparable to that in the free-living nematode Caenorhabditis elegans. These results prompted the suggestion that RQ is synthesized via a pathway nearly identical to that of UQ biosynthesis: possibly only the last reaction differs.

Animals↗