Identification of

The succinate dehydrogenases (SDH: soluble, membrane-extrinsic subunits of succinate:quinone oxidoreductases) from Escherichia coli and beef heart mitochondria each adsorb at a pyrolytic graphite 'edge' electrode and catalyse the interconversion of succinate and fumarate according to the electrochemical potential that is applied. E. coli and beef heart mitochondrial SDH share only ca. 50% homology, yet the steady-state catalytic activities, when measured over a continuous potential range, display very similar catalytic operating potentials and energetic biases (the relative ability to catalyse succinate oxidation vs. fumarate reduction). Importantly, E. coli SDH also exhibits the interesting 'tunnel-diode' behaviour previously reported for the mitochondrial enzyme. Thus as the potential is lowered below ca. -60 mV (pH 7, 38 degrees C) the rate of catalytic fumarate reduction decreases abruptly despite an increase in driving force. Since the homology relates primarily to residues associated with active site regions, the marked similarity in the voltammetry reaffirms our previous conclusions that the tunnel-diode behaviour is a characteristic property of the enzyme active site. Thus, succinate dehydrogenase is an excellent fumarate reductase, but its activity in this direction is limited to a very specific range of potential.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

Experimental evidence for proton motive force-dependent catalysis by the diheme-containing succinate:menaquinone oxidoreductase from the Gram-positive bacterium Bacillus licheniformis

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Deltap, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Deltap dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Deltap-dependent catalysis of succinate oxidation by quinone as well as for Deltap generation upon catalysis of fumarate reduction by quinol.     

New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation.

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.     

Physiological role of rhodoquinone in Euglena gracilis mitochondria

Rhodoquinone (RQ) participates in fumarate reduction under anaerobiosis in some bacteria and some primitive eukaryotes. Euglena gracilis, a facultative anaerobic protist, also possesses significant rhodoquinone-9 (RQ9) content. Growth under low oxygen concentration induced a decrease in cytochromes and ubiquinone-9 (UQ9) content, while RQ9 and fumarate reductase (FR) activity increased. However, in cells cultured under aerobic conditions, a relatively high RQ9 content was also attained together with significant FR activity. In addition, RQ9 purified from E. gracilis mitochondria was able to trigger the activities of cytochrome bc1 complex, bc1-like alternative component and alternative oxidase, although with lower efficiency (higher Km, lower Vm) than UQ9. Moreover, purified E. gracilis mitochondrial NAD+-independent D-lactate dehydrogenase (D-iLDH) showed preference for RQ9 as electron acceptor, whereas L-iLDH and succinate dehydrogenase preferred UQ9. These results indicated a physiological role for RQ9 under aerobiosis and microaerophilia in E. gracilis mitochondria, in which RQ9 mediates electron transfer between D-iLDH and other respiratory chain components, including FR.     

Fumarate reductase system of filarial parasite Setaria digitata.

In the cattle filarial parasite Setaria digitata the mitochondria like particles have been shown to possess NADH dependent fumarate reduction coupled with site I electron transport associated phosphorylation. This reduction is catalysed by the fumarate reductase system. The Km for fumarate is 1.47 mM and that for NADH is 0.33 mM. This activity is sensitive to rotenone, antimycin A and o-Hydroxy diphenyl. One ATP is produced for each pair of electrons transferred to fumarate. The fumarate reductase system consisting of NADH-coenzyme Q reductase, cytochrome b like component(s) and succinate dehydrogenase/fumarate reductase is thus very important and hence specific inhibitors of the system may prove useful in the effective control of filariasis.     

Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: Gene targeting of the Fp subunit

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite.     

Ubiquinone-binding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo.We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.     

Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase.

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV). The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop.     

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.     

Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.     

Succinate-DCPIP and NADH-fumarate oxidoreductases in fresh water snails susceptible and non susceptible to schistosoma infection.

The activities of succinate-DCPIP oxidoreductase (SO) and NADH-fumarate oxidoreductase (FR) were determined in tissue homogenate of Biomphalaria alexandrina and Bulinus truncatus, the snail vectors of Schistosomiasia. A parallel study was done on Lymnea truncatula snails which are not susceptible to Schistosoma infection. The Michaelis constant (Km) and maximum velocities (Vmax) for fumarate reduction and succinate oxidation by the tissue homogenates from the three species were determined. The results obtained showed that both susceptible species are aerobic and lactate is the sole end product of anaerobic glycolysis. Lymnea truncatula snails are facultative anaerobic producing succinate as a major end product in the glycolytic pathway.     

An Escherichia coli mutant conditionally altered in respiratory chain components

Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components. Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-). Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*). The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate. Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions. The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation. R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate. R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25. The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E. coli genome. Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.     

Fumarate reductase activity of Streptococcus faecalis

Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The K(m) value of the enzyme for reduced flavin mononucleotide was 2 x 10(-4)m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive.     

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.     

Paragonimus westermani possesses aerobic and anaerobic mitochondria in different tissues,adapting to fluctuating oxygen tension in microaerobic habitats

We previously showed that adult Paragonimus westermani, the causative agent of paragonimiasis and whose habitat is the host lung, possesses both aerobic and anaerobic respiratory chains, i.e., cyanide-sensitive succinate oxidase and NADH-fumarate reductase systems, in isolated mitochondria (Takamiya et al., 1994). This finding raises the intriguing question as to whether adult Paragonimus worms possess two different populations of mitochondria, one having an aerobic succinate oxidase system and the other an anaerobic fumarate reductase system, or whether the worms possess a single population of mitochondria possessing both respiratory chains (i.e., mixed-functional mitochondria). Staining of trematode tissues for cytochrome c oxidase activity showed three types of mitochondrial populations: small, strongly stained mitochondria with many cristae, localised in the tegument and tegumental cells; and two larger parenchymal cell mitochondria, one with developed cristae and the other with few cristae. The tegumental and parenchymal mitochondria could be separated by isopycnic density-gradient centrifugation and showed different morphological characteristics and respiratory activities, with low-density tegumental mitochondria having cytochrome c oxidase activity and high-density parenchymal mitochondria having fumarate reductase activity. These results indicate that Paragonimus worms possess three different populations of mitochondria, which are distributed throughout trematode tissues and function facultatively, rather than having mixed-functional mitochondria.     

Reconstitution of succinate-Q reductase.

Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome $\text{b-c}{}_1$ complex. The reconstituted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome $\text{c}$ reductase or submitochondrial particles) does not show “low concentration ferricyanide reductase activity” as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms.     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

NADH oxidase and fumarate reductase of Ancylostoma ceylanicum.

Ancylostoma ceylanicum, the hookworm parasite of cat, dog and man, was found to contain NADH and/or NADPH oxidase as well as fumarate reductase activities. Both the enzyme systems were predominantly located in the membranes of mitochondrial-rich preparations. The membranes also exhibited the presence of a reduced pyridine nucleotide transhydrogenase activity which transferred hydrogen from NADPH to NAD. Amongst respiratory inhibitors, rotenone (Site I inhibitor) markedly depressed both NADH oxidase and fumarate reductase while others, namely antimycin-A, KCN and azide, had a lesser effect.