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PubMed1998

Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase.

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3'-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent Km for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster.

Carboxin↗
PubMed2005

The COPG2, DCN, and SDHD genes are biallelically expressed in cattle.

Imprinted genes are preferentially expressed from either the maternally inherited allele or the paternally inherited allele. Most genes known to be imprinted have been identified and studied in the human and the mouse. There is only a small number of reported imprinted genes in cattle, which is probably because of the limited sequence and polymorphism information available for bovine genes. To study the imprinting status of cattle genes and assess their conservation among mammalian species, the expression patterns of COPG2, DCN, and SDHD genes were examined in a total of 128 fetal and adult tissues. Two single nucleotide polymorphisms (SNPs) were identified in COPG2, three SNPs in DCN, and one SNP in SDHD. These polymorphisms were used to distinguish between monoallelic and biallelic expression using a primer extension method and a sequencing-based approach. In all 128 tissues, COPG2, DCN, and SDHD transcripts showed biallelic expression. Other cattle genes examined to date have been found to be imprinted like their known counterparts in human and mouse. This is the first report of genes that are not imprinted in cattle while the corresponding genes in human or mouse are imprinted. Lack of conservation of imprinting among mammals suggests important biological, developmental, and regulatory consequences.

Alleles↗
PubMed2000

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans.

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

Animals↗
PubMed2005

Antihypoxic and antinecrotic effect of mexidol in skin ischemia.

Course treatment with mexidol in a dose of 25 mg/kg for 3 days decreased activities of aspartate transaminase and creatine phosphokinase in the plasma on day 3 after the incidence of skin ischemia (by 1.3 and 1.66 times, respectively). Under these conditions the index of cytolysis decreased by 1.3 times. Therefore, mexidol prevented progression of necrotic processes in the skin. Mexidol therapy of animals with skin ischemia restored the reserve capacity of systems for energy supply and antioxidant defense. The systems of NADH-ubiquinone reductase and succinate-ubiquinone reductase served as the targets for the action of mexidol. Mexidol significantly decreased the damaging effect of reactive oxygen species. Dermatoprotective properties of mexidol were associated with its influence on the energy supply system (regulation of enzyme activity in the electron transport chain, ubiquinone metabolism) and antioxidant defense.

Animals↗
PubMed2005

Protective effect of reamberin on functional activity of mitochondria during skin ischemia.

Reamberin in a dose of 25 mg/kg (succinate concentration) was injected intravenously for 3 days starting from the 1st hour after skin ischemia modeling. This treatment decreased activities of lactate dehydrogenase, aspartate transaminase, and creatine phosphokinase in skin homogenates by 1.6 times, 19%, and 51.3%, respectively. The index of cytolysis decreased by 18%. Reamberin had an energotropic effect, which manifested in an increase in the total ATP content and concentration of creatine phosphate (by 16 and 10%, respectively). After administration of Reamberin, activity of the succinate-ubiquinone reductase system increased by 17%. Under these conditions succinate dehydrogenase activity exceeded the normal by 21%. Reamberin had no effect on the mitochondrial NADH-ubiquinone reductase system in dermal cells during skin ischemia. Superoxide dismutase activity in the area of necrosis increased to the control level on day 3 of treatment with Reamberin. Activities of catalase and glutathione peroxidase increased by 13 and 19%, respectively. Our results indicate that the course of intravenous treatment with Reamberin for 3 days contributes to an increase in reserve capacities of the antioxidant protection system and produces a protective effect during skin ischemia.

Adenosine Triphosphate↗
PubMed1984

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes.

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.

Animals↗
PubMed1987

The inhibition site of MPP+, the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding site of NADH dehydrogenase.

The inhibition of NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) leading to ATP depletion has been proposed to explain cell death in the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Electron paramagnetic resonance studies show no effect of MPP+ on the reduction of the iron-sulfur clusters of NADH dehydrogenase. Mitochondria inhibited by MPP+ were sonicated and both the NADH oxidase and the NADH-Q reductase activities were measured. NADH oxidase activity was not fully restored to control levels, but NADH-Q reductase activity was the same as that of the control. Neither succinate-oxidase nor succinate-Q reductase activities were inhibited. These data indicate that MPP+ interaction with NADH dehydrogenase interferes with the passage of electrons from the iron-sulfur cluster of highest potential to endogenous Q10 but that the inhibition can be relieved by the addition of a small, water-soluble Q analog. Inhibition at this site is sufficient to explain the inhibition of respiration and no inhibition of other mitochondrial functions was observed.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1981

The participation of primary amino groups of succinate dehydrogenase in the formation of succinate-Q reductase.

(1) Purified succinate dehydrogenase contains about 49 mol of lysine residues per mol enzyme. Titration of succinate dehydrogenase with fluorescamine indicates that half the lysyl groups are located on the surface of the protein and the other half are buried inside. (2) The reconstitutive activity and the low Km ferricyanide reductase activity of succinate dehydrogenase decreased as the extent of alkylation of amino groups by fluorescamine increased. (3) The inhibitory effects of fluorescamine on both activities are parallel and are succinate concentration dependent. (4) Alkylation of the native succinate-Q reductase by fluorescamine does not affect the enzymatic activity or alter the enzyme kinetic parameters. This indicates that the inhibitory effect of fluorescamine on succinate dehydrogenase is due to the modification of a specific amino group(s) on succinate dehydrogenase which is essential in the interaction with QPs to form succinate-Q reductase. The participation of an ionic group in the formation of succinate-Q reductase supports the idea of the involvement of ionic interaction between succinate dehydrogenase and QPs.

Alkylation↗
PubMed1982

Kinetics of ubiquinone reduction by the resolved succinate: ubiquinone reductase.

A significant lag in the thenoyltrifluoroacetone (TTFA)-sensitive succinate: ubiquinone reductase activity was observed when a ubiquinone-deficient resolved preparation of the enzyme was assayed in the presence of exogenous ubiquinone-2 (Q2) and 2,6-dichlorophenolindophenol. No such lag was seen when the free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (Wurster's Blue) was used as the terminal electron acceptor, or when the reduction of Q2 was directly measured. The apparent Km value for exogenous Q2 was determined in the Q2-mediated TTFA-sensitive succinate: Wurster's Blue reductase reaction. When the enzyme activity was measured directly by monitoring Q2 reduction without terminal acceptors, the time course of the reaction deviated from zero-order kinetics at Q2 concentrations which were much higher than those expected from the KQ2m value determined in the presence of Wurster's Blue. The time course of Q2 reduction fits a curve describing a competitive interrelationship between oxidized and reduced Q2 at the specific binding site. The data obtained are in agreement with the Q-pool behavior of ubiquinone in mitochondrial membranes and suggest that the rate of ubiquinone reduction by succinate is dependent on the Q/QH2 ratio.

Animals↗
PubMed1985

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase.

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.

Animals↗
PubMed1986

Studies on the succinate dehydrogenating system. Interaction of the mitochondrial succinate-ubiquinone reductase with pyridoxal phosphate.

The inhibitory effect of pyridoxal phosphate on the Triton X-100 solubilized purified bovine heart succinate-ubiquinone reductase (Choudhry, Z.M., Gavrikova, E.V., Kotlyar, A.B., Tushurashvilli, P.R. and Vinogradov, A.D. (1985) FEBS Lett. 182, 171-175) was studied. The kinetics of the enzyme inactivation by pyridoxal phosphate was found to be strongly dependent both qualitatively and quantitatively on the concentration of the protein-detergent complexes. In the diluted system the inactivation of the ubiquinone-depleted enzyme was completely prevented by the saturating concentrations of Q2, carboxin, thenoiltrifluoroacetone and pentachlorophenol, i.e., by the substrate and specific inhibitors of the enzyme. The protective effects of Q2 and the inhibitors was employed to quantitate the affinities of the ligands to their specific binding sites. Strong difference in the affinity of Q2 to the reduced and oxidized enzyme was found. When the soluble reconstitutively active succinate dehydrogenase was treated with pyridoxal phosphate, the reactivity of the enzyme towards low ferricyanide concentrations and its reconstitutive activity was significantly protected against aerobic inactivation.

Animals↗
PubMed1987

Thermal inactivation of electron-transport functions and F0F1-ATPase activities.

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1988

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans.

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.

Animals↗
PubMed1988

Oxidation of malate by the mitochondrial succinate-ubiquinone reductase.

The purified succinate-ubiquinone reductase catalyzes the L- (or D-) malate: acceptor oxidoreductase reaction with Km for malate of about 2.10(-3) M and initial Vmax of 50 and 100 nmol per min per mg of protein for L- and D-stereoisomers, respectively (25 degrees C, pH 7.0). The reaction rate rapidly decreases both in the absence and presence of L-glutamate and L-glutamate-oxaloacetate transaminase added for trapping of oxaloacetate. Both keto and enol forms of oxaloacetate were found to be strong, slowly dissociating inhibitors of succinate dehydrogenase; the first-order rate constant for the enzyme inhibition by the enol form is about 3 times as high as that by the keto form. Oxidation of malate by succinate dehydrogenase in the presence of the oxaloacetate trapping system occurs at an indefinitely constant rate when enoloxaloacetate, which is an immediate product of the reaction, is rapidly converted into the keto isomer--a substrate for transaminase. A quantitative kinetic scheme for malate oxidation by succinate dehydrogenase which includes two kinetically distinct enzyme-oxaloacetate complexes is proposed, and the specific role of the mitochondrial oxaloacetate keto-enol-tautomerase (EC 5.3.2.2) in the regulation of succinate dehydrogenase is suggested.

Animals↗
PubMed1993

Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction.

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.

Animals↗
PubMed1994

The cDNA sequence of beef heart CII-3, a membrane-intrinsic subunit of succinate-ubiquinone oxidoreductase.

We provide the first full-length cDNA and amino acid sequences for beef heart CII-3, one of two hydrophobic subunits that bind succinate dehydrogenase to the mitochondrial inner membrane to form succinate-ubiquinone oxidoreductase (EC 1.3.99.1). Other low molecular weight proteins present in preparations of the isolated complex, including three possible forms of the second anchor polypeptide CII-4, have been identified by amino terminal sequencing.

Amino Acid Sequence↗