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PubMed1992

cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer.

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.

Amino Acid Sequence↗

PubMed2006

Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of Huntington's disease knock-in striatal cells.

Abstract Cystamine significantly improved motor deficits and extended survival in mouse models of Huntington's disease (HD); however, the precise mechanism(s) by which cystamine and the related compound cysteamine are beneficial remain to be elucidated. Using clonal striatal cell lines from wild-type (STHdhQ7/HdhQ7) and mutant huntingtin knock-in (STHdhQ111/HdhQ111) mice, we have tested the hypothesis that cystamine and cysteamine could be beneficial by preventing the depolarization of mitochondria in cell cultures. Treatment with 3-nitroproprionic acid (3-NP), a mitochondrial complex II inhibitor, induces mitochondrial depolarization and cell death of mutant HD striatal cells but not of wild-type cells. The 3-NP-mediated decrease in the mitochondrial membrane potential was attenuated by 50 microm cystamine and completely inhibited by 250 microm cystamine. Similar results were obtained using cysteamine (50-500 microm). In addition, both cystamine and cysteamine significantly attenuated the 3-NP-induced cell death. Treatment of mutant HD striatal cells with 3-NP resulted in a robust decrease in the cellular and mitochondrial levels of glutathione (GSH) compared with cells exposed to the vehicle alone. Pre-treatment of the cells with cystamine and cysteamine completely prevented the 3-NP-mediated decrease in cellular and mitochondrial GSH levels. Incubation with L-buthionine (S,R) sulfoximine (BSO) 250 microm in combination with cystamine (250 microm) or cysteamine (250 microm) prior to being treated with 3-NP completely prevented the beneficial effects of cystamine and cysteamine on the 3-NP-mediated mitochondrial depolarization. These results demonstrate that cystamine and cysteamine prevent the 3-NP-induced mitochondrial depolarization of HD striatal cell cultures.

Animals↗

PubMed2000

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons.

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.

Adenosine Triphosphate↗

PubMed1996

Succinate:quinone oxidoreductase (complex II) containing a single heme b in facultative alkaliphilic Bacillus sp. strain YN-2000.

Succinate:quinone oxidoreductase (EC 1.3.5.1) was first purified from the facultative alkaliphilic Bacillus sp. strain YN-2000 in the presence of Triton X-100. The isolated enzyme showed high succinate-ubiquinone oxidoreductase activity at pH 8.5. The Km for ubiquinone 1 and the Vmax of the enzyme were determined to be about 5 microM and 48 micromol of ubiquinone 1 per min per mg, respectively. The catalytic activity of the enzyme was 50% inhibited by 9 microM 2-thenoyltrifluoroacetone or 0.8 microM 2-n-heptyl-4-hydroxyquinoline- N-oxide. The enzyme consisted of three kinds of subunits with molecular masses of 66, 26, and 15 kDa, respectively, and contained 1.28 mol of covalently bound flavin adenine dinucleotide, 0.9 mol of heme b, 1.35 mol of menaquinone, 8.3 mol of nonheme iron, and 7.5 mol of inorganic sulfide per mol of enzyme. The enzyme showed symmetrical alpha absorption peaks at 556.5 and 554 nm in the reduced state at room temperature and 77 K, respectively. The potentiometric analysis of the enzyme yielded an Em,7 of heme b of about -64 mV (n = 1). Furthermore, the content of the enzyme was increased up to fivefold when the bacterium was grown at pH 10 compared with pH 7. These results indicate that the succinate:quinone oxidoreductase with a single heme b is involved in the respiratory chain of the alkaliphile at a very alkaline pH.

Bacillus↗

PubMed1990

Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II).

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.

Amino Acid Sequence↗

PubMed2003

Adenosinergic protection of dopaminergic and GABAergic neurons against mitochondrial inhibition through receptors located in the substantia nigra and striatum, respectively.

Mitochondrial dysfunction may contribute to dopaminergic (DAergic) cell death in Parkinson's disease and GABAergic cell death in Huntington's disease. In the present work, we tested whether blocking A1 receptors could enhance the damage to DAergic and GABAergic neurons caused by mitochondrial inhibition, and whether blocking A2a receptors could protect against damage in this model. Animals received an intraperitoneal injection of 8-cyclopentyl-1,3-dipropylxanthine (CPX) (A1 antagonist) or 3,7-dimethyl-1-propargylxanthine (DMPX) (A2a antagonist) 30 min before intrastriatal infusion of malonate (mitochondrial complex II inhibitor). Damage was assessed 1 week later by measuring striatal dopamine, tyrosine hydroxylase (TH), and GABA content. In mice and rats, malonate-induced depletion of striatal dopamine, TH, or GABA was potentiated by pretreatment with 1 mg/kg CPX and attenuated by pretreatment with 5 mg/kg DMPX. To determine the location of the A1 and A2a receptors mediating these effects, CPX or DMPX was infused directly into the striatum or substantia nigra of rats 30 min before intrastriatal infusion of malonate. When infused into the striatum, CPX (20 ng) potentiated, whereas DMPX (50 ng) prevented malonate-induced GABA loss, but up to 100 ng of CPX or 500 ng of DMPX did not alter malonate-induced striatal dopamine loss. Intranigral infusion of CPX (100 ng) or DMPX (500 ng), however, did exacerbate and protect, respectively, against malonate-induced striatal dopamine loss. Thus, A1 receptor blockade enhances and A2a receptor blockade protects against damage to DAergic and GABAergic neurons caused by mitochondrial inhibition. Interestingly, these effects are mediated by A1 and A2a receptors located in the substantia nigra for DAergic neurons and in the striatum for GABAergic neurons.

Adenosine↗

PubMed2005

The SDH mutation database: an online resource for succinate dehydrogenase sequence variants involved in pheochromocytoma, paraganglioma and mitochondrial complex II deficiency.

BACKGROUND: The SDHA, SDHB, SDHC and SDHD genes encode the subunits of succinate dehydrogenase (succinate: ubiquinone oxidoreductase), a component of both the Krebs cycle and the mitochondrial respiratory chain. SDHA, a flavoprotein and SDHB, an iron-sulfur protein together constitute the catalytic domain, while SDHC and SDHD encode membrane anchors that allow the complex to participate in the respiratory chain as complex II. Germline mutations of SDHD and SDHB are a major cause of the hereditary forms of the tumors paraganglioma and pheochromocytoma. The largest subunit, SDHA, is mutated in patients with Leigh syndrome and late-onset optic atrophy, but has not as yet been identified as a factor in hereditary cancer. DESCRIPTION: The SDH mutation database is based on the recently described Leiden Open (source) Variation Database (LOVD) system. The variants currently described in the database were extracted from the published literature and in some cases annotated to conform to current mutation nomenclature. Researchers can also directly submit new sequence variants online. Since the identification of SDHD, SDHC, and SDHB as classic tumor suppressor genes in 2000 and 2001, studies from research groups around the world have identified a total of 120 variants. Here we introduce all reported paraganglioma and pheochromocytoma related sequence variations in these genes, in addition to all reported mutations of SDHA. The database is now accessible online. CONCLUSION: The SDH mutation database offers a valuable tool and resource for clinicians involved in the treatment of patients with paraganglioma-pheochromocytoma, clinical geneticists needing an overview of current knowledge, and geneticists and other researchers needing a solid foundation for further exploration of both these tumor syndromes and SDHA-related phenotypes.

Codon, Nonsense↗

PubMed2002

Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera.

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.

Animals↗

PubMed2001

A high-resolution integrated map spanning the SDHD gene at 11q23: a 1.1-Mb BAC contig, a partial transcript map and 15 new repeat polymorphisms in a tumour-suppressor region.

Chromosomal region 11q22-q23 is a frequent target for deletion during the development of many solid tumour types, including breast, ovary, cervix, stomach, bladder carcinomas and melanoma. One of the most commonly deleted subregions contains the SDHD gene, which encodes the small subunit of cytochrome b (cybS) in mitochondrial complex II (succinate-ubiquinone oxidoreductase). Germline mutations in SDHD cause hereditary paraganglioma type 1 (PGL1), and suggest a tumour suppressor role for cybS. We present a high-resolution physical map spanning SDHD, covered by 19 YACs and 20 BACs. An approximate 1.1-Mb gene-rich region around SDHD is spanned by a complete BAC contig. Twenty-six new STSs are developed from the BAC clone ends. In addition to the discovery and characterisation of 15 new simple tandem repeat polymorphisms, we provide integrated positional information for 33 ESTs and known genes, including KIAA1391, POU2AF1 (OBF1), PPP2R1B, CRYAB, HSPB2, DLAT, IL-18, PTPS, KIAA0781 and KAIA4591, which is mapped by NotI site cloning. We describe full-length transcript sequence for PPP2R1B, encoding the protein phosphatase 2A regulatory subunit A beta isoform. We also discover a processed pseudogene for USA-CYP, a cyclophilin associated with U4/U6 snRPNs, and a novel gene, DDP2, encoding a mitochondrial protein similar to the X-linked deafness-dystonia protein, which is juxtaposed 5'-to-5' to SDHD. This map will help assess this gene-rich region in PGL and in other common tumours.

3' Untranslated Regions↗

PubMed1984

Effect of iron deficiency on succinate- and NADH-ubiquinone oxidoreductases in skeletal muscle mitochondria.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

Animals↗

PubMed1987

Amino acid charge distribution influences the assembly of apoprotein into light-harvesting complex II.

The light-harvesting complex II of thylakoid membranes channels light energy into the photosynthetic reaction center II. The major apoproteins of this complex are the nuclear encoded light-harvesting chlorophyll a/b-proteins (LHCP). A model for the arrangement of LHCP in the thylakoid membrane predicts three alpha-helical membrane-spanning regions. The first and third putative membrane-spanning regions include oppositely charged amino acid residues. When the first and third helices are altered to carry only positive charges, the in vitro accumulation of LHCP in the complex is reduced. This mutation is partially rescued by the introduction of a new negative charge in the third helix, an arrangement that is reversed from the wild type. An arginine in the first helix is also important in some aspect of the process leading to the successful accumulation of the LHCP in thylakoids.

Amino Acids↗

PubMed1999

CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene.

Amino Acid Sequence↗

PubMed2002

Inborn errors of complex II--unusual human mitochondrial diseases.

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.

Citric Acid Cycle↗

PubMed2000

Genotoxic and functional consequences of transplacental zidovudine exposure in fetal monkey brain mitochondria.

Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5), 10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were isolated from fetal cerebrum and cerebellum at birth and mitochondrial morphology was examined in these tissues by transmission electron microscopy (TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and quantity were determined by Southern blot and slot blot analysis. In the cerebral mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) specific activity decreased by 25% in monkeys treated with 40 mg of AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and cytochrome c reductase (complex IV)-specific activities showed dose-dependent increases (p > or = .05), compared with those in controls. In the cerebellum, no difference was seen in mitochondrial OXPHOS enzyme activities between unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues. Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a human equivalent daily dose of AZT during the last half of pregnancy, mitochondria in the fetal cerebrum appear to sustain moderate damage, while the fetal cerebellum mitochondria were not effected.

Animals↗

PubMed2001

Inhibition of mitochondrial complex II induces a long-term potentiation of NMDA-mediated synaptic excitation in the striatum requiring endogenous dopamine.

Abnormal involuntary movements and cognitive impairment represent the classical clinical symptoms of Huntington's disease (HD). This genetic disorder involves degeneration of striatal spiny neurons, but not striatal large cholinergic interneurons, and corresponds to a marked decrease in the activity of mitochondrial complex II [succinate dehydrogenase (SD)] in the brains of HD patients. Here we have examined the possibility that SD inhibitors exert their toxic action by increasing glutamatergic transmission. We report that SD inhibitors such as 3-nitroproprionic acid (3-NP), but not an inhibitor of mitochondrial complex I, produce a long-term potentiation of the NMDA-mediated synaptic excitation (3-NP-LTP) in striatal spiny neurons. In contrast, these inhibitors had no effect on excitatory synaptic transmission in striatal cholinergic interneurons and pyramidal cortical neurons. 3-NP-LTP involves increased intracellular calcium and activation of the mitogen-activated protein kinase extracellular signal-regulated kinase and is critically dependent on endogenous dopamine acting via D2 receptors, whereas it is negatively regulated by D1 receptors. Thus 3-NP-LTP might play a key role in the regional and cell type-specific neuronal death observed in HD.

Animals↗

PubMed2005

Pathology of mitochondrial encephalomyopathies.

Muscle biopsy provides the best tissue to confirm a mitochondrial cytopathy. Histochemical features often correlate with specific syndromes and facilitate the selection of biochemical and genetic studies. Ragged-red fibres nearly always indicate a combination defect of respiratory complexes I and IV. Increased punctate lipid within myofibers is a regular feature of Kearns-Sayre and PEO, but not of MELAS and MERRF. Total deficiency of succinate dehydrogenase indicates a severe defect in Complex II; total absence of cytochrome-c-oxidase activity in all myofibres correlates with a severe deficiency of Complex IV or of coenzyme-Q10. The selective loss of cytochrome-c-oxidase activity in scattered myofibers, particularly if accompanied by strong succinate dehydrogenase staining in these same fibres, is good evidence of mitochondrial cytopathy and often of a significant mtDNA mutation, though not specific for Complex IV disorders. Glycogen may be excessive in ragged-red zones. Ultrastructure provides morphological evidence of mitochondrial cytopathy, in axons and endothelial cells as well as myocytes. Abnormal axonal mitochondria may contribute to neurogenic atrophy of muscle, a secondary chronic feature. Quantitative determinations of respiratory chain enzyme complexes, with citrate synthase as an internal control, confirm the histochemical impressions or may be the only evidence of mitochondrial disease. Biological and technical artifacts may yield falsely low enzymatic activities. Genetic studies screen common point mutations in mtDNA. The brain exhibits characteristic histopathological alterations in mitochondrial diseases. Skin biopsy is useful for mitochondrial ultrastructure in smooth erector pili muscles and axons; skin fibroblasts may be grown in culture. Mitochondrial alterations occur in many nonmitochondrial diseases and also may be induced by drugs and toxins.

Axons↗

PubMed1997

ADP-regulation of mitochondrial free radical production is different with complex I- or complex II-linked substrates: implications for the exercise paradox and brain hypermetabolism.

In agreement with classic studies, succinate-supplemented rat and pigeon heart and nonsynaptic brain mitochondrial free radical production is stopped by ADP additions causing the stimulation of respiration from State 4 to State 3. Nevertheless, with Complex I-linked substrates, mitochondria produce free radicals in State 3 at rates similar or somewhat higher than during resting respiration. The absence of sharp increases in free radical production during intense respiration is possible due to strong decreases of free radical leak in State 3. The results indicate that Complex I is the main mitochondrial free radical generator in State 3, adding to its already known important generation of active oxygen species in State 4. The observed rate of mitochondrial free radical production with Complex I-linked substrates in the active State 3 can help to explain two paradoxes: (a) the lack of massive muscle oxidative damage and shortening of life span due to exercise, in spite of up to 23-fold increases of oxygen consumption together with the very low levels of antioxidants present in heart, skeletal muscle, and brain; (b) the presence of some degree of oxidative stress during exercise and hyperactivity in spite of the stop of mitochondrial free radical production by ADP with succinate as substrate.

Adenosine Diphosphate↗

PubMed1999

Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli.

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.

Anilides↗