ubiquinone and 2-3-dimethoxy-5-methyl-6-decyl-1-4-benzoquinone

The coenzyme Q (CoQ) concentration in the inner membrane of beef heart mitochondria is not kinetically saturating for NADH oxidation inasmuch as the K(m) of NADH oxidation for endogenous CoQ10 is in the mM range in membrane lipids. Using CoQ1 as an electron acceptor from complex I, we have found additional evidence that the high Km of NADH oxidase for CoQ is not an artifact due to the use of organic solvents in reconstitution studies. We have also obtained experimental evidence that CoQ concentration may be rendered more rate-limiting for NADH oxidation either by a decrease of CoQ content (as in liver regeneration or under an acute oxidative stress), or by a possible increase of the Km for CoQ, as in some mitochondrial diseases and ageing. The possibility of enhancing the rate of NADH oxidation by CoQ therapy is hindered by the fact that the CoQ concentration in mitochondria appears to be regulated by its mixability with the membrane phospholipids. Nevertheless CoQ10 incorporated into heart submitochondrial particles by sonication enhances NADH oxidation (but not succinate oxidation) up to twofold. Nontoxic CoQ homologs and analogs having shorter side-chains with respect to CoQ10 can be incorporated in the mitochondrial membrane without sonication, supporting an enhancement of NADH oxidation rate above 'physiological' values. It is worth investigating whether this approach can have a therapeutical value in vivo in mitochondrial bioenergetic disorders.

Topics: Aging; Animals; Cattle; Coenzymes; Electron Transport Complex I; Electron Transport Complex II; Heart Failure; Intracellular Membranes; Kinetics; Lipid Bilayers; Liver Regeneration; Membrane Lipids; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Succinate Dehydrogenase; Ubiquinone

Mitochondrial permeability transition pore (mPTP) closure triggers cardiomyocyte differentiation during development while pathological opening causes cell death during myocardial ischemia-reperfusion and heart failure. Ubiquinone modulates the mPTP; however, little is known about its mechanistic role in health and disease. We previously found excessive proton leak in newborn Fmr1 KO mouse forebrain caused by ubiquinone deficiency and increased open mPTP probability. Because of the physiological differences between the heart and brain during maturation, we hypothesized that developing Fmr1 KO cardiomyocyte mitochondria would demonstrate dissimilar features.. Newborn male Fmr1 KO mice and controls were assessed. Respiratory chain enzyme activity, ubiquinone content, proton leak, and oxygen consumption were measured in cardiomyocyte mitochondria. Cardiac function was evaluated via echocardiography.. In contrast to controls, Fmr1 KO cardiomyocyte mitochondria demonstrated increased ubiquinone content and decreased proton leak. Leak was cyclosporine (CsA)-sensitive in controls and CsA-insensitive in Fmr1 KOs. There was no difference in absolute mitochondrial respiration or cardiac function between strains.. These findings establish the newborn Fmr1 KO mouse as a novel model of excess ubiquinone and closed mPTP in the developing heart. Such a model may help provide insight into the biology of cardiac development and pathophysiology of neonatal heart failure.. Ubiquinone is in excess and the mPTP is closed in the developing FXS heart. Strengthens evidence of open mPTP probability in the normally developing postnatal murine heart and provides new evidence for premature closure of the mPTP in Fmr1 mutants. Establishes a novel model of excess CoQ and a closed pore in the developing heart. Such a model will be a valuable tool used to better understand the role of ubiquinone and the mPTP in the neonatal heart in health and disease.

Topics: Animals; Atractyloside; Cyclosporine; Disease Models, Animal; Electron Transport; Fetal Heart; Fragile X Mental Retardation Protein; Fragile X Syndrome; Guanosine Diphosphate; Male; Mice; Mice, Knockout; Mitochondria, Heart; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Oxygen Consumption; Proton-Motive Force; Single-Blind Method; Ubiquinone

Proguanil in combination with its synergistic partner atovaquone has been used for malaria treatment and prophylaxis for decades. However its mode of action is not fully understood. Here we used yeast to investigate its activity. Proguanil inhibits yeast growth, causes cell death and acts in synergy with atovaquone. It was previously proposed that the drug would target the system that maintains the mitochondrial membrane potential when the respiratory chain is inhibited. However our data did not seem to validate that hypothesis. We proposed that proguanil would not have a specific target but accumulate in the mitochondrial to concentrations that impair multiple mitochondrial functions leading to cell death. Selection and study of proguanil resistant mutants pointed towards an unexpected resistance mechanism: the decrease of CoQ level, which possibly alters the mitochondrial membrane properties and lowers proguanil intramitochondrial level.

Topics: Antimalarials; Atovaquone; Drug Resistance, Fungal; Drug Synergism; Drug Therapy, Combination; Membrane Potential, Mitochondrial; Mutation; Oxygen; Proguanil; Pyrimidines; Strobilurins; Ubiquinone; Vitamin K 3; Yeasts

Topics: Animals; Breast Neoplasms; Chick Embryo; Female; Humans; MCF-7 Cells; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53; Ubiquinone

In the present work we studied action of several inhibitors of respiratory complex II (CII) of mitochondrial electron transport chain, namely malonate and thenoyltrifluoroacetone (TTFA) on Cd

Topics: Animals; Apoptosis; Cadmium; Cell Line, Tumor; Cell Survival; Electron Transport Complex II; Malonates; Mitochondria; PC12 Cells; Rats; Thenoyltrifluoroacetone; Ubiquinone

Here we present a simple in vivo microtiter plate assay using lead acetate [Pb(OAc)2]-soaked filter paper to detect H2S released by Escherichia coli metabolizing cysteine. The released H2S precipitates as brown lead sulfide (PbS) on Pb(OAc)2 soaked filter paper. The PbS stain quantitated by ImageJ software is proportional to the amount of H2S released from the culture. Expression of recombinant Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) converts the H2S to sulfur, resulting in less PbS formation. The in vivo H2S oxidation activity of SQR was calculated based on the density of the PbS stain formed by E. coli expressing SQR compared with cells harboring the empty vector pLM1. The results are consistent with the in vitro activity of SQR measured by decylubiquinone (DUQ) reduction. This assay can be applied to sulfide metabolizing enzymatic studies, mutant screening and high-throughput inhibitor screens.

Topics: Biological Assay; Blotting, Western; Calibration; Chemical Precipitation; Escherichia coli; Hydrogen Sulfide; Lead; Sulfides; Ubiquinone

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.

Topics: Cell Proliferation; Cytochromes c; Gene Expression Regulation, Bacterial; Hydrostatic Pressure; Oxidoreductases; Shewanella; Transcription, Genetic; Ubiquinone

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

Topics: Amino Acid Sequence; Antiporters; Catalysis; Electron Transport Complex II; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Isoenzymes; Metabolomics; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Molecular Sequence Data; Phenotype; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Succinate Dehydrogenase; Ubiquinone

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.

Topics: Antimalarials; Atovaquone; Cells, Cultured; Dihydroorotate Dehydrogenase; Drug Resistance; Electron Transport; Fungal Proteins; Humans; Mitochondria; Organisms, Genetically Modified; Oxidoreductases Acting on CH-CH Group Donors; Parasitemia; Plasmodium falciparum; Recombinant Proteins; Ubiquinone

The effects of decylubiquinone, a ubiquinone analogue, on mitochondrial function and inhibition thresholds of the electron transport chain enzyme complexes in synaptosomes were investigated. Decylubiquinone increased complex I/III and complex II/III activities by 64 and 80%, respectively, and attenuated reductions in oxygen consumption at high concentrations of the complex III inhibitor myxothiazol. During inhibition of complex I, decylubiquinone attenuated reductions in synaptosomal oxygen respiration rates, as seen in the complex I inhibition threshold. Decylubiquinone increased the inhibition thresholds of complex I/III, complex II/III, and complex III over oxygen consumption in the nerve terminal by 25-50%, when myxothiazol was used to inhibit complex III. These results imply that decylubiquinone increases mitochondrial function in the nerve terminal during complex I or III inhibition. The potential benefits of decylubiquinone in diseases where complex I, I/III, II/III, or III activities are deficient are discussed.

Topics: Animals; Antimycin A; Electron Transport; Female; Methacrylates; Mitochondria; Models, Biological; Neurodegenerative Diseases; Oxygen Consumption; Rats; Rats, Wistar; Rotenone; Synaptosomes; Thiazoles; Ubiquinone; Uncoupling Agents

Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I). Majority of investigators agree that superoxide is generated at the flavin site. Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ), like a "tug-of-war" through electrons. All preparations of bovine heart complex I, which have been isolated at Yoshikawa's laboratory, have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al., Biochemistry, 49 (2010) 487-492). Using these preparations, we measured (i) EPR signals of the SF, the SQ and iron-sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method. Our experimental evidence was (1) without added decylubiquinone (DBQ), no catalytic oxidation of NADH occurs. The NADH addition produced mostly SF and it generated superoxide as reported by Kussmaul and Hirst (PNAS, 103 (2006) 7607-7612). (2) During catalytic electron transfer from NADH to DBQ, the superoxide generation site was mostly shifted to the SQ. (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ, and it enhances the formation of SF and increases the overall superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased.

Topics: Animals; Benzoquinones; Binding Sites; Biocatalysis; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex I; Flavins; Free Radicals; Hydrogen Peroxide; Mitochondria, Heart; Myocardium; NAD; Oxidation-Reduction; Pyridines; Quinones; Rotenone; Superoxides; Ubiquinone; Uncoupling Agents

Prolonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet.. The effects of ubiquinone 0 (Ub(0)), ubiquinone 5 (Ub(5)), ubiquinone 10 (Ub(10)) and decyl-ubiquinone (DUb) were studied in freshly isolated rat hepatocytes, cultured rat liver Clone-9 cells and cancerous rat liver MH1C1 cells. PTP regulation by ubiquinones differed significantly in permeabilized Clone-9 and MH1C1 cells from that previously reported in liver mitochondria. Ub(0) inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub(5) did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub(10) regulated PTP in isolated hepatocytes, whereas it did not affect PTP opening in Clone-9 and MH1C1 cells. Only DUb displayed the same effect on PTP regulation in the three hepatocyte lines tested. Despite such modifications in PTP regulation, competition between ubiquinones still occurred in Clone-9 and MH1C1 cells. As expected, Ub(5) induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub(0) induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism.. We found that various ubiquinone analogs regulate PTP in different ways depending on the cell studied. We took advantage of this unique property to develop a PTP opening-targeted strategy that leads to cell death specifically in cells where the ubiquinone analog used induces PTP opening, while sparing the cells in which it does not induce PTP opening.

Topics: Animals; Benzoquinones; Calcium; Cell Death; Cell Line; Cell Line, Tumor; Cell Survival; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Rats; Reactive Oxygen Species; Ubiquinone

Here we report effect of ischemia-reperfusion on mitochondrial Ca2+ uptake and activity of complexes I and IV in rat hippocampus. By performing 4-vessel occlusion model of global brain ischemia, we observed that 15 min ischemia led to significant decrease of mitochondrial capacity to accumulate Ca2+ to 80.8% of control whereas rate of Ca2+ uptake was not significantly changed. Reperfusion did not significantly change mitochondrial Ca2+ transport. Ischemia induced progressive inhibition of complex I, affecting final electron transfer to decylubiquinone. Minimal activity of complex I was observed 24 h after ischemia (63% of control). Inhibition of complex IV activity to 80.6% of control was observed 1 h after ischemia. To explain the discrepancy between impact of ischemia on rate of Ca2+ uptake and activities of both complexes, we performed titration experiments to study relationship between inhibition of particular complex and generation of mitochondrial transmembrane potential (DeltaPsi(m)). Generation of a threshold curves showed that complex I and IV activities must be decreased by approximately 40, and 60%, respectively, before significant decline in DeltaPsi(m) was documented. Thus, mitochondrial Ca2+ uptake was not significantly affected by ischemia-reperfusion, apparently due to excess capacity of the complexes I and IV. Inhibition of complex I is favourable of reactive oxygen species (ROS) generation. Maximal oxidative modification of membrane proteins was documented 1 h after ischemia. Although enhanced formation of ROS might contribute to neuronal injury, depressed activities of complex I and IV together with unaltered rate of Ca2+ uptake are conditions favourable of initiation of other cell degenerative pathways like opening of mitochondrial permeability transition pore or apoptosis initiation, and might represent important mechanism of ischemic damage to neurones.

Topics: Adaptor Protein Complex 1; Adaptor Protein Complex 4; Animals; Azides; Brain Ischemia; Calcium; Ferricyanides; Hippocampus; Male; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Diseases; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Rotenone; Spectrometry, Fluorescence; Ubiquinone; Uncoupling Agents

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations. Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K(m) value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.

Topics: Amino Acid Sequence; Amino Acid Substitution; Escherichia coli; Escherichia coli Proteins; Humans; Kinetics; Malates; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation, Missense; NADH Dehydrogenase; Protein Structure, Quaternary; Proton Pumps; Ruthenium Compounds; Ubiquinone

Coenzyme Q10 (which is also designated as CoQ10, ubiquinone-10, UQ10, CoQ, UQ or simply as Q) plays an important role in energy metabolism. For NADH-Q oxidoreductase (complex I), Ohnishi and Salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a Q-binding protein, and that the bound form of semiquinone (SQ) serves as its gate [FEBS Letters 579 (2005) 45-55]. This was based on the following experimental results: (i) EPR signals of the fast-relaxing SQ anion (designated as QNf(.-)) are observable only in the presence of the proton electrochemical potential (DeltamuH+); (ii) iron-sulfur cluster N2 and QNf(.-) are directly spin-coupled; and (iii) their center-to-center distance was calculated as 12angstroms, but QNf(.-) is only 5angstroms deeper than N2 perpendicularly to the membrane. After the priming reduction of Q to QNf(.-), the proton pump operates only in the steps between the semiquinone anion (QNf(.-)) and fully reduced quinone (QH2). Thus, by cycling twice for one NADH molecule, the pump transports 4H+ per 2e(-). This hypothesis predicts the following phenomena: (a) Coupled with the piericidin A sensitive NADH-DBQ or Q1 reductase reaction, DeltamuH+ would be established; (b) DeltamuH+ would enhance the SQ EPR signals; and (c) the dissipation of DeltamuH+ with the addition of an uncoupler would increase the rate of NADH oxidation and decrease the SQ signals. We reconstituted bovine heart complex I, which was prepared at Yoshikawa's laboratory, into proteoliposomes. Using this system, we succeeded in demonstrating that all of these phenomena actually took place. We believe that these results strongly support our hypothesis.

Topics: Animals; Benzoquinones; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cattle; Electron Transport Complex I; Membrane Potentials; Mitochondria, Heart; Models, Molecular; Oxidation-Reduction; Protein Conformation; Proteolipids; Proton Pumps; Ubiquinone; Uncoupling Agents

Mitochondrial reactive oxygen species (ROS) are mainly produced by the respiratory chain enzymes. The sites for ROS production in mitochondrial respiratory chain are normally ascribed to the activity of Complex I and III. The presence of specific inhibitors modulates reactive oxygen species production in Complex I: inhibitors such as rotenone induce a strong ROS increase, while inhibitors such as stigmatellin prevent it. We have investigated the effect of hydrophilic quinones on Complex I ROS production in presence of different inhibitors. Some short chain quinones are Complex I inhibitors (CoQ2, idebenone and its derivatives), while CoQ1, decylubiquinone~ (DB) and duroquinone (DQ) are good electron acceptors from Complex I. Our results show that the ability of short chain quinones to induce an oxidative stress depends on the site of interaction with Complex I and on their physical-chemical characteristics. We can conclude that hydrophilic quinones may enhance oxidative stress by interaction with the electron escape sites on Complex I while more hydrophobic quinones can be reduced only at the physiological quinone reducing site without reacting with molecular oxygen.

Topics: Animals; Antimycin A; Cattle; Electron Transport Complex I; Models, Biological; Oxidative Stress; Reactive Oxygen Species; Rotenone; Submitochondrial Particles; Ubiquinone

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.

Topics: Amino Acid Sequence; Binding Sites; Cell Respiration; Conserved Sequence; Cytochromes b; Cytochromes c1; Electron Transport; Glutamic Acid; Hydrogen-Ion Concentration; Hydroquinones; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes; Mutagenesis, Site-Directed; Mutant Proteins; Oxidation-Reduction; Protein Binding; Protons; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Ubiquinone

We have studied the interaction of decylubiquinone, an effective substrate for respiratory chain complexes III and II, with complex I in mouse and human tissues. We found that its reduced form, decylubiquinol, severely impedes complex I activity, while the oxidized form, decylubiquinone acts as a potent acceptor for complex I electrons. This observation has obvious incidence on the assay conditions for complex I. In keeping with that, we found that the inhibition by the reduced form can be avoided by maintaining decylubiquinone under an oxidized form. Under these experimental conditions, a high complex I activity could be measured allowing to detect partial complex I deficiency. Use of these conditions is however restricted to tissues/cells with limited contaminating NADH dehydrogenase activities that are prone to react with redox active compounds.

Topics: 2,6-Dichloroindophenol; Animals; Cell Extracts; Electron Transport Complex I; Fibroblasts; Humans; Male; Mice; Mice, Inbred C57BL; Mitochondria, Heart; Multienzyme Complexes; Myocardium; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Rotenone; Superoxides; Tissue Distribution; Ubiquinone; Uncoupling Agents

Decylubiquinone treatment in vitro has demonstrated a potent inhibitor effect on reactive oxidative species production. However, the effectin vivo has not been demonstrated yet. Thus, rats SHRSP male were divided in two groups: treated and controls (n=6, each). The treated group received 10 mg/Kg(-)/body weight of decylubiquinone diluted in coconut oil by oral gavage during four weeks. Control rats just received the vehicle. Body weight, diuresis, food and water intake, systolic blood pressure, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, blood glucose levels and malondialdehyde were determined. There were a significant (p<0.05) reduction on systolic blood pressure, plasma malondialdehyde, total cholesterol and LDL-cholesterol in the treated group. Additionally, HDL-cholesterol also increased significantly. However, body weight, diuresis, food and water intake, blood glucose levels and triglycerides did not alter after treatment. Thus, decylubiquinone can be a new antihypertensive, hypolipidemic and antioxidant agent on the prevention and treatment of diseases linked to oxidative stress.

Topics: Animals; Antihypertensive Agents; Antioxidants; Blood Glucose; Blood Pressure; Disease Models, Animal; Hypolipidemic Agents; Lipids; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Inbred SHR; Ubiquinone

Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC50 = 12, 11, and 13 microm, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.

Topics: Antitubercular Agents; Binding, Competitive; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Kinetics; Mycobacterium tuberculosis; NAD; Phenothiazines; Quinone Reductases; Quinones; Ubiquinone

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Butylene Glycols; Cell Membrane; Chloride Channels; Cross-Linking Reagents; Cysteine; Cytoplasm; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fish Proteins; Hydrogen-Ion Concentration; Immunoblotting; Immunoprecipitation; Ligands; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Pepsin A; Phosphoproteins; Phosphorylation; Potassium; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Sodium; Sodium-Potassium-Exchanging ATPase; Squalus acanthias; Sulfhydryl Compounds; Ubiquinone

Since synthetic analogs of 1,4-anthraquinone (AQ code number), such as AQ8, AQ9 and AQ10, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these anti-tumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential (/Deltapsim) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various AQ analogs to induce the /Deltapsim in wild-type and MDR HL-60 cells are rapid (within 2.5-10 min), irreversible after drug removal, concentration dependent in the 0.256-10 micromol/l range and generally related to their anti-tumor activities in vitro. The /Deltapsim caused by AQ9 and AQ10, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activations of these apical caspases upstream of mitochondria are not involved in this process. Antitumor AQ analogs (0.256-10 micromol/l) also mimic the abilities of the known depolarizing agents CCCP, alamethicin, gramicidin A and 100 micromol/l CaCl2 to directly induce within 15 min the /Deltapsim in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 micromol/l Ca2+, which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of AQ9 in isolated mitochondria suggests that anti-tumor AQ analogs might interact with the PTP to alter its conformation and increase its Ca2+ sensitivity. Indeed, such Ca2+-dependent /Deltapsim of isolated mitochondria treated with 1.6 micromol/l AQ9 or 100 micromol/l Ca2+ are blocked by ruthenium red. Daunorubicin (DAU) is unable to mimic the rapid /Deltapsim caused by anti-tumor AQ analogs within 2.5-40 min of treatment in HL-60 cells or isolated mitochondria. Moreover, the /Deltapsim caused by 1.6 micromol/l AQ9 or 100 micromol/l Ca2+ in isolated mitochondria are similarly blocked by cyclosporin A (CsA), bongkrekic acid and decylubiquinone, which prevent PTP opening, suggesting that, in contrast to DAU, anti-tumor AQ analogs that directly target mitochondria to trigger the Ca2+-dependent and CsA-sensitive /Deltapsim, might induce PTP opening and the mitoch

Topics: Alamethicin; Animals; Anthraquinones; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bongkrekic Acid; Calcium Chloride; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Cysteine Proteinase Inhibitors; Daunorubicin; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Female; Gramicidin; HL-60 Cells; Humans; Intracellular Membranes; Ion Channels; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitoxantrone; Ruthenium Red; Staurosporine; Ubiquinone

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six "human" mutations, namely G33S, S152P, G252D, Y279C, G291D, and Delta252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and Delta252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc(1) complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc(1) complex. Y279C caused a significant decrease of the bc(1) function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Q(o) site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Q(o) site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.

Topics: Aspartic Acid; Binding Sites; Blotting, Western; Cytochromes b; Cytochromes c; Cytochromes c1; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Fungal Proteins; Genetic Diseases, Inborn; Humans; Immunoblotting; Intracellular Membranes; Iron-Sulfur Proteins; Kinetics; Lipids; Magnetics; Mitochondria; Models, Molecular; Mutation; Saccharomyces cerevisiae Proteins; Spectrophotometry; Suppression, Genetic; Temperature; Ubiquinone

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.

Topics: Antimycin A; Cytochromes b; Cytochromes c; Dimerization; Electron Transport Complex III; Fungal Proteins; Heme; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Mutation; Oxidation-Reduction; Oxygen; Spectrophotometry; Time Factors; Ubiquinone; Ultraviolet Rays

The fragment of tau containing the first and third tubulin-binding motifs, involved in self-assembly of tau, was phosphorylated by protein kinase A (PKA). In the presence of hydroxynonenal (HNE) or in the presence of quinones such as juglone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q(0) or DMM), or menadione, the polymerization of this phosphorylated tau fragment is catalyzed, whereas polymerization of the unmodified fragment takes place in a lesser extent. The quinones coenzyme Q(0) and menadione are found in every cell, including neural cells, and may interact with tau protein to facilitate its assembly into filamentous structures. These tau filaments, assembled in the presence of quinones, have a fibrillar morphology very similar to that of paired helical filaments present in the brains of patients with Alzheimer's disease.

Topics: Amino Acid Motifs; Amino Acid Sequence; Benzoquinones; Cyclic AMP-Dependent Protein Kinases; Humans; Molecular Sequence Data; Naphthoquinones; Neurofibrillary Tangles; Peptide Fragments; Phosphorylation; Polymers; Protein Binding; Protein Processing, Post-Translational; Quinones; Recombinant Proteins; tau Proteins; Tubulin; Ubiquinone; Vitamin K 1; Vitamin K 3

Quinone derivatives are among the rare compounds successfully used as therapeutic reagents to fight mitochondrial diseases. However, their beneficial effect appears to depend on their side chain which presumably governs their interaction with the respiratory chain. The effect of four quinone derivatives was comparatively studied on NADH- and succinate-competitive oxidation by a sub-mitochondrial fraction. Under our experimental conditions, the less hydrophobic derivatives (menadione, duroquinone) poorly affected electron flow from either NADH or succinate to oxygen, yet readily diverting electrons from isolated complex I. This latter effect was abolished by succinate addition. More hydrophobic derivatives (idebenone, decylubiquinone) stimulated oxygen uptake from succinate. But while NADH oxidation was slightly inhibited by idebenone, it was somewhat increased by decylubiquinone. As a result, idebenone strongly favoured succinate over NADH oxidation. This study therefore suggests that any therapeutic use of quinone analogues should take into account their specific effect on each respiratory chain dehydrogenase.

Topics: Animals; Benzoquinones; Binding, Competitive; Cell Respiration; Cells, Cultured; Dose-Response Relationship, Drug; Homeostasis; Mice; Mice, Inbred C57BL; Mitochondria, Liver; NAD; Oxidation-Reduction; Oxygen; Oxygen Consumption; Quinones; Substrate Specificity; Ubiquinone; Vitamin K 3

The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Cell Membrane; Codon, Initiator; Electron Transport Complex I; Escherichia coli; Escherichia coli Proteins; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; NADH, NADPH Oxidoreductases; Plasmids; Protein Structure, Secondary; Protein Subunits; Recombinant Proteins; Ubiquinone

Friedreich Ataxia (FRDA), the most common inherited ataxia, arises from defective expression of the mitochondrial protein frataxin, which leads to increased mitochondrial oxidative damage. Therefore, antioxidants targeted to mitochondria should be particularly effective at slowing disease progression. To test this hypothesis, we compared the efficacy of mitochondria-targeted and untargeted antioxidants derived from coenzyme Q10 and from vitamin E at preventing cell death due to endogenous oxidative stress in cultured fibroblasts from FRDA patients in which glutathione synthesis was blocked. The mitochondria-targeted antioxidant MitoQ was several hundredfold more potent than the untargeted analog idebenone. The mitochondria-targeted antioxidant MitoVit E was 350-fold more potent than the water soluble analog Trolox. This is the first demonstration that mitochondria-targeted antioxidants prevent cell death that arises in response to endogenous oxidative damage. Targeted antioxidants may have therapeutic potential in FRDA and in other disorders involving mitochondrial oxidative damage.

Topics: Antioxidants; Benzoquinones; Cell Death; Drug Delivery Systems; Fibroblasts; Friedreich Ataxia; Humans; Mitochondria; Models, Biological; Organophosphorus Compounds; Oxidative Stress; Ubiquinone; Vitamin E

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.

Topics: Electron Transport Complex III; Enzyme Inhibitors; Glutathione; HL-60 Cells; Humans; Oxidation-Reduction; Polyenes; Reactive Oxygen Species; Ubiquinone

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ-DQH2-NAD+. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH2 formation in the active site and releases of DQH2 and NAD+ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc1 complex. Physiological significances of these results are discussed.

Topics: Animals; Cattle; Electron Transport Complex III; Electron Transport Complex IV; Hydrogen-Ion Concentration; Kinetics; Myocardium; NADH Dehydrogenase; Oxidation-Reduction; Protein Binding; Substrate Specificity; Ubiquinone

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.

Topics: Androstadienes; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line; Cell Survival; Cells, Cultured; Chloride Channels; Cycloheximide; Cytochrome c Group; Cytosol; Diuretics; DNA Damage; Enzyme Inhibitors; Etoposide; Fibroblasts; Furosemide; Mice; Mitochondria; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Protein Synthesis Inhibitors; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Time Factors; Transfection; Tumor Suppressor Protein p53; Ubiquinone; Up-Regulation; Wortmannin

The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.

Topics: Bacterial Proteins; Centrifugation, Density Gradient; Chromatography, Ion Exchange; Electron Transport Complex I; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli; Kinetics; NADH Dehydrogenase; NADH, NADPH Oxidoreductases; Osmolar Concentration; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ubiquinone

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.

Topics: Amino Acid Chloromethyl Ketones; Amino Acids, Aromatic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carrier Proteins; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fas Ligand Protein; fas Receptor; Humans; Ligands; Melanoma; Membrane Glycoproteins; Mitochondria; Models, Biological; Phenylalanine; Time Factors; Tumor Cells, Cultured; Tyrosine; Ubiquinone

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.

Topics: 1-Methyl-4-phenylpyridinium; Binding Sites; Electron Transport Complex I; Enzyme Inhibitors; Enzyme Stability; Hot Temperature; Molecular Structure; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Photoaffinity Labels; Polyenes; Pyridazines; Rotenone; Structure-Activity Relationship; Tritium; Ubiquinone

An electrometric technique was used to investigate the effect of coenzyme Q(10) (UQ), substitution by decylubiquinone (dQ) at the Q(B) binding site of reaction centers (UQ-RC and dQ-RC, respectively) on the electrogenic proton transfer kinetics upon Q(B) reduction in Rhodobacter sphaeroides chromatophores. Unlike dQ-RC, the kinetics of the second flash-induced proton uptake in UQ-RC clearly deviated from the mono-exponential one. The activation energy (about 30 kJ/mol) and the pH profile of the kinetics in dQ-RC were similar to those in UQ-RC, with the power law approximation used in the latter case. The interpretation of the data presumed the quinone translocation between the two binding positions within the Q(B) site. It is proposed that the native isoprenyl side chain (in contrast to decyl chain) favors the equilibrium binding of neutral quinone at the redox-active 'proximal' position, but causes a higher barrier for the hydroquinone movement from 'proximal' to 'distal' position.

Topics: Bacterial Chromatophores; Binding Sites; Coenzymes; Hydrogen-Ion Concentration; Kinetics; Protons; Rhodobacter sphaeroides; Temperature; Thermodynamics; Ubiquinone

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Topics: Amino Acid Substitution; Antimycin A; Blotting, Western; Cytochrome b Group; DNA Mutational Analysis; DNA, Mitochondrial; Electron Transport Complex III; Gene Frequency; Genetic Variation; Haplotypes; Humans; Methacrylates; Mitochondrial Myopathies; Mutation; Point Mutation; Thiazoles; Ubiquinone

The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 micromol (mg protein)(-1) min(-1) at 20 degrees C. Rates of 220 nmol (mg protein)(-1) min(-1)] for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)(-1) min(-1) for the oxidation of sulfide at 20 C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3+/-0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.

Topics: Amino Acid Sequence; Bacteria; Cell Membrane; Electron Transport Complex III; Molecular Sequence Data; Oxidation-Reduction; Oxygen Consumption; Quinone Reductases; Sequence Analysis, DNA; Sulfides; Temperature; Ubiquinone

Ubiquinone 0 and decylubiquinone have been reported to inhibit the mitochondrial permeability transition pore (PTP) [Fontaine, E., Ichas, F. and Bernardi, P. (1998) J. Biol. Chem. 273, 25734-257401, offering a new clue to its molecular composition. In patch-clamp experiments on rat liver mitochondria we have observed that these compounds also inhibit the previously described mitochondrial megachannel (MMC), confirming its identification as the PTP. Inhibition can be reversed by increasing [Ca2+], in analogy to the behavior observed with several other disparate PTP/MMC inhibitors. To rationalize the ability of Ca2+ to overcome inhibition by various quite different compounds we propose that it acts via the phospholipid bilayer.

Topics: Animals; Benzoquinones; Calcium; Cations, Divalent; Ion Channels; Membrane Proteins; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Rats; Ubiquinone

The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.

Topics: Capsicum; Carotenoids; Escherichia coli; Isomerism; Kinetics; Lycopene; Oxidoreductases; Plants, Medicinal; Plastoquinone; Recombinant Proteins; Ubiquinone

We have investigated the regulation of the mitochondrial permeability transition pore (PTP) by ubiquinone analogues. We found that the Ca2+-dependent PTP opening was inhibited by ubiquinone 0 and decylubiquinone, whereas all other tested quinones (ubiquinone 5, 1,4-benzoquinone, 2-methoxy-1,4-benzoquinone, 2,3-dimethoxy-1, 4-benzoquinone, and 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone) were ineffective. Pore inhibition was observed irrespective of the method used to induce the permeability transition (addition of Pi or atractylate, membrane depolarization, or dithiol cross-linking). Inhibition of PTP opening by decylubiquinone was comparable with that exerted by cyclosporin A, whereas ubiquinone 0 was more potent. Ubiquinone 5, which did not inhibit the PTP per se, specifically counteracted the inhibitory effect of ubiquinone 0 or decylubiquinone but not that of cyclosporin A. These findings define a ubiquinone-binding site directly involved in PTP regulation and indicate that different quinone structural features are required for binding and for stabilizing the pore in the closed conformation. At variance from all other quinones tested, decylubiquinone did not inhibit respiration. Our results define a new structural class of pore inhibitors and may open new perspectives for the pharmacological modulation of the PTP in vivo.

Topics: Animals; Arsenicals; Atractyloside; Binding Sites; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cyclosporine; Intracellular Membranes; Ion Channels; Mitochondria, Liver; Molecular Structure; Permeability; Porins; Quinones; Rats; Ubiquinone

Radical anions and cations of the biologically important coenzymes Q-6 and Q-10, which have 6 and 10 unsaturated isoprene units in their side chains, respectively, have been generated in various solvents, and the results compared with those obtained for Q-0, a ubiquinone with no isoprene units, and for decylubiquinone Q-2 which has a saturated side chain. Hyperfine splitting constants (hfsc) of methyl and methoxy protons of the substituents in the quinone ring, and beta and gamma protons of the side chain were measured by EPR and ENDOR spectroscopy for both the radical anions and cations of Q-0, Q-6 and Q-10, and for the radical anion of Q-2. The relative signs of the hfsc were determined by general TRIPLE resonance spectroscopy. TRIPLE induced EPR (TIE) spectra were used for identification of the primary and secondary radicals of Q-10. The temperature dependence of the hfsc of the beta protons of Q-2 was different from those of Q-6 and Q-10. Fully optimised structures of Q-3 and Q-7 were obtained by performing semiempirical PM3 molecular orbital (MO) calculations for both neutral molecules and radical anions, neutral radicals and radical cations. Partial optimisation of the molecules was carried out for the side chain in a planar conformation. The folded conformation always had the minimum energy. Folding was so complete in the Q-7 series that the end of the side chain came into contact with the quinone ring, and small hfsc were detected in the PM3 calculations.

Topics: Butadienes; Electron Spin Resonance Spectroscopy; Free Radicals; Hemiterpenes; Models, Molecular; Molecular Conformation; Molecular Structure; Pentanes; Ubiquinone

We have undertaken a study of the role of coenzyme Q (CoQ) in glycerol-3-phosphate oxidation in mitochondrial membranes from hamster brown adipose tissue, using either quinone homologs, as CoQ1 and CoQ2, or the analogs duroquinone and decylubiquinone as artificial electron acceptors. We have found that the most suitable electron acceptor for glycerol-3-phosphate:CoQ reductase activity in situ in the mitochondrial membrane is the homolog CoQ1 yielding the highest rate of enzyme activity (225 +/- 41 nmol x min(-1) x mg(-1) protein). With all acceptors tested the quinone reduction rates were completely insensitive to Complex III inhibitors, indicating that all acceptors were easily accessible to the quinone-binding site of the dehydrogenase preferentially with respect to the endogenous CoQ pool, in such a way that Complex III was kept in the oxidized state. We have also experimentally investigated the saturation kinetics of endogenous CoQ (1.35 nmol/mg protein of a mixture of 70% CoQ9 and 30% CoQ10) by stepwise pentane extraction of brown adipose tissue mitochondria and found a K(m) of the integrated activity of glycerol-3-phosphate cytochrome c reductase for endogenous CoQ of 0.22 nmol/mg protein, indicating that glycerol-3-phosphate-supported respiration is over 80% of V(max) with respect to the CoQ pool. A similar K(m) of 0.19 nmol CoQ/mg protein was found in glycerol-3-phosphate cytochrome c reductase in cockroach flight muscle mitochondria.

Topics: Adipose Tissue, Brown; Animals; Benzoquinones; Cricetinae; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Glycerolphosphate Dehydrogenase; Kinetics; Mitochondria; NADH Dehydrogenase; Oxidation-Reduction; Ubiquinone

The trypanosome alternative oxidase (TAO) is an attractive target for chemotherapy for the diseases caused by African trypanosomes because there is no equivalent enzyme in mammalian hosts. Many inhibitors of this enzyme have been described, but there have been no data on the mechanism of inhibition. In the present study, reduced 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-CoQ-H2) was used as a substitute for the natural substrate CoQ9-H2 to allow direct measurements of the TAO in crude mitochondrial preparations from Trypanosoma brucei brucei. A Km value of 3.8 microM was obtained for this substrate. The following five compounds that have alkyl side chains from 1 to 4 carbons and belong to three classes of inhibitors showed a competitive inhibition pattern with respect to decyl-CoQ-H: p-methoxybenzhydroxamic acid, p-ethoxybenzhydroxamic acid,p-n-butyloxybenzhydroxamic acid, methyl 3,4-dihydroxybenzoate and N-n-butyl-3,4-dihydroxybenzamide. The following four compounds belonging to the same chemical classes but having alkyl side chains from 10 to 12 carbons showed uncompetitive inhibition patterns: p-n-dodecyloxybenzhydroxamic acid, n-decyl 3,4-dihydroxybenzoate, n-dodecyl 3,4-dihydroxybenzoate, and N-n-decyl-3,4-dihydroxybenzamide. Clearly, the first group of inhibitors compete with CoQ-H2 for the active site of the TAO. We propose that the uncompetitive patterns produced by the second group of inhibitors are due to the greater lipophilicity of these compounds and the resulting change in the interaction of the inhibitors and the membrane containing the TAO, thus affecting the local concentration of the inhibitors at the active site.

Topics: Animals; Antiparasitic Agents; Binding Sites; Enzyme Inhibitors; Female; Kinetics; Mitochondrial Proteins; Oxidoreductases; Plant Proteins; Rats; Rats, Sprague-Dawley; Trypanocidal Agents; Trypanosoma brucei brucei; Ubiquinone

Topics: Bacterial Proteins; Chemical Phenomena; Chemistry, Physical; Electrochemistry; Enzymes, Immobilized; Escherichia coli; Glucosides; Gold; Membranes, Artificial; Micelles; Oxaloacetates; Oxidation-Reduction; Solubility; Succinate Dehydrogenase; Succinates; Succinic Acid; Ubiquinone

The recently detected Rieske iron-sulfur center in the membrane of the thermoacidophilic archaeon Sulfolobus acidocaldarius (Anemüller et al., 1993, FEBS Lett. 318, 61-64) was further characterized by EPR spectroscopy, coupled to redox-potentiometry and functional studies. The reduction potential is pH-dependent above pH 6, revealing the influence of two ionization equilibria in the oxidized form, with pKaox-values of 6.2 and 8.5. Above pH 9, the slope of the curve is--120 mV/pH-unit. A partially purified fraction exerted a ubiquinol-cytochrome c oxidoreductase activity. To our knowledge, for the first time, in a membrane bound Rieske iron-sulfur protein, unequivocal evidence for a two proton dependent redox equilibrium is presented.

Topics: Animals; Ascorbic Acid; Cell Membrane; Cytochrome c Group; Electron Transport Complex III; Horses; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Membrane Proteins; Myocardium; Oxidation-Reduction; Sulfolobus acidocaldarius; Ubiquinone

The coenzyme quinone (CoQ) binding region of mitochondrial NADH:CoQ reductase (complex-I) was investigated by the fluorescent probes erythrosine-5'-iodoacetamide (ER) and 3,3'-diethyloxadicarbocyanine iodide (DODCI). Both steady-state and time-resolved fluorescence was used in these experiments. Both probes competed for the binding site of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), an analogue of CoQ. The fluorescence lifetimes of the complex-I bound probes were approximately 600 ps and approximately 1.7 ns in the cases of ER and DODCI, respectively. Binding of the probes was not affected by the binding of the inhibitor rotenone. However, rotenone binding caused some changes in the lifetime of the bound probes. Reduction of the enzyme caused an increase in the level of binding of ER and a decrease in the level of binding of DODCI. The level of binding of cationic DODCI increased with the increase in pH, and in the case of anionic of ER the trend was reverse. Binding of Ca2+ to complex-I resulted in an increase in the level of binding of ER and a decrease in the level of binding of DODCI. Reaction with N,N'-dicyclohexylcarbodiimide (DCCD) resulted in alterations in the time-resolved fluorescence profiles of dye: complex-I system. All these results were interpreted as due to the presence of carboxyl group(s) with pKa approximately 6 in the probe/CoQ binding region. The rotational correlation time (tau r) of DODCI bound at the CoQ region was 2-3 ns.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acids; Amines; Animals; Binding Sites; Binding, Competitive; Carbocyanines; Cations, Divalent; Cattle; Electron Transport; Electron Transport Complex I; Erythrosine; Fluorescence Polarization; Fluorescent Dyes; Mitochondria, Heart; Models, Chemical; NADH, NADPH Oxidoreductases; Spectrometry, Fluorescence; Ubiquinone

Decyl-ubiquinone and decyl-plastoquinone were used as model compounds to test the potential effect of quinone derivatives on two enzymes of the vitamin K cycle in vitro. Substantial inhibition of gamma-glutamate carboxylase was found, whereas vitamin K-epoxide reductase was inhibited to a much lesser extent. The inhibitory effect of both decylquinones was eliminated in a time-dependent way by solubilized microsomes, but not by purified carboxylase. Since a wide variety of prenylquinones occur as micronutrients, these results are of potential relevance for the effects of natural quinones in the human diet.

Topics: Animals; Carbon-Carbon Ligases; Cattle; Kinetics; Ligases; Liver; Microsomes, Liver; Mixed Function Oxygenases; Plastoquinone; Ubiquinone; Vitamin K; Vitamin K Epoxide Reductases

A microbicidal system, mediated by neutrophil myeloperoxidase, inhibits succinate-dependent respiration in Escherichia coli at rates that correlate with loss of microbial viability. Succinate dehydrogenase, the initial enzyme of the succinate oxidase respiratory pathway, catalyzes the reduction of ubiquinone to ubiquinol, which is reoxidized by terminal oxidase complexes. The steady-state ratio of ubiquinol to total quinone (ubiquinol + ubiquinone) reflects the balance between dehydrogenase-dependent ubiquinone reduction and terminal oxidase-dependent ubiquinol oxidation. Myeloperoxidase had no effect on total quinone content of E. coli but altered the steady-state ratio of ubiquinol to total quinone. The ratio doubled for organisms incubated with the myeloperoxidase system for 10 min, suggesting decreased ubiquinol oxidase activity, which was confirmed by observation of a 50% decrease in oxidation of the ubiquinol analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol. Despite inhibition of ubiquinol oxidase, overall succinate oxidase activity remained unchanged, suggesting that succinate dehydrogenase activity was preserved and that the dehydrogenase was rate limiting. Microbial viability was unaffected by early changes in ubiquinol oxidase activity. Longer (60 min) exposure of E. coli to the myeloperoxidase system resulted in only modest further inhibition of the ubiquinol oxidase, but the ubiquinol to total quinone ratio fell to 0%, reflecting complete loss of succinate dehydrogenase activity. Succinate oxidase activity was abolished, and there was extensive loss of microbial viability. Early myeloperoxidase-mediated injury to ubiquinol oxidase appeared to be compensated for by higher steady-state levels of ubiquinol which sustained electron turnover by mass effect. Later myeloperoxidase-mediated injuries eliminated succinate-dependent ubiquinone reduction, through inhibition of succinate dehydrogenase, with loss of succinate oxidase activity, effects which were associated with, although not clearly causal for, microbicidal activity.

Topics: Animals; Cytochrome b Group; Cytochromes; Dogs; Electron Transport Chain Complex Proteins; Escherichia coli; Escherichia coli Proteins; Kinetics; Models, Theoretical; Neutrophils; Oxidoreductases; Oxygen Consumption; Peroxidase; Succinate Dehydrogenase; Ubiquinone