Mitochondrial Electron Transfer Chain Research Articles

The participation of coenzyme Q in free radical production and antioxidation

Published experimental data pertaining to the participation of coenzyme Q as a site of free radical formation in the mitochondrial electron transfer chain and the conditions required for free radical production have been reviewed critically. The evidence suggests that a component from each of the mitochondrial NADH-coenzyme Q, succinate-coenzyme Q, and coenzyme QH 2-cytochrome c reductases (complexes I, II, and III, most likely a nonheme iron-sulfur protein of each complex, is involved in free radical formation. Although the semiquinone form of coenzyme Q may be formed during electron transport, its unpaired electron most likely serves to aid in the dismutation of superoxide radicals instead of participating in free radical formation. Results of studies with electron transfer chain inhibitors make the conclusion dubious that coenzyme Q is a major free radical generator under normal physiological conditions but may be involved in superoxide radical formation during ischemia and subsequent reperfusion. Experiments at various levels of organization including subcellular systems, intact animals, and human subjects in theclinical setting, support the view that coenzyme Q, mainly in its reduced state, may act as an antioxidant protecting a number of cellular membranes from free radical damage.

Effect of Ca2+-homopantothenate and mild hypoxia on some enzyme activities evaluated in subcellular fractions from different rat brain regions

The effect of Ca2+-homopantothenate (HOPA) treatment (250 mg/kg for 5 d) has been studied by evaluating the specific activity of enzymes related to: glycolytic pathway (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), tricarboxylic acid cycle (citrate synthase, malate dehydrogenase), mitochondrial electron transfer chain (succinate dehydrogenase, cytochrome oxidase), NADH redox state (NADH cytochrome c reductase), acetylcholine metabolism (acetylcholinesterase), and glutamate metabolism (glutamate dehydrogenase). The enzymatic activity assays were performed on homogenate in toto, nonsynaptic mitochondria and synaptosomes isolated from: cerebral cortex, hippocampus, striatum, hypothalamus, medulla oblongata, and cerebellum of normoxic rats and rats submitted to intermittent normobaric hypoxia (90:10, N2:O2). In normoxic rats, HOPA was unable to induce any modification. Hypoxia per se induced a decrease in the activity of synaptosomal cytochrome oxidase in cerebral cortex, hippocampus, and cerebellum.

Photodependent generation of reactive oxygen species by a phytotoxic pyridine derivative

LS 82556 [(α methyl benzyl) carbamoyl 5 propionyl -2,6 lutidine] is an experimental herbicide discovered by Rhōne-Poulenc. This compound induces symptoms quite similar to those of diphenyl ethers (1,2) and is active only in the light (3,4). It induces photodependent pigment bleaching and rapidly destroys the aerial parts of higher plants (1,5,6). Its action seems associated to insatured fatty acid peroxidation leading to membrane disruption (1,5,7). Several authors have found some effects on the redox photosynthetic reaction (8,9) while others suggest a requirement of the mitochondrial electron transfer chain (2,10). However, LS 82556 and DPE are powerful inhibitors of tetrapyrrole biosynthesis, leading to porphyrin accumulation and to at least one part of their phytotoxic effect under light (11,12). From the point of view of photochemistry the phytotoxic effect of this compound could be associated either with the photodependent formation of active oxygen species (10,13) or with the degradation of endogenous antioxidants such as vitamin C or E (5,7,10,14,15,16). The aim of this work was to study the in vitro formation of superoxide and singlet oxygen, by direct irradiation of LS 82556 used as a photosensitizer.

Changes of synaptosomal energy metabolism induced by hypoxia during aging.

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO2 = 17-19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24 degrees C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the "in vivo" hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochrome c and a contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (delta Gox-red) and the phosphorylation state of adenine nucleotide system (delta GATP). The free-energy change (delta delta G) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggregation and diffusion in the mitochondrial electron-transfer chain: role in electron flow and energy transfer

Aggregation and diffusion in the mitochondrial electron-transfer chain: role in electron flow and energy transfer

3] Structure of beef heart cytochrome-c oxidase obtained by combining studies of two-dimensional crystals with biochemical experiments

Publisher Summary This chapter describes the mammalian cytochrome- c oxidase by combining the data of biochemical studies with a low-resolution structure provided by electron microscopy and image reconstruction. Cytochrome- c oxidase is the terminal enzyme in the mitochondrial electron transfer chain, catalyzing the four electron reduction of molecular oxygen and coupling this reaction to the generation of a proton gradient across the mitochondrial inner membrane. As purified under certain conditions, the beef heart enzyme is arranged in 2D crystals, and this allows for detailed structural analysis. 2D crystals of beef heart cytochrome- c oxidase are made by three different procedures that involve (1) purifying the enzyme in Triton X-114 followed by Triton X-100, (2) using deoxycholate alone as the detergent, and (3) using cholate followed by deoxycholate. The electron microscopy done using ice rather than phosphotungstate or uranylacetate as the contrast stain for water against protein, allows higher resolution imaging of the protein and allows direct visualization of a cytochrome c -cytochrome- c oxidase complex.

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex.

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.

Detection of iron-sulfur center-containing subunits of mitochondrial NADH dehydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by high-performance gel permeation chromatography

Soluble NADH dehydrogenase resolved from Complex I of the mitochondrial electron-transfer chain was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate at 4°C, and then the gel was stained for iron with bathophenanthroline disulfonate and thioglycolic acid. The 23,000-dalton subunit was markedly stained, and the 51,000-dalton subunit was also stained, but only slightly. High-performance gel permeation chromatography using an eluant containing 0.1% sodium dodecyl sulfate also demonstrated that these subunits contain an iron-sulfur center: the elution pattern recorded by light absorption at 400 nm gave two peaks corresponding to the positions of the subunits.

Effect of photosynthesis on dark mitochondrial respiration in green cells

Green plant cells can generate ATP in both chloroplasts and mitochondria. Hence the effect of photosynthesis on dark mitochondrial respiration can be considered at a variety of levels. Turnover of ceitric acid cycle dehydrogenases, which is essential for supply of carbon skeletons for amino acid synthesis, seems to be largely unaffected during photosynthesis. The source of carbon for the anaplerotic function of the citric acid cycle in light is however, not known with certainty. NADH generated in these reactions is probably not oxidised via the mitochondrial electron transfer chain coupled to ATP synthesis. However, it may be oxidised by the alternative cyanide-insensitive pathway, exported to the cytosol via the oxaloacetate-malate dicarboxylate shuttle or directly utilised for cytosolic nitrate reduction. Oxidation of succinate via cytochrome oxidase may also be similarly inhibited in light. Whether increase in the cytosolic ATP/ADP ratio in light is responsible for the inhibition of mitochondrial electron transfer to O 2 is not clearly established, because the ATP/ADP ratio is reported to be already quite high in the dark. Effective collaboration between photophosphorylation and oxidative phosphorylation in order to maintain the cytosolic energy charge at a present high level is discussed.

Crystallization of cytochromes in the mitochondrial electron-transfer chain.

Crystallization of cytochromes in the mitochondrial electron-transfer chain.

Crystallization of the middle part of the mitochondrial electron transfer chain: cytochrome bc1-cytochrome c complex.

Complex III (cytochrome bc1 particle; ubiquinol:ferricytochrome c oxidoreductase, EC 1.10.22) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were depleted by washing the particle with detergent while it still was on the affinity column. The particle first was mixed with an excess of cytochrome c in 1.5% cholate (wt/vol); slow removal of the detergent from the mixture was achieved by dialysis. Freezing of the mixture resulted in crystallization of the cytochrome bc1 particle in the form of a 1:2 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. The same crystallization maneuver used in the case of cytochrome oxidase has been demonstrated to be effective in crystallizing the middle part of the mitochondrial electron-transfer chain.

Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild-type and carboxin-resistant mutant strains of Ustilago maydis

A variety of thiophene carboxamide compounds have been synthesized and tested on the succinate dehydrogenase complex (SDC) in mitochondria from a wild-type strain and carboxin-resistant strains of Ustilago maydis (corn smut). The site of action of thiophene carboxamides is identical to that of carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and thenoyltrifluoroacetone, that is, the succinate-ubiquinone reductase (complex II) span in the mitochondrial electron transfer chain. This investigation reveals new molecular structures which are strong inhibitors of wild-type and carboxin-resistant SDCs. The 5-amino analog of the parent anilide, 3-methylthiophene-2-carboxanilide (I), proved to be an especially potent inhibitor of the wild-type SDC (I 50, 0.019 μ M). Analogs of (I) such as 4′-carboethoxy, 4′- nbutyl, 4′-phenyl, and 4′-benzoyl were negatively correlated in activity to the carboxanilide (I) with respect to resistance level. A number of structures showed considerable selectivity for mutated SDCs from both highly and (particularly) moderately carboxin-resistant SDCs of U. maydis, markedly lowering the resistance level, i.e., the degree of resistance. Thus, in addition to the oxathiins, specific structural groups of thiophene carboxamides can also alleviate or reverse the effect of carboxin-selected mutation with reference to inhibition of the SDC. Of important significance was the finding that molecular selectivity for mutated, carboxin-resistant SDCs can be influenced by replacement of an oxathiin by a thiophene heterocyclic ring as well as by the substitutive group on the amide nitrogen, permitting different categories of mutant types and even mutants within a single category to be distinguished from one another. With all the structural combinations available, it appears quite possible, in terms of inhibition, to overcome any type of mutation in a fungal SDC which arises through selection by carboxin or other carboxamide compounds. A reasonable correlation generally exists between inhibition by thiophene carboxamides of the SDC and sporidial growth of wild-type and carboxin-resistant strains of U. maydis. A permeability barrier to 4′-substituted analogs of (I) was encountered in the wild-type strain but not mutant strains. Excellent protectant activity against bean rust ( Uromyces phaseoli) was obtained with the 3′- nhexyl, 3′- nhexyloxy, and 4′-phenoxy analogs of (I).

Crystallization of part of the mitochondrial electron transfer chain: cytochrome c oxidase--cytochrome c complex.

Cytochrome c oxidase (ferrocytochrome c oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were removed by washing the oxidase with detergent on the affinity column; 1 mole of cardiolipin remained per mole of heme a. The oxidase was mixed with excess cytochrome c in 1.5% (wt/vol) cholate to form a complex. Slow removal of the detergent from the mixture by dialysis resulted in crystallization of cytochrome oxidase in the form of a 1:1 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. Increasing the solubility of a hydrophobic membrane protein by combination with hydropholic ligand is demonstrated as a maneuver for crystallizing the membrane protein.

Resolution of complex III of the mitochondrial electron transfer chain into two component complexes.

Complex III can be resolved into an electron transfer complex and an ion transfer complex. The electron transfer complex contains exclusively the oxidation-reduction proteins of complex III, and the ion transfer complex the capability for augmenting cation-induced fluorescence of 8-anilino-1-naphthalenesulfonic acid. This finding has important implications for the molecular mechanism of mitochondrial energy coupling.

Oxathiin carboxamides highly active against carboxin-resistant succinic dehydrogenase complexes from carboxin-selected mutants of Ustilago maydis and Aspergillus nidulans

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and structurally related analogs are powerful inhibitors of succinate oxidation in mitochondria isolated from a variety of sources. The site of action which is, apparently, also that for thenoyltrifluoracetone, is in the complex II (succinate-ubiquinone reductase) region of the mitochondrial electron transfer chain. The succinic dehydrogenase complex (SDC) in mitochondria from carbon-resistant mutant strains of Ustilago maydis and Aspergillus nidulans is resistant to the fungicide in vitro. The current study shows that certain oxathiin carboxamides are selectively active against particular mutated SDCs of U. maydis and A. nidulans. Molecular structures affecting the phenotypic expression of mutation to carboxin resistance in U. maydis do not appear to affect similarly such expression in A. nidulans and vice versa. Of particular interest was the discovery of oxathiin carboxamides, e.g., 4′-phenylcarboxin, which were more inhibitory to the enzyme complex from one category of carboxin-resistant mutants of U. maydis than from the wild-type strain. Although such negative correlation between carboxin and other carboxin analogs has not been observed in studies with other categories of mutants, structures which drastically lower the resistance level were found in all cases. It appears that for any given mutation affecting carboxin sensitivity of the SDC in fungi, a specific structural group of carboxamides (or even a specific carboxamide) may be found which will alleviate or reverse the effect of the mutation in terms of inhibition of the SDC. If the mutations alter a protein receptor site for carboxamides, such mutations might be expected to influence the binding of carboxins of different structure. In essence, then, different molecular structures can “recognize” different alterations in the mutated enzyme complex and inhibit effectively. With few exceptions, the inhibition by carboxamides of cell growth of wild-type and carboxin-resistant strains of U. maydis and A. nidulans closely paralleded the inhibition of their respective SDCs. Although the few analogs tested were found unable to control corn smut systemically in seedlings artificially inoculated with compatible carboxin-resistant strains, control of naturally occurring carboxin-resistant strains of pathogenic fungi may be possible using particular structural analogs of carboxin which selectively inhibit the mutant organisms.

Respiration of wild type and extrachromosomal mutants of Neurospora crassa.

The specific rates of respiration of cells of wild type and four extrachromosomal mutants of Neurospora crassa were measured throughout the vegetative growth cycle. Two forms of respiration were observed: (i) cyanide sensitive; and (ii) cyanide resistant, salicyl hydroxamate sensitive. These two forms are called terminal and alternate, respectively. The former proceeds by the mitochondrial electron transfer chain and involves the cytochromes; the latter apparently proceeds by the initial portion of the electron transfer chain and does not involve cytochromes. Large and rapid changes of both the terminal and alternate respiratory activities occurred during the vegetative growth cycle. The kinetics of these changes in wild type were compared under some conditions which inhibit protein synthesis and others in which the nitrogen source was varied. The kinetics of the changes of the two forms of respiration of mutants differed from those normally exhibited by wild type, but with varied experimental conditions wild type could be made to resemble the mutants. The results of these studies are discussed in terms of a dynamic model of regulation of mitochondrial biogenesis in the coordination of the synthesis of mitochondrial proteins encoded by nuclear and mitochondrial genomes.

Rôle des lipides dans diverses activités enzymatiques de la chaîne de transport des électrons d'une fraction mitochondriale isolée d'inflorescences de chou-fleur

Evidence is presented that lipids play a role in the mitochondrial electron-transfer chain of Cauliflower inflorescences. Extraction of mitochondrial lipids with aqueous acetone causes an important decrease of NADH—cytochrome c-reductase, NADH-dehydrogenase, succinate—cytochrome c-reductase and succinate-dehydrogenase activities. The activities of the two first complexes can be restored by addition of lipids in a micellar state.

シロサケ親魚の溯河前後における臓器ユビキノン量の比較

Ubiquinone is a natural benzoquinone, which is now recognized as an essential component in the electron transfer chain of mitochondria for many forms of life. In order to get some insight into the biochemical basis underlying the physiological adaptation of the chum salmon during its return to spawn in the river, tissue levels of ubiquinone in the salmon were examined. The dominant ubiquinone in chum salmon was identified as ubiquinone-10 by chromatographic comparison with standard ubiquinone-9 and ubiquinone-10. The concentrations of ubiquinone in the tissue of salmon captured by a trap at a designated sea spot (Otsuchi Bay) were 82.7, 36.2, 27.3 and 12.4 μg per gram of wet tissue in heart, liver, kidney and gills respectively, while the values obtained from the salmon captured in the river (Otsuchi river) were 81.2, 49.9, 28.7 and 18.0 μg respectively. Of the tissues examined, a significant increment in the ubiquinone concentration (ca. 45%) was observed only in the gills of the salmon captured in the river, suggesting the biochemical significance of gills in sea water-fresh water transition of the chum salmon.

The effect of metabolic inhibitors on transferrin and iron uptake and transferrin release from reticulocytes

Metabolic inhibitors were broadly classified into three groups on the basis of their effect on the uptake of [ 125I]transferrin and 59Fe and the release of [ 125I]transferrin by rabbit reticulocytes in vitro: 1. 1. Inhibitors which had no effect on transferrin or iron uptake (sodium malonate and ouabain). 2. 2. Those which blocked iron uptake more than transferrin uptake (retonone, 2,4-dinitrophenol, oligomycin, NaCN, NaN 3, NaF and cycloheximide). The effect of these compounds was attributed to inhibition of the mitochondrial electron transfer chain. 3. 3. Those which inhibited iron and transferrin uptake to approximately the same degree (sodium arsenite, iodoacetamide, N-ethylmaleimide, p-chloromercuribenzoate (PCMB), p-chloromercuribenzenesulfonate (PCMBS) and lead acetate). The onset of inhibition by all the inhibitors studied was immediate, but the effects of iodoacetamide, N-ethylmaleimide, PCMB and PCMBS were largely prevented by prior addition of cysteine in 1–4 times the molar concentration of inhibitor. Sodium arsenite, iodoacetamide and N-ethylmaleimide also inhbited transferrin release. These compounds (all sulfhydryl reagents) may act by altering reticulocyte membrane structure, reducing transferrin and hence iron uptake.

Oxido-Reduction States and Natural Homologue of Ubiquinone (Coenzyme Q) in Submitochondrial Particles From Etiolated Mung Bean (Phaseolus aureus) Seedlings

A procedure for the isolation of submitochondrial particles in quantity from etiolated Mung bean (Phaseolus aureus) seedlings is described. Using a combination of acetone extraction and 2 systems of thin layer chromatography ubiquinone has been isolated. The isolated ubiquinone migrates coincident with authentic ubiquinone-10 in reversed phase thin layer partition chromatography, gives a positive Craven's test, and has oxidized and reduced spectra characteristic of ubiquinone. The quinone is partially reduced under steady-state electron transfer conditions with both succinate and NADH as substrates and is almost completely reduced under anaerobic conditions with either substrate. The concentration of ubiquinone in the particle is of the order of 4.4 mmumoles per mg particle protein, approximately equal to that found in similar submitochondrial particles from beef heart. It is tentatively concluded that ubiquinone-10 is a functional member of the mitochondrial electron transfer chain of Phaseolus aureus.