The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria.

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

The absence of coupling between the formation of membrane potential and electron transfer via the alternative pathway of cyanide-resistant mitochondria

The estimation of membrane potential of cyanide-resistant mitochondria of Candida lipolytica yeast was carried out using positively charged dye phenosafranine. The electron transfer via alternative pathway of cynide resistant mitochondria was shown not to be coupled with the formation of potential on membrane mitochondria.     

Cause of the activity loss of the alternative pathway of electron transport in cyanide-resistant mitochondria of the yeast Candida lipolytica

The cause of the activity loss of alternative pathway of electron transport in mitochondria of the yeast Candida lipolytica has been investigated. Incubation of cyanide-resistant mitochondria at 25 degrees was shown to cause the loss by mitochondria of their ability to oxidize substrates in the presence of 1 mM cyanide. This suggests that in the course of incubation the alternative pathway loses its activity. Repeated washing of mitochondria with a solution containing 2,5 mM EDTA inhibits, while Ca2+, Mn2+, Cu2+ and Zn2+ (but not Sr2+) enhance the process of the activity loss of the alternative pathway. The loss of the cyanide-resistant respiration is also observed during incubation of mitochondria in the presence of phospholipases A, C and D or lysolecithin. In all cases studied the reactivation of the cyanide-resistant respiration of mitochondria is attained by addition of azolectin. The loss of cyanide-resistant respiration is accompanied by the activity reduction of the main respiratory chain, which is restored by addition of cytochrome c and Mg2+. These data indicate that the activity loss of the alternative pathway is not related to inactivation of any components in the alternative pathway itself or in the main respiratory chain. The most probable cause of the activity loss in the destruction of reducing equivalents in the alternative pathway of a donor as a result of a break of the structural entity of the internal membrane of mitochondria due to the detersive action of the phospholipid lysoforms produced either by endogenic or exogenic phospholipases.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.     

Isolation and some properties of mitochondria from yeast Candida lipolytica 695]

Mitochondria is obtained from yeast Candida lipolytica 695 grown in the presence of glucose, lactate or citrate. Yeast mitochondria were shown to be practically indistinguishable from animal tissue mitochondria in [ADP]/[O] values and in their sensitivity to electron transport inhibitors, to inhibitors and uncoupling agents of oxidative phosphorylation. The only exception was more low value of the respiration control under succinate oxidation. Mitochondria from yeast, grown in the presence of lactate or citrate were capable of the reduction of endogenous pyridine nucleotides under succinate oxidation for the expense of the reverse electron transport. No reverse electron transport from succinate to NAD(P) was observed in mitochondria from yeast grown in the presence of glucose, but it was found under oxidation of alpha-glycerophosphate. All three types of yeast mitochondria were not capable of the reverse electron transport coupled with the pyridine nucleotides reduction under lactate oxidation.     

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.     

Effect of quinocitrinines from the fungus Penicillium citrinum on the respiration of yeasts and bacteria

We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.     

水生动物和大鼠线粒体的呼吸链比较

用陆生哺乳动物线粒体呼吸链与水生动物线粒体呼吸链相比较的研究方法,探讨了呼吸链的功能与环境相适应的关系。研究了淡水中生活的草鱼肝丝线粒体,观察到琥珀酸脱氢酶的活性非常低,而ＮＡＤＨ脱氢酶和泛醌细胞色素Ｃ还原酶的活性较高。但海洋生物海绵的线粒体ＮＡＤＨ脱氢酶和琥垢酸脱氢酶的活性都非常低。     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C(5) and C(6)) displayed significantly higher IC(50) values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Properties of mitochondria from cells of the "fermentative" variant of Endomyces magnusii]

The properties of mitochondria from the cells of the "fermentative" variant of End. magnusii were studied. The induced fermentative transformation was brought about by a non-balanced vitamin cultivation. It was shown that the "fermentative" variant of End. magnusii represents an interesting model, in which the energy required for the cell functioning is provided for by a high fermentative activity and a normally functioning respiratory chain. The "fermentative" variant mitochondria were tightly coupled and possessed theoretical efficiency during oxidation of NAD-dependent substrates, which suggested the existence of all the three sites of energy coupling and phosphorylation at the substrate level. A specificity of energy regulation of the End. magnusii "fermentative" variant mitochondria, e. g. tight coupling during oxidation of succinate and lack of tight coupling during oxidation of exogenous NADH, is discussed. The tight coupling during succinate oxidation is confirmed by the observation of reverse electron transfer. Thus, the energy-dependent reduction of NAD during succinate oxidation has been firstly demonstrated for the mitochondria of yeast grown on a fermentable substrate.     

Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain

Antimycin, a specific and highly potent inhibitor of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain, does not inhibit reduction of cytochrome c1 by succinate in isolated succinate-cytochrome c reductase complex under conditions where the respiratory chain complex undergoes one oxidation-reduction turnover. If a slight molar excess of cytochrome c is added to the isolated reductase complex in the presence of antimycin, there is rapid reduction of one equivalent of c type cytochrome by succinate, after which reduction of the remaining c type cytochrome is inhibited. Antimycin fully inhibits succinate-cytochrome c reductase activity of isolated succinate-cytochrome c reductase complex in which the b-c1 complex undergoes multiple turnovers in a catalytic fashion. In addition, when antimycin is added to isolated reductase complex in the presence of cytochrome c plus cytochrome c oxidase, the inhibitor causes a "crossover" in the steady state level of reduction of the cytochromes b and c1 comparable to this classical effect in mitochondria. On the basis of these results, it is suggested that linear schemes of electron transfer are not adequate to account for the site of antimycin inhibition and the mechanism of electron transfer in the cytochrome b-c1 segment of the respiratory chain. The effects of antimycin are consistent with cyclic electron transfer mechanisms such as the protonmotive Q cycle.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C₁₂TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C₁₂TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C₁₂TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are “relatively weak, mild uncouplers”, do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.     

Bioenergetics of Yarrowia lipolytica cells grown at alkaline conditions

Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4-3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.     

The role of respiratory chain in paraquat toxicity in yeast

Yeast respiratory deficient mutants are resistant to paraquat. Similar resistance is caused by lowering the respiration by physiological mechanisms, as well as by some inhibitors of electron transfer chain of mitochondria. Presented results suggest that the major contribution of mitochondria to overall toxicity of paraquat in yeast is a consequence of very low level of cytochrome P-450, and presumably its presumably its reductase in aerobic yeast cells. In mammalian cells this enzyme is considered as the most important factor involved in paraquat toxicity. Mitochondrial cytochromes seem to be the first targets of damaging effects of paraquat.     

Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase)

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.     

"Oxygen regulation" of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide- and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by "the hypoxia genes" and activated (induced) under microaerobic conditions.     

A unique respiratory adaptation in Drosophila independent of supercomplex formation

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane in mammals and yeast, as well as in some bacterial membranes. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanolamine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation.