Relationships of respiratory chain and ATP-synthetase in energized mitochondria

The present study reveals that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of respiratory chain functioning may be observed at different absolute values of delta psi on the mitochondrial membrane. This fact points out that the membrane potential is not a unique regulator in coupling of ATP-synthetase and respiratory chain activities. We found, using the double-inhibitor titration technique, that ATP-synthetase inhibition induces proportional inhibition of respiratory chain enzymes and vice versa respiratory chain inhibition induces proportional inhibition of ATP-synthetase. This effect is shown to exist only when osmolarity is close to 150-300 (mosM) (in the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. Under conditions of high osmolarity (400-600 mosM) the respiratory chain and ATP-synthetase behave as if they were coupled by bulk phase delta -mu H+, from the kinetic point of view.     

Effect of dehydrocorrine-cobalt complex on mitochondria

The effects of the decamethyloctadehydrocorrine-cobalt complex (Co-C) on respiration and the ATP-synthetase activity of rat liver mitochondria were investigated. The Co-C complex was found to be an effective shunt of the respiratory chain. It accepts electrons from ubiquinone and donates them directly to O2. The Co-C complex inhibits the ATPase and ATP-synthetase activities of mitochondria.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

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.     

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.     

Energetics of swelling in isolated hepatocytes: A comprehensive study

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Regulation of ion transport in mitochondria by respiratory chain enzymes and ATPase

The effects of the respiratory chain inhibitors as well as those of the inhibitors and substrates of ATP-synthetase in Ca2+ and K+ transport induced in the mitochondria upon the medium acidification in the presence of phosphate or arsenate, were investigated. Evidence has been obtained suggesting that under the experimental conditions used the transmembrane fluxes of K+ and Ca2+ are paralleled with H+ leakage through the proton channel of ATPase. It was found also that the system inducing cation fluxes at low pH values included peroxidation and hydrolysis of phospholipids. A scheme of regulation of ion transport in the mitochondria involving oxidative phosphorylation and oxidation and hydrolysis of lipids is proposed.     

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.     

Competition between hydrogen peroxide and nitrate for electrons from the respiratory chains of Thiosphaera pantotropha and Rhodobacter capsulatus

Abstract Thiosphaera pantotropha and some strains of Rhodobacter capsulatus express both a periplasmic nitrate reductase and cytochrome c peroxidase when grown under aerobic conditions. Harvested cell suspensions of either species can respire nitrate in the presence of 200 μM O₂ (∼ 80% air saturation), at 70–80% of the anaerobic rate. Addition of hydrogen peroxide to such cells causes a 90% inhibition of nitrate reduction under anaerobic or aerobic conditions. The duration of the inhibition is proportional to the concentration of hydrogen peroxide added and can be ascribed to the expression of periplasmic peroxidases that compete with the nitrate reductase for electrons from the respiratory chain. The results reveal a hitherto unrecognised interaction between reactions of denitrification and the reduction of hydrogen peroxide by a periplasmic peroxidase that may have implications for the denitrification in microaerobic environments. The creation of aerobic conditions in bacterial cultures by addition of hydrogen peroxide, and relying on the generation of oxygen by endogenous catalase activity, is a commonly used technique for studying respiratory processes. The observations presented here demonstrate that results derived from such experiments should be interpreted with caution.     

Glutamine effect on cultured granule neuron death induced by glucose deprivation and chemical hypoxia

Using a specific fluorescent probe of mitochondrial membrane potential (tetramethylrhodamine ethyl ester), we have shown that glucose deprivation (GD) of cultured cerebellar granule neurons (CGN) for 3 h lowers mitochondrial membrane potential in these cells. Longer glucose starvation (24 h) causes CGN death that is not prevented by blockers of ionotropic glutamate receptors (MK-801 (10 μM) and NBQX (10 μM)). Glutamine or pyruvate (2 mM) maintain membrane potential of mitochondria and decrease CGN death under GD conditions. In the presence of glucose the mitochondrial respiratory chain blocker rotenone induces neuron death potentiated by glutamine. The potentiation effect is completely prevented by blockers of ionotropic glutamate receptors. These results show that glutamine under conditions of GD can be utilized by mitochondria as substrate, but at the same time, in the case of mitochondrial function deterioration, metabolism of this amino acid results in glutamate accumulation to toxic level.     

Metabolic activity of Siberian permafrost isolates, <Emphasis Type="Italic">Psychrobacter arcticus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium sibiricum</Emphasis>, at low water activities

The Siberian permafrost is an extreme, yet stable environment due to its continuously frozen state. Microbes maintain membrane potential and respiratory activity at average temperatures of -10 to -12 degrees C that concentrate solutes to an a (w) = 0.90 (5 osm), The isolation of viable Psychrobacter arcticus sp. 273-4 and Exiguobacterium sibiricum sp. 255-15 from ancient permafrost suggests that these bacteria have maintained some level of metabolic activity for thousands of years. Permafrost water activity was simulated using (1/2) TSB + 2.79 m NaCl (5 osm) at and cells were held at 22 and 4 degrees C. Many cells reduced cyano-tetrazolium chloride (CTC) indicating functioning electron transport systems. Increased membrane permeability was not responsible for this lack of electron transport, as more cells were determined to be intact by LIVE/DEAD staining than were reducing CTC. Low rates of aerobic respiration were determined by the slope of the reduced resazurin line for P. arcticus, and E. sibiricum. Tritiated leucine was incorporated into new proteins at rates indicating basal level metabolism. The continued membrane potential, electron transport and aerobic respiration, coupled with incorporation of radio-labeled leucine into cell material when incubated in high osmolarity media, show that some of the population is metabolically active under simulated in situ conditions.     

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

Studies with a reconstituted muscle glycolytic system. The rate and extent of glycolysis in simulated post-mortem conditions

The stimulation of succinate-cytochrome c reductase in Jerusalem artichoke mitochondria by lowering osmolarity was found to be associated with conformational changes in the inner membrane rather than with rupture of the outer membrane. This conclusion is based on the following evidence. (1) When the activation of succinate dehydrogenase was measured by using either K(3)Fe(CN)(6) or exogenous cytochrome c as an electron acceptor, electron flow to cytochrome c was always 7% of that to K(3)Fe(CN)(6) throughout the activation process. (2) The rate of exogenous cytochrome c reduction by succinate and NADH was directly related to the maximum rate of electron flow as determined by oxygen utilization. These two observations are not consistent with the low rate of succinate-cytochrome c reductase being limited by a permeability barrier at the outer membrane. (3) In addition to stimulating the succinate-cytochrome c reductase, lowering the osmolarity caused simultaneous changes in the permeability of the inner membrane to ferricyanide and NADH. The data show that lowering the osmolarity results in progressive changes in the permeability of the inner membrane. The first change detected was an increased permeability to K(3)Fe(CN)(6), then a simultaneous increase in accessibility of the respiratory chain to exogenous cytochrome c and an increased permeability to NADH, followed finally by rupture as measured by the release of malate dehydrogenase.     

Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite

The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa.     

Quantum-mechanical descriptions of proton transfer in biosystems containing hydrogen-bonded chains

A mechanism of proton transfer along the proton channel of (F0) ATP-synthetase of a membrane is suggested. In the small polaron model the charged fault (an excess of protons or a proton hole) transfer is considered in a longitudinal electric field along an assumed chain which is formed by hydroxyl groups connected by strong H-bonded chains. A number of kinetic parameters are estimated. The theoretical data are compared with the experimental results.     

ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.     

Hemoglobin enhances the self-association of spectrin heterodimers in human erythrocytes

Spectrin in isolated erythrocyte membranes is known to undergo tetramer to dimer transformation upon hypotonic incubation at 37 degrees C. In the present study, we detect no such transformation in intact erythrocytes in which hypotonicity is achieved by valinomycin treatment followed by hypotonic swelling. The inhibition of spectrin tetramer to dimer transformation is attributable to intracellular hemoglobin, since the addition of hemoglobin to isolated membranes or spectrin extracts blocks a similar spectrin transformation. However, the inhibitory effect is not limited to hemoglobin; other proteins including heme-containing proteins and basic proteins such as cytochrome c, ribonuclease, and albumin are also effective. The magnitude of their effect is proportional to the increased pI value of these proteins. We conclude that the stabilizing effect of these proteins on spectrin tetramers under hypotonic conditions is partly due to their non-ideality, which excludes water from spectrin and thus increases the effective concentration of spectrin, and to their electrostatic interactions with spectrin. In addition, promotion of spectrin self-association by hemoglobin under hypotonic conditions increases the stability of membrane skeletons against mechanical shearing. More importantly, the hemoglobin effect on spectrin self-association is demonstrable at physiological hemoglobin concentration, pH, and osmolarity, suggesting that in intact red cells the spectrin dimer-dimer association, as well as the membrane skeletal structure, is strengthened by intracellular hemoglobin.     

"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.     

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.