The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F₁F₀-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K_0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.     

The nicotinamide nucleotide specificity of glutamate dehydrogenase in intact RAT-liver mitochondria

1. The kinetics of oxidation of intramitochondrial reduced nicotinamide nucleotides by -oxoglutarate plus ammonia in intact rat-liver mitochondria have been reinvestigated. It is demonstrated that the preferential oxidation of NADPH observed on addition of ammonia to mitochondria, preincubated under energized conditions in the presence of -oxoglutarate, is due to a transhydrogenation catalysed by glutamate dehydrogenase rather than to an energy-dependent modification of the nicotinamide nucleotide specificity of the enzyme in intact mitochondria.

2. When mitochondria are preincubated at 25 °C under energized conditions in the presence of respiratory inhibitors with the substrates of glutamate dehydrogenase, an oxidation of NADPH, but not of NADH, is brought about by decreasing the reaction temperature. Both the rate of NADPH oxidation and the final steady-state mass-action ratio of nicotinamide nucleotides are dependent on the concentration of ammonia and on the final reaction temperature. A similar effect is observed when rhein is added to the reaction medium at 25 °C in order to inhibit the energy-linked transhydrogenase reaction.

3. In the presence of the substrates of glutamate dehydrogenase, intact ratliver mitochondria catalyse an ATPase reaction due to the simultaneous activity of the energy-linked transhydrogenase and the non-energy-linked transhydrogenation catalysed by glutamate dehydrogenase.

4. These findings are discussed in relation to the nicotinamide nucleotide specificity of glutamate dehydrogenase and to a possible compartmentation of nicotinamide nucleotides in intact rat-liver mitochondria.     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

Significance of respirasomes for the assembly/stability of human respiratory chain complex I

We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.     

Effect of substrates on reconstitution of the mitochondrial respiratory chain under various conditions

The reconstitution of NADH oxidase, succinate oxidase and the complete respiratory chain from NADH: cytochrome c oxidoreductase, succinate: coenzyme Q oxidoreductase, cytochrome oxidase and cytochrome c was studied under various conditions. The formation of these multi-enzyme systems was prevented by cobra venom phospholipase. Reconstitution was possible in the presence of cobra venom only if the medium contained NADH (or succinate) and O₂. Bovine serum albumin prevented the formation of NADH oxidase at low temperatures but hardly affected this process at 38–42°. It also increased the thermal stability of the reconstituted system. Reconstitution of NADH oxidase did not occur in the presence of potassium oleate, and bovine serum albumin completely eliminated the effect of the latter. However, bovine serum albumin did not protect the respiratory chain from the action of phospholipase. Therefore, the presence of NADH was necessary for the reconstitution of NADH oxidase at 38° in a medium containing bovine serum albumin and cobra venom.

Thus, the natural agents indicated above have a substantial effect on the reconstitution of the respiratory chain. Reconstitution becomes possible with a strictly definite ratio between the effects of different external factors. A special part in the formation of the respiratory chain is played by substrates having a specific influence on its structure.     

Cardiac Metabolic Pathways Affected in the Mouse Model of Barth Syndrome

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F₀F₁-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

The effect of constant and changing temperatures on the thermal resistance of Lymnaea peregra (Müller)

The effect of constant and changing temperatures on both high and low lethal temperatures of Lymnaea peregra and the time course of acclimation were investigated. Snails acclimated to 6.5°, 11.5° and 16.5°C showed paradoxical ‘adaptation’ of their high letal temperature and reasonable ‘adaptation’ of the lowlethal temperature. This combination is thus observed for the first time in molluscs. Seasonal variation in the upper lethal temperature showed parallel changes and the snails became less resistant to heat during spring and summer. Acclimation to changing temperature conditions did not extend their temperature tolerance range. During the course of acclimation of 6.5°C snails to 16.5°C, about 70% of the acclimation was completed within the first 24 h and the rest was completed within 360 h at 16.5°C.     

Demonstration of the existence of an organo-specific NADH dehydrogenase in heart mitochondria

Experimental evidence is presented showing the existence of an NADH-consuming enzyme in heart mitochondria, in addition to the NADH--ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo-specific. The "exogenous NADH dehydrogenase" of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy-dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin.     

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD⁺ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD⁺ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

The concentration dependence of the ‘zero-shear’ specific viscosity for a commercial hydroxyethylmethylcellulose (HEMC) in water at several temperatures

This short paper presents preliminary results on the ‘zero-shear’ specific viscosity η_sp0 of a commercial hydroxyethylmethylcellulose (Tylose MH-4000) in water, at the temperatures 10, 25 and 40·5°C, over a wide range of concentrations. At the two higher temperatures, two regions are found in the plot of logC[η]₀ against logη_sp0 with a C^*[η]₀ value of about 2·5. This is consistent with the behaviour of other random-coil polymers. At 10°C however, there is an interesting ‘upward shift’ in this plot in the dilute region. It is suggested that this is related to the different degree of hydration of the oligo(ethyleneoxide) side chains at this temperature.     

Proton permeation, adenine nucleotide translocation and respiratory control in mitochondria cross-linked by dimethylsuberimidate

Rat liver mitochondria or isolated mitoplasts were treated with the cross-linking agent, dimethylsuberimidate, under conditions (pH 7.5; 0°C) which were not detrimental for the coupling quality of the mitochondria and the effect was evaluated on a kinetic basis. When about 25% of the NH₂-groups reacted, the mitochondria or the mitoplasts acquired complete osmotic stability. Succinate oxidation in state 4 was inhibited by about 30–35%. This effect was also observed when the organelles were amidinated by methylacetimidate, a monofunctional imidate which caused no osmotic stabilization. Uncouplers stimulated succinate oxidation in cross-linked mitochondria to the same extent as in the control, in contrast stimulation by ADP was suppressed. Accordingly, the rate of decay of the respiration-dependent cross-membrane proton gradient was only decreased by 25%, whereas the ATPase and adenine nucleotide translocase were strongly inhibited. In the cross-linked mitochondria, the extent of inhibition of the ATPase and of the translocase was found to be the same whether the assays were performed at 30°C (like the respiratory assay) or at 0°C. The effect of methylacetimidate treatment on these activities at the two temperatures was different. At 30°C, the ATPase was not inhibited and the extent of inhibition of ATP translocation was small. At 0°C, the two activities were nearly as much inhibited as in cross-linked mitochondria. Our results suggest that a considerable rigidity can be introduced in the coupling membrane by cross-linking, without a major loss in the initial step of energy conservation. However, the energy conserved in the proton gradient cannot be utilized for ATP synthesis, probably because of the restricted mobility of adenine nucleotide translocase in the cross-linked mitochondria.     

Oxidative phosphorylation in human muscle in patients with ocular myopathy and after general anaesthesia

The fuel preference of human muscle mitochondria has been given. Substrates which are oxidized with low velocity cannot be used to detect defects in oxidative phosphorylation. After general anaesthesia, the oxygen uptake with the different substrates is much lower than after local analgesia. The latter was therefore used in the subsequent study. In 15 out of 18 patients with ocular myopathy, defects in oxidative phosphorylation could be detected in isolated muscle mitochondria prepared from freshly biopsied tissue. Measurement of the activity of segments of the respiratory chain in homogenate from frozen muscle showed no, or minor defects. In two of these patients showing exercise intolerance, decreased oxidation of NAD(+)-linked substrates and apparently normal mitochondrial DNA, further study revealed deficiency of pyruvate dehydrogenase in a girl with ptosis and a high Km of complex I for NADH in a man. Both patients responded to vitamin therapy.     

Functional and molecular analysis of mitochondria in thyroid oncocytoma

We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes I (NADH dehydrogenase), II (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.     

Pumpkin pectin: gel formation at unusually low concentration

The gel properties of high-methoxy pectin from pumpkins have been investigated to assess the potential of this material as a hard-currency export from the former Soviet Union. Comparison was made with commercial slow-set, medium-set and rapid-set pectins from citrus peel. Gels were formed by cooling pectin solutions (pH 3·0; 60% (w/w) sucrose; 5% (w/w) corn syrup) from 95°C to 25°C, and the time-temperature course of network formation was monitored by small-deformation oscillatory measurements of storage modulus (G′). At concentrations above 1% (w/w) the pumpkin pectin gave weaker gels than the other three samples, but its minimum critical gelling concentration (c₀) was found to be much lower (by at least a factor of five). Compression testing gave similar results, with pumpkin pectin giving useful breaking-stress (‘hardness’) at concentrations down to 0·5% (w/w), about a factor of two lower than for the citrus samples. Its gelation was also less rapid, giving G′ values below those of the other three samples at temperatures down to 60°C, but then setting sharply; this behaviour could be useful in avoiding ‘pregelation’ in commercial processing. The commercial slow-set pectin showed typical ‘weak gel’ properties in the solution state at 95°C, with systematic reduction in gel-like character with increasing ester content in the other samples. The rigidity of the final gels also decreased systematically through the series: rapid-set < medium-set < slow-set. These observations are tentatively ascribed to stable association of unesterified galacturonate chain segments at low pH, where electrostatic repulsion is suppressed.     

The oxygen reaction of the cytochrome d-terminated respiratory chain of Escherichia coli at sub-zero temperatures: Kinetic resolution by EPR spectroscopy of two high-spin cytochromes

The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy. Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes. At −120 to −100°C, a rhombic signal appears, attributable to cytochrome d, followed at above −100°C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters. The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen.

Cytochrome oxidase Escherichia coli Cytochrome b-d complex Bacterial electron transport Low-temperature technique     

The mitochondrial respiratory chain of Ustilago maydis

Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca²⁺, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation.The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool.     

'P' solubilization potential of plant growth promoting Pseudomonas mutants at low temperature

Pseudomonas fluorescens strain GRS₁, PRS₉ and their cold tolerant mutants were examined for their tricalcium phosphate (TCP) solubilizing activity in NBRIP (broth) media at 10°C and 25°C. Invariably, all the cold tolerant mutants of GRS₁ and PRS₉ were found more efficient than their respective wild type counterparts for ‘P’ solubilization activity at 10°C as compared to 25°C. ‘P’ solubilization potential of CRM was found maximum among all the strains followed by CRPF₆ and CRPF₄. To the best of out knowledge, this is the first report regarding low temperature ‘P’ solubilization activity.