Metabolic changes during phosphate deprivation in Englena in air and in oxygen

1. Euglena cells were grown in culture media containing either 20mm-phosphate or 20mum-phosphate, with ethanol or glucose as the sole source of carbon, and gassed with either air+carbon dioxide (95:5) or oxygen+carbon dioxide (95:5) at atmospheric pressure. 2. After growth in low-phosphate medium with ethanol as substrate, the cells developed signs of oxygen toxicity, as indicated by a decreased rate of respiration, a decreased net synthesis of paramylum and a failure to resume growth on replenishment of phosphate. 3. After growth in low-phosphate medium with glucose as substrate, the signs of oxygen toxicity were less apparent. 4. During phosphate deprivation the carotenoid content of Euglena increased more than threefold. This increase was largely prevented by exposure of the cells to oxygen+carbon dioxide (95:5) during growth. Oxygenation appears to interfere with ring closure of the common carotenoid precursor. 5. Mitochondria obtained from Euglena exposed to oxygen during phosphate deprivation, i.e. when signs of oxygen toxicity were evident, had greatly decreased activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and NADH-cytochrome c oxidoreductase, compared with mitochondria obtained from Euglena exposed to oxygen in medium containing 20mm-phosphate.     

Carbohydrate energy metabolism in Fasciola gigantica (trematoda)

Umezurike G. M. and Anya A. O. 1980. Carbohydrate energy metabolism in Fasciola gigantica (Trematoda). International Journal for Parasitology10: 175–180. Adult Fasciola gigantica contained 4.49 ± 0.06 % (mean ± S.D.) wet weight glycogen. Tissue homogenates contained high levels of malate dehydrogenase (MDH), NAD-linked malic enzyme (ME), Phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDH). MDH, PEPCK and ME activities appeared to be localized in both cytosolic and mitoehondrial fractions, fumarase activity appeared to be predominantly mitochondrial whereas LDH and pyruvate kinase activities were cytosolic in distribution. Polyacrylamide gel electrophoresis revealed the predominance of LDH-1, LDH-2 and LDH-3 but only traces of LDH-4 and LDH-5 isoenzymes in the crude cytosolic fraction. LDH activity in the crude sample was inhibited by excess substrate (pyruvate). The mitoehondrial system showed NADH -cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, NADH oxidase and some cytochrome c-oxygen oxidoreductase activities. Under anaerobic conditions, NADH-fumarate oxidoreductase and succinate-NAD + oxidoreductase activities of mitoehondrial preparations were stimulated in the presence of ADP and ATP respectively. Isolated mitochondria contained rhodoquinone and no ubiquinone, and isolated rhodoquinone was readily reduced by succinate in the presence of submitochondrial particles. Hydrogen peroxide was produced by submitochondrial particles in the presence or absence of KCN or in the presence of fumarate.     

Changes in respiratory activities during the cell-cycle of the fission yeast Schizosaccharomyces pombe 972h? growing in the presence of glycerol

1. The specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, and NADPH-cytochrome c oxidoreductase in mid-exponential-phase batch cultures of glycerol-grown Schizosaccharomyces pombe indicated that the organisms were catabolite-de-repressed. 2. In cultures growing synchronously in the presence of glycerol as sole carbon source, the respiration rate showed two abrupt increases at about 0.45 and 0.95 of the cell-cycle and remained constant in the periods between successive rises. 3. Catalase, succinate dehydrogenase, NADH-cytochrome c oxidoreductase and acid p-nitrophenyl-phosphatase all showed peak patterns of expression in synchronous cultures. 4. Cytochrome c oxidase and cytochromes a+a(3) both showed step patterns of expression with two rises per cell-cycle. 5. Cytochromes c(548), b(554) and b(560) all followed similar time-courses in step patterns of expression, but these were distinct from, and more complex than, that of cytochromes a+a(3). 6. These results are compared with those previously obtained with glucose-grown cultures, and the part played by catabolite repression in the expression of respiratory activities in the cell-cycle is assessed.     

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.     

Effect of long-wave ultraviolet light (313 nm) on the survivability and respiration of Candida utilis yeasts

Irradiation of Candida utilis with near-UV (313 nm) was found to inhibit respiration; the degree of the inhibition correlated with the number of killed cells. Irradiation of the yeast mitochondria inhibited the activity of succinate oxidase; the dependence of the inhibition on the dose of near-UV (313 nm) was close to the dose dependence of the photoinhibition of respiration in whole cells. The action spectrum for the inhibition of the activity of succinate oxidase was recorded; it corresponded to the absorption spectrum of ubiquinone isolated from the yeast cells. The data obtained suggest that ubiquinone is a target molecule in the effects of inhibition of respiration and death of yeast cells induced with near-UV (313 nm).     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain

In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti-Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q2, Q4, Q6 and Q10 and an analogue, 2,3-dimethoxyl-5-n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1-6) or both, of QH2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

Effects of carbon–dioxide-bicarbonate mixtures on oxidative phosphorylation by cauliflower mitochondria

1. Carbon dioxide-bicarbonate mixtures markedly inhibited oxidation and phosphorylation rates of mitochondria prepared from cauliflower. Inhibition occurred with succinate, malate, citrate, isocitrate and NADH as substrates. 2. Indophenol-reductase systems with malate, succinate, isocitrate and NADH as substrates were inhibited by 5% and 15% carbon dioxide. Cytochrome c oxidase was not inhibited by 15% carbon dioxide.     

The respiratory chain in a ubiquinone-deficient mutant of Saccharomyces cerevisiae.

1. Two allelic mutants of Saccharomyces cerevisiae with a deficiency in the biosynthesis of ubiquinone have been isolated. The properties of one particular mutant strain were investigated. Submitochondrial particles of this strain contain maximally 3% of the amount of ubiquinone in wild-type particles; the amounts of other components of the respiratory chain are essentially normal. 2. The respiratory rates of mutant cells, mitochondria and submitochondrial particles are low with ubiquinone-dependent substrates, but are restored to normal levels by addition of Q-1; the restored respiration is antimycin sensitive. Intact cells and mitochondria show respiratory control both in the absence and presence of Q-1. 3. The NADH:Q-1 oxidoreductase of submitochondrial particles of the mutant followspseudo first-order kinetics in [Q-1]. QH2-1 inhibits competitively with respect to Q-1, the Ki for QH2-1 being equal to the Km for Q-1. 4. Succinate dehydrogenase in both wild-type and mutant submitochondrial particles can be activated by NADH. 5. The turnover number of succinate dehydrogenase in the mutant, measured with phenazine methosulphate as primary electron acceptor, is about one-half that of wild-type particles. The turnover numbers measured with Q-1 as electron acceptor are about the same in the two types of particles. 6. The kinetics of redox changes in cytochrome b, in the presence of antimycin and oxygen, are distinctly different in the mutant and wild-type particles. They indicate that ubiquinone plays an important role in the phenomenon of the increased reducibility of cytochrome b induced by antimycin plus oxygen.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Cadmium inhibits the electron transfer chain and induces reactive oxygen species

Recent research indicates that cadmium (Cd) induces oxidative damage in cells; however, the mechanism of the oxidative stress induced by this metal is unclear. We investigated the effects of Cd on the individual complexes of the electron transfer chain (ETC) and on the stimulation of reactive oxygen species (ROS) production in mitochondria. The activity of complexes II (succinate:ubiquinone oxidoreductase) and III (ubiquinol:cytochrome c oxidoreductase) of mitochondrial ETC from liver, brain, and heart showed greater inhibition by Cd than the other complexes. Cd stimulated ROS production in the mitochondria of all three tissues mentioned above. The effect of various electron donors (NADH, succinate, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol) on ROS production was tested separately in the presence and in the absence of Cd. ESR showed that complex III might be the only site of ROS production induced by Cd. The results of kinetic studies and electron turnover experiments suggest that Cd may bind between semiubiquinone and cytochrome b566 of the Q0 site of cytochrome b of complex III, resulting in accumulation of semiubiquinones at the Q0 site. The semiubiquinones, being unstable, are prone to transfer one electron to molecular oxygen to form superoxide, providing a possible mechanism for Cd-induced generation of ROS in mitochondria.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals.

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the 'Q cycle' [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.     

Studies on the effects of copper deficiency on rat liver mitochondria. II. Effects on oxidative phosphorylation

Effects of dietary copper deficiency in rats on respiratory enzymes of isolated rat liver mitochondria have been studied. After 2 weeks of Cu-depletion, cytochrome c oxidase (EC 1.9.3.1) activity had declined by 42% and between 4 and 8 weeks exhibited between 20 and 25% of the activity of control mitochondria. Activities of NADH cytochrome c reductase (EC 1.6.99.3) and succinate cytochrome c reductase (EC 1.3.99.1), were unaffected initially but declined by 32 and 46%, respectively, after 8 weeks of Cu-depletion. After 4 weeks there was a significant (34%) decline in succinate supported state 3 respiration with only a modest (18%) decline in state 4 respiration. The ADP:O ratio was unaffected by Cu-depletion after 6 and 8 weeks of dietary Cu-restriction. State 3 respiration was significantly reduced after 6 weeks when glutamate/malate or beta-hydroxybutyrate were used as substrates, whereas state 4 respiration and ADP:O ratios were unaffected. The fall in state 3 respiration was of sufficient magnitude at 8 weeks to cause a significant decline in the respiratory control ratio with all substrates. Comparisons between the relative activities of cytochrome c oxidase and reductase activities in Cu-deficient preparations, the relatively specific effect of the deficiency on state 3 respiration with all substrates tested and the ability to increase significantly oxygen consumption in excess of maximal state 3 respiration by the uncoupler 2,4-dinitrophenol suggest that the defect in Cu-deficient mitochondria cannot be attributed solely to the decreased activity of cytochrome c oxidase.     

Hepatocyte mitochondrion respiratory chain in rats with experimental toxic hepatitis

The purpose of this study was to examine hepatocyte mitochondrion respiratory chain in rats subjected to ethanol and CCl4 administration within 4 weeks to induce an experimental hepatitis. Oxygen consumption was determined as a measure of mitochondrion respiration chain function. The development of liver pathology was accompanied by fat accumulation, fibrosis, triglycerides and lipid peroxidation increase. Respiratory chain characteristics damage was found. Endogenous oxygen consumption by hepatocytes isolated from pathological liver was found 34% higher compared to control. Exogenous malate and pyruvate substrates delivery didn't stimulate cell respiration. Rotenone (the inhibitor of the I complex) decreased 27% oxygen consumption by pathological hepatocytes while dinitrophenol produced 37% cell respiration increase. States 3 (V3) and 4 (V4) mitochondrial respiration with malate + glutamate as substrates were found to be 70 and 56% higher accordingly compared to control level. V3 and Vd (dinitrophenol respiration) for mitochondria from pathological liver didn't differ from control when being tested with malate + glutamate or succinate as substrates. Cytochrome c oxidase activity increased (+ 80%) as compared to control. Administration of hypolipidemic agent simvastatin simultaneously with ethanol and CC14 resulted in decrease liver fat accumulation, fibrosis and peroxidation products. Simvastatin administration caused hepatocyte endogenous respiration decrease while malate + pyruvate, dinitrophenol or rotenone delivery produced oxygen consumption alterations similar to control. However, when isolated mitochondria from liver of simvastatin treated animals being tested the decrease of oxidative phosphorylation coupling for substrates malate + glutamate was found. While simvastatin did not cause changes in cytochrome c oxidase activity. We propose the hypothesis that the NCCR complex in rat mitochondria with experimental toxic hepatitis works extensively on superoxydanion production. Alterations of SCCR, Coenzyme Q-cytochrome c-reductase, cytochrome c oxidase and ATP-synthase activities have an adaptive nature to compensate for impaired NCCR function.     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction

Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins.