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Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart michondria

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Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart michondria

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The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.

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... The CoQ pool is certainly required for electron transfer from Complex II to Complex III: in bovine heart mitochondria and submitochondrial particles, Complex II kinetically follows pool behaviour in reconstitution experiments [66] and also in intact mitochondria [40]; this is in complete accordance with the lack of Complex II-containing supercomplexes as found by both BN-PAGE [25]. Flux control analysis [61] confirmed that Complex II is the only rate-limiting step in succinate oxidation, and both Complex III and Complex IV have low flux control coefficients at difference with NADH oxidation (Section 3.2). ...
... The relation between electron transfer rate and CoQ concentration was seen for NADH and succinate oxidation in reconstituted systems [66] and in phospholipid-enriched mitochondria [73]. Direct titrations of CoQ-depleted mitochondria reconstituted with different CoQ supplements yielded a "K m " of NADH oxidation for Q t in the range of 2-5 nmol/mg mitochondrial protein [66], corresponding to a Q t concentration of 4-10 mM in the lipid bilayer. ...
... The relation between electron transfer rate and CoQ concentration was seen for NADH and succinate oxidation in reconstituted systems [66] and in phospholipid-enriched mitochondria [73]. Direct titrations of CoQ-depleted mitochondria reconstituted with different CoQ supplements yielded a "K m " of NADH oxidation for Q t in the range of 2-5 nmol/mg mitochondrial protein [66], corresponding to a Q t concentration of 4-10 mM in the lipid bilayer. The K m for CoQ 10 of NADHcytochrome c reductase was found to be much higher than that of succinate-cytochrome c reductase. ...
Article
Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based upon specific interactions between individual respiratory complexes. These supercomplexes were found to be functionally relevant by flux control analysis and to confer a kinetic advantage to NAD-linked respiration (channelling). However, the Coenzyme Q pool is still required for FAD-linked oxidations and for the proper equilibrium with Coenzyme Q bound in the supercomplex. Channelling in the cytochrome c region probably also occurs but does not seem to confer a particular kinetic advantage. The supramolecular association of individual complexes strongly depends on membrane lipid amount and composition and is affected by lipid peroxidation; it also seems to be modulated by membrane potential and protein phosphorylation. Additional properties of supercomplexes are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or IV, and prevention of excessive generation of reactive oxygen species. The dynamic character of the supercomplexes allows their involvement in metabolic adaptations and in control of cellular signalling pathways. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.
... An increase in respiratory parameters can be attributed to an increased ETC. Estornell et al., 1992 reported a correlation between CoQ 10 concentration and respiratory rate that led to the assumption that physiological CoQ 10 concentrations do not saturate the respiratory chain (Estornell et al., 1992;Littarru and Tiano, 2007). ...
... An increase in respiratory parameters can be attributed to an increased ETC. Estornell et al., 1992 reported a correlation between CoQ 10 concentration and respiratory rate that led to the assumption that physiological CoQ 10 concentrations do not saturate the respiratory chain (Estornell et al., 1992;Littarru and Tiano, 2007). ...
... It is assumed that the addition of CoQ 10 leads to improved electron transport and thus to an increase in respiratory parameters. A correlation between CoQ 10 concentration and respiratory rate has already been reported in the literature, which led to the assumption that physiological CoQ 10 concentrations alone are not able to saturate the respiratory chain (Estornell et al., 1992;Littarru et al., 2007). It was therefore expected that administration of additional CoQ 10 would induce an increase in mitochondrial respiration, which was confirmed here. ...
... Rat liver and heart mitochondria were prepared as described by Fleischer et al. (1979;Fleischer and Kervina 1974). CoQ-depleted mitochondria were obtained by pentane extraction after lyophilization (Szarkowska 1966) and reconstitution with CoQj0 or other homologs was accomplished as described (Norling et ah 1974, Estornell et al. 1992, unless otherwise specified. ...
... Vmo, Vmr are the maximal velocities of oxidation and reduction, respectively, of the CoQ pool; K~o, Ksr are the respective dissociation constants of CoQ for the oxidase and the reductase. The term in the denominator is an operational Km of the system for Qt in pentane-extracted and reconstituted beef heart mitochondria (Estornell et al. 1992, confirming the hyperbolic behavior expected and already found in previous research (Norling et al. 1974). Table2 exhibits the kinetic constants for NADH cytochrome c reductase and succinate cytochrome c reductase in mitochondrial preparations extracted and reconstituted with different levels of CoQ10. ...
... Table 2 also reports data for glycerol-3-phosphate dehydrogenase from hamster brown fat mitochondria. The average Km for CoQl0 of NADH cytochrome c reductase of 2.4 nmol/mg protein (Estornell et al. 1992) is close to the CoQ concentration in beef heart mitochondria (Battino et al. 1990), therefore this concentration does not saturate NADH oxidation. On the other hand, in succinate cytochrome c reductase, the Km for CoQ10 is ten times lower, indicating that the natural quinone content is almost saturating; actually, the Km is close to the average content of mitochondrial respiratory enzymes (Capaldi 1982); the Km for CoQ10 of glycerol-3-phosphate cytochrome c reductase is in the same range of that of succinate oxidation. ...
Article
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In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.
... (b) Electron transfer in the CoQ and cytochrome c region obeys pool behavior according to the equation developed for CoQ by Kröger and Klingenberg [1973]. (c) Electron transfer follows saturation kinetics with respect to CoQ and cytochrome c concentrations [Mac-Lennan et al., 1966;Estornell et al., 1992]. ...
... The relation between electron transfer rate and CoQ concentration was seen for NADH and succinate oxidation in reconstituted systems [Estornell et al., 1992] and in phospholipid-enriched mitochondria [Schneider et al., 1982]. Direct titrations of CoQ-depleted mitochondria reconstituted with different CoQ supplements yielded a K m of NADH oxidation for Q t in the range of 2-5 nmol/ mg mitochondrial protein [Estornell et al., 1992], corresponding to a Q t concentration of 4-10 m M in the lipid bilayer. ...
... The relation between electron transfer rate and CoQ concentration was seen for NADH and succinate oxidation in reconstituted systems [Estornell et al., 1992] and in phospholipid-enriched mitochondria [Schneider et al., 1982]. Direct titrations of CoQ-depleted mitochondria reconstituted with different CoQ supplements yielded a K m of NADH oxidation for Q t in the range of 2-5 nmol/ mg mitochondrial protein [Estornell et al., 1992], corresponding to a Q t concentration of 4-10 m M in the lipid bilayer. The K m for CoQ 10 of NADH-cytochrome c reductase was found to be much higher than that of succinate-cytochrome c reductase. ...
Article
Two alternative models of organization of the mitochondrial electron transport chain (mETC) have been alternatively favored or questioned by the accumulation evidences of different sources, the solid model or the random collision model. Both agree in the number of respiratory complexes (I-IV) that participate in the mETC, but while the random collision model proposes that Complexes I-IV do not interact physically and that electrons are transferred between them by coenzyme Q and cytochrome c, the solid model proposes that all complexes super-assemble in the so-called respirasome. Recently, the plasticity model has been developed to incorporate the solid and the random collision model as extreme situations of a dynamic organization, allowing super-assembly free movement of the respiratory complexes. In this review, we evaluate the supporting evidences of each model and the implications of the super-assembly in the physiological role of coenzyme Q.
... The soluble saturatedstraight-chain decylubiquinone was used as ubiquinone analogue. Succinate±CoQ reductase (complex II) and succinate±cytochrome c reductase (integrated complex II and III) were assayed as described previously (Degli Esposti & Lenaz, 1982;Estornell et al. 1992;Lenaz et al. 1995b) after 10 min preincubation with succinate (1´25 mM) to activate complex II. Ubiquinol±cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) activities were measured as have been previously reported (Degli Esposti & Lenaz, 1982). ...
... This is in accordance with our previous data and with the kinetic behaviour of the CoQ pool. In kinetic terms, it is possible to increase respiratory-chain activity from NADH oxidation by increasing CoQ content over the normal level due to its lack of saturation for enzyme activities (Estornell et al. 1992). Thus, partial enzyme deficiencies can be counteracted by higher CoQ levels (Lenaz et al. 1995b;Estornell et al. 1997). ...
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The aim of this study was to investigate comparative effects of vitamin A deficiency on respiratory activity and structural integrity in liver and heart mitochondria. Male rats were fed a liquid control diet (control rats) or a liquid vitamin A-deficient diet (vitamin A-deficient rats) for 50 days. One group of vitamin-A deficient rats was refed a control diet for 15 days (vitamin A-recovered rats). To assess the respiratory function of mitochondria the contents of coenzyme Q (ubiquinone, CoQ), cytochrome c and the activities of the whole electron transport chain and of each of its respiratory complexes were evaluated. Chronic vitamin A deficiency promoted a significant increase in the endogenous coenzyme Q content in liver and heart mitochondria when compared with control values. Vitamin A deficiency induced a decrease in the activity of complex I (NADH–CoQ reductase) and complex II (succinate–CoQ reductase) and in the levels of complex I and cytochrome c in heart mitochondria. However, NADH and succinate oxidation rates were maintained at the control levels due to an increase in the CoQ content in accordance with the kinetic behaviour of CoQ as an homogeneous pool. On the contrary, the high CoQ content did not affect the electron-transfer rate in liver mitochondria, whose integrity was preserved from the deleterious effects of the vitamin A deficiency. Ultrastuctural assessment of liver and heart showed that vitamin A deficiency did not induce appreciable alterations in the morphology of their mitochondria. After refeeding the control diet, serum retinol, liver and heart CoQ content and the activity of complex I and complex II in heart mitochondria returned to normality. However, the activities of both whole electron transfer chain and complex I in liver were increased over the control values. The interrelationships between physiological antioxidants in biological membranes and the beneficial effects of their administration in mitochondrial diseases are discussed.
... The existence of this equilibrium is widely confirmed by a series of studies, such as the previously reported findings by Heron et al. [77] on CI to CIII association, by the saturation kinetics for CoQ exhibited by the integrated activity of CI and CIII (NADH-cyt. c oxidoreductase) [101] and by the decrease in respiration in mitochondria fused with phospholipids causing subsequent dilution of the CoQ pool [102]. ...
... All evidence converges in the statement that electron transfer from CII to CIII takes place only through the CoQ pool. Accordingly, succinate oxidation kinetically follows pool behavior after extraction and reconstitution [101] and in intact mitochondria [106] in accordance with the notion that CII does not participate in SC formation (see previous sections). For the same reason, also energy-dependent reverse electron transfer from succinate to NAD + , taking place through sequential activity of CII and CI connected by CoQ, must take place by collisional interactions in the CoQ pool. ...
Article
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Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F1FO-ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F1FO-ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.
... Here, we have used proteoliposomes (PLs) to determine the kinetics of complex I catalysis with a series of ubiquinone substrates of varying isoprenoid tail length, from ubiquinone-1 (Q1) to ubiquinone-10 (Q10). Previous attempts to investigate the effects of quinone tail length used native membranes supplemented with exogenous quinones, following removal of the endogenous Q10 by lyophilization and pentane extraction (20)(21)(22). However, these studies were compromised because (i) the quinone exchange procedures were detrimental to catalysis (the specific activities were 10-fold lower than observed here); and (ii) effects on complex I activity were obscured by the catalysis of other enzymes: Lenaz et al. (22) reconstituted Q1 to Q10 into lyophilized pentane-extracted mitochondria and measured O 2 consumption, which requires complexes III and IV, whereas Estornell et al. (20) similarly reconstituted Q10 and measured cytochrome c reduction, which requires complex III. ...
... Previous attempts to investigate the effects of quinone tail length used native membranes supplemented with exogenous quinones, following removal of the endogenous Q10 by lyophilization and pentane extraction (20)(21)(22). However, these studies were compromised because (i) the quinone exchange procedures were detrimental to catalysis (the specific activities were 10-fold lower than observed here); and (ii) effects on complex I activity were obscured by the catalysis of other enzymes: Lenaz et al. (22) reconstituted Q1 to Q10 into lyophilized pentane-extracted mitochondria and measured O 2 consumption, which requires complexes III and IV, whereas Estornell et al. (20) similarly reconstituted Q10 and measured cytochrome c reduction, which requires complex III. More recently, Fato et al. (21) assayed lyophilized pentane-extracted bovine mitochondria reconstituted with Q3, Q5, and Q10 with an NADH-Q1 reductase assay. ...
Article
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Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy- transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Å isoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/ dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4–7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.
... Nevertheless, to account for intersample variations using the same protocol, CoQ4 still is a valuable option. 159 and a PL density of ~ 1 mg/µL, 160,161 CoQ10 concentration can be calculated 160 to be between 13 and 33 mM in the present sample. Taking into account the highest detected CoQ10 concentration and assuming a HO-CoQ10 portion of 0.3%, its concentration in mitochondria was calculated to be 100 µM. ...
... 11 This may also explain the reported`essential cofactor' role of CoQ 10 in uncoupling proteins from brown adipose tissue, which by their regulated transmembrane proton transport is able to uncouple mitochondrial oxidative phosphorylation, producing heat rather than ATP. 12 As the MRC may not be saturated with CoQ 10 , a small decrease in CoQ 10 concentration may depress ATP production and cause organ dysfunction. 13 Another major role of CoQ 10 is its antioxidant function in protecting the cell from free-radicalinduced oxidation. 14 The antioxidant capacity of CoQ 10 has been attributed to CoQ 10 H 2 , 15 and the ratio CoQ 10 H 2 /CoQ 10 has been proposed as a possible marker of oxidative stress. ...
Article
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Coenzyme Q(10) (CoQ(10)) is the predominant form of ubiquinone in man. CoQ(10) functions as an electron carrier in the mitochondrial respiratory chain as well as serving as an important intracellular antioxidant, Lowered blood and tissue concentrations of CoQ(10) have been reported in a number of diseases, although whether this deficiency is the cause or an effect of the disease remains largely unresolved. Some studies have reported lowered plasma CoQ(10) concentrations after statin drug treatment of hypercholesterolaemic patients. However, a deficiency in CoQ(10) status has yet to be demonstrated in patients experiencing the rare myotoxic side-effects of these drugs. Most clinical investigations assessing the therapeutic potential of CoQ(10) have focused on cardiovascular disease, specifically congestive heart failure. Although a number of studies have reported clinical improvement in congestive heart failure patients after CoQ(10) supplementation to standard therapy, concerns about the design of these studies coupled to the small number of patients involved have limited their acceptance. Assessment of CoQ(10) status is generally based on plasma measurements. As plasma concentrations are influenced by a number of physiological factors and may not represent cellular concentrations, platelets, lymphocytes and fibroblasts may provide suitable alternatives for these measurements.
... Nonetheless the CoQ 10 treatment improves endothelial function and blood flow; thus, long-term treatment may be effective by improving oxygenation of the peripheral nerves (74). An increase in the concentration of CoQ 10 might affect mitochondrial respiratory function and early supplementation should be administrated in cases of deficiency (77). Since these events are due to mitochondrial PTP opening, Papucci et al. suggested the antiapoptotic activity of CoQ 10 could be related to its ability to prevent PTP opening and thus apoptosis (70). ...
Article
Full-text available
Neuropathic pain (NP) is one of the most suffering medical conditions that often fail to respond to certain pain therapy. Although its exact etiology is still unknown the role of reactive oxygen species (ROS) and oxidative stress were explored by many researchers. Neuropathies either central or peripheral lead to painful condition as well as social and economic isolation, thus various therapies were used to treat or reduce the pain. Laser therapy and antioxidant drugs have separately considered as treatment for NP, but the combination of them have not been used yet. In order to study the combination effects of Low Level Laser Therapy (LLLT) and Coenzyme Q10 (CoQ10) the present study was designed. Sixty adult male rats (230-320g) were used in this experimental study that divided into six groups (n=10). Chronic constriction injury (CCI) was used to induce neuropathic pain. The CoQ10 or vehicle, a low level laser of 980nm was used for two consecutive weeks. Thermal and mechanical paw withdrawal thresholds were assessed before and after surgery on 7(th) and 14(th) days. As we expected CCI decreased the pain threshold, whereas CoQ10 administration for two weeks increased mechanical and thermal threshold. The same results obtained for laser therapy using the CCI animals. Combination of laser 980nm with CoQ10 also showed significant differences in CCI animals. Based on our findings the combination of CoQ10 with LLLT showed better effects than each one alone. In this regard we believe that there might be cellular and molecular synergism in simultaneous use of CoQ10 and LLLT on pain relief.
... The normal content of coenzyme Q in mitochondrial membranes has been reported to be below that required for kinetic saturation [21]. This finding strongly suggests that coenzyme Q may be the rate-limiting component in the respiratory chain, especially in the mitochondria of compromised or incapacitated tissues. ...
Article
Illness is nothing else than a break of body's harmony that has the endogenous tendency to reconstitute itself, an endeavor that the physician can only support by appropriate measures. Hippocrates, 460–377 B . C .
... Moreover, inhibitor-titration studies of yeast mitochondria indicated that complexes II-IV behaved as a functional unit suggesting a physical interaction (143). There is also evidence for a complex-II-III-interaction in yeast and mammals derived from other approaches (e.g., 144,145). It can be speculated that mitochondrial complex II exists as a homotrimer like the homologous complex in bacteria (146) which interacts with III-IV-supercomplexes. ...
Chapter
The supramolecular organisation of the four respiratory chain complexes as well as the FOF1-ATP synthase (complex V), comprising together the oxidative phosphorylation (OXPHOS) system, has been a matter of debate since the beginning of their biochemical investigation some decades ago. The still generally accepted view found in the textbooks is the “random collision model” regarding the five oxidative phosphorylation complexes as independent entities in the mitochondrial inner membrane. Meanwhile, the experimental evidences favouring the existence of specific respiratory supercomplexes of complexes I, III and IV as well as of ATP synthase oligomers in most eukaryotic mitochondria provide a strong collection of arguments which is not less valid than that supporting the existence of individual respiratory complexes in vivo. Of particular note is the recent determination of single particle structures of OXPHOS supercomplexes from mammals, plants, algae and yeast. Although the number of studies supporting the existence of respiratory supercomplexes has been increasing at an accelerating pace, the functional significance of respiratory supercomplexes like conceivable substrate channeling is poorly understood. Besides enzymatic advantages, an emerging crucial role appears to be the assembly/stabilisation of individual complexes. In particular, the biogenesis and/or stabilisation of complex I, the largest and most complicated respiratory complex, seem to rely on the presence of other proteins (in particular complex III and/or IV), which may integrate complex I into supramolecular structures in the inner membrane. Herein a concise but broad overview about the recent advances in the research on respiratory supercomplexes and ATP synthase oligomers is presented, attempting to put the existence of oxidative phosphorylation superstructures into perspective with the overall supramolecular organisation of interrelated mitochondrial pathways and the sophisticated subcompartmentalisation of mitochondria.
... A third Q-pool is suggested as an exo-mtCoQ-pool, reduced by NADH added to intact pigeon heart mitochondria and located on the outer face of the mtIM (Jørgensen et al 1985). Considering that lateral diffusion of Q is high in the lipid bilayer and not rate-limiting for electron transfer, the inhomogeneity can be explained by SCInIIInIVn supercomplex formation (NADH oxidation through the CI-Q-CIII branch) in contrast to homogenous 'Qpool behavior' between CII (and other dehydrogenases) and CIII (succinate oxidation; Bianchi et al 2004;Estornell et al 1992;Rauchová et al 1997;Stoner et al 1984;Enriquez, Lenaz 2014). According to the solid-state model (Rich 1984), Q-intermediates are transferred in currently considered supercomplexes QSC by substrate channeling preventing equilibration with the free Q-pool Qfree. ...
Article
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Redox states of the mitochondrial coenzyme Q pool, which reacts with the electron transfer system, reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to O2, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force. A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor continuously the redox state of CoQ2 added as a Q-mimetic simultaneously with O2 consumption. The Q-Module was optimized for high signal-to-noise ratio, minimum drift, and minimum oxygen diffusion. CoQ2 equilibrates in the same manner as Q at Complexes CI, CII and CIII. The CoQ2 redox state is monitored amperometrically with the working electrode, which is poised at CoQ2 redox peak potentials determined by cyclic voltammetry. The voltammogram also provides quality control of the Q-sensor and reveals chemical interferences. The CoQ2 redox state and O2 consumption were measured simultaneously in isolated mouse cardiac and brain mitochondria. CoQ2 ― and by implication mitochondrial Q ― was more oxidized when O2 flux was stimulated by coupling control: when energy demand increased from LEAK to OXPHOS and electron transfer capacities in the succinate pathway. In contrast, CoQ2 was more reduced when O2 flux was stimulated by pathway-control of electron input capacities, increasing from the NADH (N)- to succinate (S)-linked pathway which converge at the Q-junction, with CI-Q-CIII and CII-Q-CIII segments, respectively. N- and S- respiratory pathway capacities were not completely additive, compatible with partitioning of Q intermediary between the solid-state and liquid-state models of supercomplex organization. The direct proportionality of CoQ2 reduction and electron input capacities through the CI-Q-CIII and CII-Q-CIII segments suggests that CoQ2 is accurately mimicking mitochondrial Q-redox changes.
... The discovery of POD (EC 1.11.1.7) associated to thylakoid membranes, which has a high affinity for hydroquinone (HQ) (Zapata et al. 1998, Casano et al. 2000, and the substratelike properties of the mitochondrial coenzyme Q (Estornell et al. 1992) prompted us to examine whether hydroquinones could also be natural substrates for mitochondrial POD(s). Oxidation of HQ with H 2 O 2 in the presence of mitochondria isolated from maize roots and the identification of possible systems involved in HQ regeneration, such as enzymatic oxidation of NAD(P)H and nonenzymatic cooxidation of ascorbate, were investigated in vitro in the present study. ...
Article
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Plasma membranes were isolated and purified from 14- day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD+-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD § and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P~, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.
... Besides the chronological aging process, occasional external stress events inside the epidermis may have an impact on the levels of both quinones as demonstrated by Podda et al. [20] in human skin equivalents after UV-irradiation. In skin, both causes of Q10 decline (age-dependent and UV-induced) may be of significant physiological importance given that even small changes in Q10 concentration could result in substantial alterations in the respiratory rate as shown by the saturation kinetics of Q10-dependent enzymes [34]. ...
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Ubiquinone (coenzyme Q10, Q10) represents an endogenously synthesized lipid-soluble antioxidant which is crucial for cellular energy production but is diminished with age and under the influence of external stress factors in human skin. Here, it is shown that topical Q10 treatment is beneficial with regard to effective Q10 replenishment, augmentation of cellular energy metabolism, and antioxidant effects. Application of Q10-containing formulas significantly increased the levels of this quinone on the skin surface. In the deeper layers of the epidermis the ubiquinone level was significantly augmented indicating effective supplementation. Concurrent elevation of ubiquinol levels suggested metabolic transformation of ubiquinone resulting from increased energy metabolism. Incubation of cultured human keratinocytes with Q10 concentrations equivalent to treated skin showed a significant augmentation of energy metabolism. Moreover, the results demonstrated that stressed skin benefits from the topical Q10 treatment by reduction of free radicals and an increase in antioxidant capacity. © 2015 BioFactors, 2015.
... Because of the insufficient nutrient databases, we cannot assess coenzyme Q10 intake from 24- h dietary recall, but our CAD subjects had significantly lower antioxidants intake (such as vitamins A and E) than the control . An increase in the concentration of coenzyme Q10 may somehow affect the mitochondrial respiratory function [34] and increase the antioxidants activities [35, 36]; as a result, early supplementation should be administrated in cases of deficiency [36]. Our study has two limitations. ...
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A higher oxidative stress may contribute to the pathogenesis of coronary artery disease (CAD). The purpose of this study was to investigate the relationship between coenzyme Q10 concentration and lipid peroxidation, antioxidant enzymes activities and the risk of CAD. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery were assigned to the case group (n = 51). The control group (n = 102) comprised healthy individuals with normal blood biochemical values. The plasma coenzyme Q10, malondialdehyde (MDA) and antioxidant enzymes activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx)) were measured. Subjects with CAD had significant lower plasma coenzyme Q10, CAT and GPx activities and higher MDA and SOD levels compared to those of the control group. The plasma coenzyme Q10 was positively correlated with CAT and GPx activities and negatively correlated with MDA and SOD. However, the correlations were not significant after adjusting for the potential confounders of CAD with the exception of SOD. A higher level of plasma coenzyme Q10 (≥ 0.52 μmol/L) was significantly associated with reducing the risk of CAD. Our results support the potential cardioprotective impact of coenzyme Q10.
... The discovery of POD (EC 1.11.1.7) associated to thylakoid membranes, which has a high affinity for hydroquinone (HQ) (Zapata et al. 1998, Casano et al. 2000, and the substratelike properties of the mitochondrial coenzyme Q (Estornell et al. 1992) prompted us to examine whether hydroquinones could also be natural substrates for mitochondrial POD(s). Oxidation of HQ with H 2 O 2 in the presence of mitochondria isolated from maize roots and the identification of possible systems involved in HQ regeneration, such as enzymatic oxidation of NAD(P)H and nonenzymatic cooxidation of ascorbate, were investigated in vitro in the present study. ...
Article
Full-text available
The oxidation of hydroquinone with H2O2 in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent K m for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H2O2 scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed.
... In fact, Rodriguez-Hernandez et al. [65] recently reported stimulation of mitophagy, and consequently mitochondrial degradation, in patients with coenzyme Q deficiency. Coenzyme Q10 has been shown to be rate-limiting in the electron transport chain in beef heart mitochondria [66], so an age-related decrease in content would have a large effect. Coenzyme Q10 is also a scavenger of superoxide, so the hypothesis of its deficiency lends to the abundant evidence that excess ROS plays a role in neurodegeneration with age [67,68]. ...
Article
Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.
... This redox activity underlies different biological functions possibly related to maintenance of mitochondrial homeostasis and prevention of free radical generation . In particular, being the physiological concentration of CoQ 10 close to the K m of respiratory complexes, supplementation is able to increase the velocity of the respiratory chain [37]. Minimal variations in the intra-mitochondrial concentration of CoQ 10 could determine considerable changes in terms of efficiency of the mitochondrial respiratory chain. ...
Article
Structural changes and abnormal function of mitochondria have been documented in Down's syndrome (DS) cells, patients, and animal models. DS cells in culture exhibit a wide array of functional mitochondrial abnormalities including reduced mitochondrial membrane potential, reduced ATP production, and decreased oxido-reductase activity. New research has also brought to central stage the prominent role of oxidative stress in this condition. This review focuses on recent advances in the field with a particular emphasis on novel translational approaches involving the utilization of coenzyme Q(10) (CoQ(10) ) to treat a variety of clinical phenotypes associated with DS that are linked to increased oxidative stress and energy deficits. CoQ(10) has already provided promising results in several different conditions associated with altered energy metabolism and oxidative stress in the CNS. Two studies conducted in Ancona investigated the effect of CoQ(10) treatment on DNA damage in DS patients. Although the effect of CoQ(10) was evidenced only at single cell level, the treatment affected the distribution of cells according to their content in oxidized bases. In fact, it produced a strong negative correlation linking cellular CoQ(10) content and the amount of oxidized purines. Results suggest that the effect of CoQ(10) treatment in DS not only reflects antioxidant efficacy, but likely modulates DNA repair mechanisms.
... Coenzyme Q10 supplementation might be beneficial in patients with CAD. An increase in the concentration of coenzyme Q10 might affect mitochondrial respiratory function [42] , and early supplementation should be administrated in cases of deficiency. Coenzyme Q10 is a well-tolerated and safe supplementation [43] and has a greater synergistic effect than other antioxidant vitamins such as vitamins A, C, and E [23,24,44]. ...
Article
The purpose of this study was to investigate the effect of coenzyme Q10 supplementation on oxidative stress and antioxidant enzyme activity in patients with coronary artery disease (CAD). This was an intervention study. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery or receiving percutaneous transluminal coronary angioplasty (n = 51) were randomly assigned to the placebo group (n = 14) or one of the two coenzyme Q10-supplemented groups (60 mg/d, n = 19 [Q10-60 group]; 150 mg/d, n = 18 [Q10-150 group]). Intervention was administered for 12 wk. Patients' blood samples were analyzed every 4 wk for plasma coenzyme Q10 concentrations, malondialdehyde (MDA), and antioxidant enzyme (catalase [CAT], superoxide dismutase [SOD], glutathione peroxidase) activity. Forty-three subjects with CAD completed intervention study. Plasma coenzyme Q10 concentration increased significantly after coenzyme the Q10-150 intervention (P < 0.01). The MDA levels were significantly lower than baseline in the Q10-150 group at week 4 (P = 0.03). The Q10-150 group had significantly lower MDA levels than the placebo group at week 8 (P = 0.03). With respect to antioxidant enzyme activity, subjects in the Q10-150 group had significantly higher CAT (P = 0.03) and SOD (P = 0.03) activity than the placebo group at week 12. The plasma coenzyme Q10 concentration was significantly correlated with MDA levels (r = -0.35, P = 0.02) and CAT (r = 0.43, P = 0.01) and SOD activity (r = 0.39, P = 0.01). The ratio of plasma coenzyme Q10 to total cholesterol was significantly correlated with SOD activity (r = 0.39, P = 0.02). The ratio of plasma coenzyme Q10 to low-density lipoprotein was significantly correlated with CAT (r = 0.35, P = 0.04) and SOD (r = 0.45, P = 0.01) activity. However, there was no relation between coenzyme Q10 concentration and glutathione peroxidase activity. Coenzyme Q10 supplements at a dose of 150 mg can decrease oxidative stress and increase antioxidant enzyme activity in patients with CAD. A higher dose of coenzyme Q10 supplements (>150 mg/d) might promote rapid and sustainable antioxidation in patients with CAD.
... Previously, elegant single-turnover kinetics on Rhodobacter complex III in chromatophores defined K M (Q 10 H 2 ) = 3-5 Q 10 H 2 per complex, but using a "collisional mechanism" in which the rate is determined by [Q 10 H 2 ] and unaffected by [Q 10 ]. [26] For complex I, a value of K M (Q 10 ) = 2.4 AE 1.7 nmol/ mg protein (consistent with our value) was reported using pentane-extracted mitochondria (but with the redox state of the Q pool undefined). [27] Figure 2 also shows our value for complex I turnover in B. taurus heart mitochondrial membranes that contain approximately 12 nmol Q 10 /mg phospholipid (ca. 60 Q 10 per complex I; see Table S2). ...
Article
Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone-10 (Q10 ) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10 , and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10 , and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10 H2 to Q10 . AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined KM value for Q10 . The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
... Previously,e legant single-turnover kinetics on Rhodobacter complex III in chromatophores defined K M (Q 10 H 2 ) = 3-5 Q 10 H 2 per complex, but using a" collisional mechanism" in which the rate is determined by [Q 10 H 2 ]a nd unaffected by [Q 10 ]. [26] Forc omplex I, av alue of K M (Q 10 ) = 2.4 AE 1.7 nmol/ mg protein (consistent with our value) was reported using pentane-extracted mitochondria (but with the redox state of the Qp ool undefined). [27] Figure 2also shows our value for complex Iturnover in B. taurus heart mitochondrial membranes that contain approximately 12 nmol Q 10 /mg phospholipid (ca. 60 Q 10 per complex I; see Table S2). ...
Article
Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone-10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined KM value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
... An increase can be attributed to an enhanced ETC. Estornell et al. reported a correlation between CoQ10 concentration and respiratory rate which led to the assumption that physiological CoQ10 concentrations do not saturate the respiratory chain [33,34]. us, CoQ10 administration was expected to induce an increase of mitochondrial respiration. ...
Article
Full-text available
The process of aging is characterized by the increase of age-associated disorders as well as severe diseases. Due to their role in the oxidative phosphorylation and thus the production of ATP which is crucial for many cellular processes, one reason for this could be found in the mitochondria. The accumulation of reactive oxygen species damaged mitochondrial DNA and proteins can induce mitochondrial dysfunction within the electron transport chain. According to the “mitochondrial theory of aging,” understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general, but the measurement of mitochondrial respiration in skin cells from cell cultures cannot completely reflect the real situation in skin. Here, we describe a new method to measure the mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using a XF24 extracellular flux analyzer to evaluate the effect of coenzyme Q10. We observed a decrease in mitochondrial respiration and ATP production with donor age corresponding to the “mitochondrial theory of aging.” For the first time ex vivo in human epidermis, we could show also a regeneration of mitochondrial respiratory parameters if the reduced form of coenzyme Q10, ubiquinol, was administered. In conclusion, an age-related decrease in mitochondrial respiration and ATP production was confirmed. Likewise, an increase in the respiratory parameters by the addition of coenzyme Q10 could also be shown. The fact that there is a significant effect of administered coenzyme Q10 on the respiratory parameters leads to the assumption that this is mainly caused by an increase in the electron transport chain. This method offers the possibility of testing age-dependent effects of various substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.
... In contrast, in the detergent-solubilized bc 1 complex, the enzyme is homogenously dispersed in the aqueous phase, and no strong enrichment of lipophilic compounds is expected. The membrane concentration of native UQ-10 was approximated around 5-10 mM (40,41). ...
... Their endogenous production has been shown to be limited in different diseased conditions and therapeutic supplementation represent an obvious means of re-equilibrating these levels (Miles et al. 2007;Tiano et al. 2012). In particular, for Coenzyme Q10 it is important to take into consideration that the physiological levels within the mitochondrial membranes are in the same concentration-range of the Michaelis-Menten constant (Km) of NADH oxidation at the respiratory complexes, so that even a slight decrease in CoQ10 content could result in a remarkable decrease in the ETC functionality (Estornell et al. 1992). On the other hand, this also means that the physiological concentration is not saturating and even a small increase in the CoQ10 content of mitochondrial membranes could lead to an increased respiratory rate. ...
Chapter
Fanconi Anemia (FA) is a heterogeneous genetic disorder, with 16 genes so far characterized that function in a common pathway for the maintenance of genomic stability. The clinical manifestations are also heterogeneous, although all patients share progression to bone marrow failure (BMF) and increased predisposition to cancer, particularly acute myeloid leukemia. At the cellular level chromosome instability (CI) is the hallmark of FA, and hypersensitivity of FA cells to the clastogenic effect of DNA crosslinking agents, such as diepoxybutane (DEB), provides a specific diagnostic marker. FA cells are characterized by abnormal accumulation of reactive oxygen species (ROS) and dysfunctional response to oxidative stress (OS). They are in a permanent pro- oxidant state, demonstrated by oxidative DNA damage, increased lipid peroxidation, free iron levels, ROS overproduction, mitochondrial dysfunction, and glutathione (GSH) depletion. The hypersensitivity of FA cells to DEB is a consequence of its OS-related mechanism of cytotoxicity. DEB induces oxidative damage by forming DNA-DNA and DNA-protein crosslinks, associated with GSH depletion and activated mitochondrialapoptotic pathway.The progressive BMF and the increased risk of malignancy associated with FA highlights the importance of understanding how the mechanisms involved in cellular defense against DNA damage fail. It was also shown that dysfunction of FA proteins increases OS damage by down-regulation of antioxidant defense genes. Thus, OS-related CI should be counteracted by other sources of antioxidants, independently of the FA pathway.Recently, it was demonstrated that a cocktail with N-acetylcysteine, a GSH repletor and α-lipoic acid, a mitochondrial nutrient, improved genetic stability in vitro, decreasing CI in cultured lymphocytes from FA patients. These two active thiol antioxidants have already been used in many diseases as pro-glutathione dietary supplements. The clinical relevance of this study is suggested, considering the potential use of small molecules as a prophylactic approach to improve the defense against OS-induced cell damage and, consequently, to delay the clinical symptoms associated with this damage.
... This last observation offers a starting point to develop a protective strategy based on CoQ 10 supplementation. In our previous work we showed that the K m value for CoQ 10 in NADH-Cyt c reductase activity approaches the natural CoQ content of BHM, meaning that Complex I works in nonsaturating conditions (Estornell et al. 1992). Thus, in normal conditions, the increase of the mitochondrial ubiquinone content could result in higher oxygen consumption capacity and energy production. ...
Article
Full-text available
Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.
... Their endogenous production has been shown to be limited in different diseased conditions and therapeutic supplementation represent an obvious means of re-equilibrating these levels (Miles et al. 2007; Tiano et al. 2012). In particular, for Coenzyme Q10 it is important to take into consideration that the physiological levels within the mitochondrial membranes are in the same concentration-range of the Michaelis–Menten constant (Km) of NADH oxidation at the respiratory complexes, so that even a slight decrease in CoQ10 content could result in a remarkable decrease in the ETC functionality (Estornell et al. 1992). On the other hand, this also means that the physiological concentration is not saturating and even a small increase in the CoQ10 content of mitochondrial membranes could lead to an increased respiratory rate. ...
... This increment in the values of mitochondrial respiration parameters can be associated to an enhanced electron transport chain (ETC). In fact, a correlation between CoQ concentration and mitochondrial respiratory rate has been reported, since physiological CoQ concentrations do not saturate the MRC [117]. Moreover, ROS accumulation in MERRF iNs suggests a MRC disruption and a leak of electrons that generates superoxide anion with the interaction with O 2 [118]. ...
Article
Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening. https://www.sciencedirect.com/science/article/abs/pii/S0925443920300715?via%3Dihub
... An increase can be attributed to an enhanced ETC. Estornell et al. reported a correlation between CoQ10 concentration and respiratory rate which led to the assumption that physiological CoQ10 concentrations do not saturate the respiratory chain [33,34]. us, CoQ10 administration was expected to induce an increase of mitochondrial respiration. ...
Article
The process of aging is characterized by the increase of age-associated disorders as well as severe diseases. Due to their role in the oxidative phosphorylation and thus the production of ATP which is crucial for many cellular processes, one reason for this could be found in the mitochondria. The accumulation of reactive oxygen species damaged mitochondrial DNA and proteins can induce mitochondrial dysfunction within the electron transport chain. According to the “mitochondrial theory of aging”, understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general, but the measurement of mitochondrial respiration in skin cells from cell cultures cannot completely reflect the real situation in skin. Here, we describe a new method to measure the mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using a XF24 extracellular flux analyzer to evaluate the effect of coenzyme Q10. We observed a decrease in mitochondrial respiration and ATP production with donor age corresponding to the “mitochondrial theory of aging”. For the first time ex vivo in human epidermis, we could show also a regeneration of mitochondrial respiratory parameters if the reduced form of coenzyme Q10, ubiquinol, was administered. In conclusion, an age-related decrease in mitochondrial respiration and ATP production was confirmed. Likewise, an increase in the respiratory parameters by the addition of coenzyme Q10 could also be shown. The fact that there is a significant effect of administered coenzyme Q10 on the respiratory parameters leads to the assumption that this is mainly caused by an increase in the electron transport chain. 'is method offers the possibility of testing age-dependent effects of various substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.
Chapter
Full-text available
For a number of years, coenzyme Q (CoQ10 in humans), was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in plasma, and also extensively investigated its antioxidant role. This chapter discusses the relationship between the acknowledged bioenergetic role of CoQ10 and some clinical effects. The antioxidant properties of CoQ10 are then analyzed especially for their consequences on protection of circulating human low-density lipoproteins and prevention of atherogenesis. The relationship between CoQ10 and statins is also discussed in the light of possible involvement of CoQ10 deficiency in the issue of statin side effects. New aspects of the antioxidant involvement of coenzyme Q are also discussed together with their relevance in cardiovascular disease. Data are reported on the efficacy of CoQ10 in ameliorating endothelial dysfunction in patients affected by ischemic heart disease. Many of the effects of CoQ10, which were classically ascribed to its bioenergetic properties, are now considered as the result of its biochemical interaction with nitric oxide (NO), NO synthase and reactive oxygen species capable of inactivating NO. Clinical studies are reported highlighting the effect of CoQ10 on extracellular SOD, which is deeply involved in endothelial dysfunction. Previous studies have shown decreased levels of CoQ10 in the seminal plasma and sperm cells of infertile men with different kinds of asthenospermia. Research has been extended to supplementation with CoQ10 of infertile men affected by idiopathic asthenozoospermia. CoQ10 levels increased significantly in seminal plasma and sperm cells after 6 months of treatment with concomitant improvement of sperm cell motility.
Article
Coenzyme Q10 (CoQ10) serves as an electron carrier within the mitochondrial respiratory chain (MRC), where it is integrally involved in oxidative phosphorylation and consequently ATP production. It has recently been suggested that phenylketonuria (PKU) patients may be susceptible to a CoQ10 deficiency as a consequence of their phenylalanine‐restricted diet, which avoids foods rich in CoQ10 and its precursors. Furthermore, the high phenylalanine level in PKU patients not on dietary restriction may also result in impaired endogenous CoQ10 production, as previous studies have suggested an inhibitory effect of phenylalanine on HMG‐CoA reductase, the rate‐controlling enzyme in CoQ10 biosynthesis. We investigated the effect of both dietary restriction and elevated plasma phenylalanine concentration on blood mononuclear cell CoQ10 concentration and the activity of MRC complex II+III (succinate:cytochrome‐c reductase; an enzyme that relies on endogenous CoQ10) in a PKU patient population. The concentrations of CoQ10 and MRC complex II+III activity were not found to be significantly different between the PKU patients on dietary restriction, PKU patients off dietary restriction and the control group, although plasma phenylalanine levels were markedly different. The results from this investigation suggest that dietary restriction and the elevated plasma phenylalanine levels of PKU patients do not effect mononuclear cell CoQ10 concentration and consequently the activity of complex II+III of the MRC.
Article
This review considers the interaction of Complex I with different redox acceptors, mainly homologs and analogs of the physiological acceptor, hydrophobic Coenzyme Q. After examining the physical properties of the different quinones and their efficacy in restoring mitochondrial respiration, a survey ensues of the advantages and drawbacks of the quinones commonly used in Complex I activity determination and of their kinetic properties. The available evidence is then displayed on structure–activity relationships of various quinone compounds in terms of electron transfer activity and proton translocation, and the present knowledge is discussed in terms of the nature of multiple quinone-binding sites in the Complex.
Chapter
In this chapter we provide a review with a focus on the function of Coenzyme Q (CoQ, ubiquinone) in mitochondria. The notion of a mobile pool of CoQ in the lipid bilayer as the vehicle of electrons from respiratory complexes has somewhat changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising Complexes I and III; in such assembly the electron transfer is thought to be mediated by direct channelling, and we provide evidence for a kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound CoQ, besides being required for electron transfer from other dehydrogenases to Complex III. CoQ bound to Complex I and to Complex III is also involved in proton translocation; although the mechanism of the Q-cycle is well established for Complex III, the involvement of CoQ in proton translocation by Complex I is still debated. This review also briefly examines some additional roles of CoQ, such as the antioxidant effect of its reduced form and its postulated action at the transcriptional level.
Article
A phenomenon associated with the aging process is a general age-dependent decline in cellular bioenergetic capacity that varies from tissue to tissue and even from cell to cell within the same tissue. This variation eventually forms a tissue bioenergy mosaic. Recent evidence by our group suggests that the accumulation of mitochondrial DNA mutations, in conjunction with a concurrent decrease in full-length mtDNA in tissues such as skeletal and cardiac muscle, strongly correlates with decreased mitochondrial function and accounts for the bioenergy mosaic. Evidence is also presented suggesting that amelioration with coenzyme Q10 may restore some of the age-associated decline in bioenergy function, in effect providing the potential for a “redox therapy”. Coenzyme Q is a naturally occurring material that is present in the membranes of all animal cells. Its primary function is to act as an electron carrier in the mitochondrial electron transport chain enabling the energy from substrates such as fats and sugars (in the form of reducing equivalents) to be ultimately captured in the form of ATP, which in turn may be utilised as a source of cellular bioenergy. Coenzyme Q10 has no known toxic effects and has been used in a limited number of animal studies and human clinical trials; however, the mechanism of action of coenzyme Q10 remains unclear. A series of experiments by this group aimed at determining the efficacy of coenzyme Q10 treatment on ameliorating the bioenergy capacity at the organ and cellular level will also be reviewed.
Article
The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidorectuctase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined. An increase in Complex I-III activity was detected during development (142 ± 10 vs. 447 ± 23 μmol cyt. c/mg/min, p < 0.001) ang ageing (447 ± 23 vs. 713 ± 45 μmol cyt. c/mg//min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity.
Article
In extreme conditions ketosis can progress to ketoacidosis, a dangerous and potentially life-threatening condition. Ketoacidosis is most common in new or poorly treated type 1 diabetes. The acidosis is usually attributed to the ‘acidic’ nature of the ketone bodies (acetoacetate, 3-hydroxybutyrate, and acetone). However, acetoacetate and 3-hydroxybutyrate are produced not as acids but as their conjugate bases, and acetone is neither an acid nor a base. This raises the question of why severe ketosis is accompanied by acidosis. Here, we analyze steps in ketogenesis and identify four potential sources: adipocyte lipolysis, hydrolysis of inorganic pyrophosphate generated during synthesis of fatty acyl-coenzyme A (CoA), the reaction catalyzed by an enzyme in the β-oxidation pathway (3-hydroxyacyl-CoA dehydrogenase), and increased synthesis of CoA.
Article
Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb 561. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.
Article
Ubiquinone (UQ; also known as coenzyme Q; CoQ) is a mobile component of the mitochondrial electron transport chain, where it acts as a pro-oxidant in its ubisemiquinone state. Despite this, UQ is also believed to be a membrane antioxidant. These properties place UQ at the center of hotly debated questions about how mitochondria and reactive oxygen species (ROS) impact aging and disease. New studies using transgenic mouse models have provided unexpected insights into whether, and how, UQ is required in various processes, cell types, and subcellular locations. These studies have not only shed light on the role of mitochondria and ROS in the aging process, but also question the mechanisms of action by which UQ might function as a therapeutic agent.
Article
Methoxymethylation of altholactone (1) led to the corresponding O-methoxymethyl derivative (3) in addition to the unexpected 6,7-dihydro-7-methoxy analogue (4), and two original tetrahydrofuranic (THF) alkyl esters ( 5,6). Moreover, when we accomplished a new method for the preparation of the furano-pyrone goniofupyrone (7) through 7-hydroxylation of 1 in acid medium, a minor compound (8) with an identical skeleton to that of compounds 5 and 6 was identified. Careful examination of the published spectral data of the reported styryl-lactones with an heptolide skeleton reveals that those structures possess also a THF alkyl ester skeleton. The revision of those structures was confirmed by chemical correlation. All altholactone derivatives assayed proved to be specific inhibitors of the mitochondrial complex I.
Article
Coronary artery disease (CAD) is the leading cause of death worldwide. The purpose of this study was to investigate the relationship between plasma levels of coenzyme Q10 and vitamin B-6 and the risk of CAD. Patients with at least 50% stenosis of one major coronary artery identified by cardiac catheterization were assigned to the case group (n = 45). The control group (n = 89) comprised healthy individuals with normal blood biochemistry. The plasma concentrations of coenzyme Q10 and vitamin B-6 (pyridoxal 5'-phosphate) and the lipid profiles of the participants were measured. Subjects with CAD had significantly lower plasma levels of coenzyme Q10 and vitamin B-6 compared to the control group. The plasma coenzyme Q10 concentration (β = 1.06, P = .02) and the ratio of coenzyme Q10 to total cholesterol (β = .28, P = .01) were positively correlated with vitamin B-6 status. Subjects with higher coenzyme Q10 concentration (≥516.0 nmol/L) had a significantly lower risk of CAD, even after adjusting for the risk factors for CAD. Subjects with higher pyridoxal 5'-phosphate concentration (≥59.7 nmol/L) also had a significantly lower risk of CAD, but the relationship lost its statistical significance after adjusting for the risk factors of CAD. There was a significant correlation between the plasma levels of coenzyme Q10 and vitamin B-6 and a reduced risk of CAD. Further study is needed to examine the benefits of administering coenzyme Q10 in combination with vitamin B-6 to CAD patients, especially those with low coenzyme Q10 level.
Article
The development of new anti-neoplastic drugs is a key issue for cancer chemotherapy due to the reality that, most likely, certain cancer cells are resistant to current chemotherapy. The past two decades have witnessed tremendous advances in our understanding of the pathogenesis of cancer. These advances have allowed identification new targets as oncogenes, tumor supressor genes and the possible implication of the mitochondria (Fulda et al. Nat Rev Drug Discov 9:447–464, 2010). Annonaceous Acetogenins (ACGs) have been described as the most potent inhibitors of the respiratory chain because of their interaction with mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116–120, 1994; Zafra-Polo et al. Phytochemistry 42:253–271, 1996; Miyoshi et al. Biochim Biophys Acta 1365:443–452, 1998; Tormo et al. Arch Biochem Biophys 369:119–126, 1999; Motoyama et al. Bioorg Med Chem Lett 12:2089–2092, 2002). To explore a possible application of natural products from Annonaceous plants to cancer treatment, we have selected four bis-tetrahydrofuranic ACGs, three from Annona cherimolia (cherimolin-1, motrilin and laherradurin) and one from Rollinia mucosa (rollinianstatin-1) in order to fully describe their mechanisms responsible within the cell (Fig. 1). In this study, using a hepato-carcinoma cell line (HepG2) as a model, we showed that the bis-THF ACGs caused cell death through the induction of the apoptotic mitochondrial pathway. Their potency and behavior were compared with the classical mitochondrial respiratory chain Complex I inhibitor rotenone in every apoptotic pathway step. Fig. 1 ACGs structures
Article
It was discovered over 60 years ago that the mitochondrial respiratory chain is constituted of a series of protein complexes imbedded in the inner mitochondrial membrane. Experimental evidence has more recently ascertained that the major respiratory complexes involved in energy conservation are assembled as supramolecular units (supercomplexes, SCs) in stoichiometric ratios. The functional role of SCs is less well defined, and still open to discussion. Several lines of evidence favour the concept that electron transfer from Complex I to Complex III operates by channelling of electrons through Coenzyme Q molecules bound to the SC I1III2IVn , in contrast with the previously accepted hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. On the contrary, electron transfer from Complex III to Complex IV seems to operate, at least in mammals, by random diffusion of cytochrome c molecules between the respiratory complexes even if assembled in SCs. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I, that might be important in the regulation of signal transduction from mitochondria. This review discusses physiological and pathological implications of the supercomplex assembly of the respiratory chain.
Article
Abstract Substantial evidence exists that the mitochondrial respiratory chain is organized in supramolecular units called supercomplexes or respirasomes. While the structural evidence of the supercomplexes is overwhelming, fewer studies were addressed to their functional relevance. Although the presence of Coenzyme Q channelling between Complexes I and III is ascertained, no such demonstration has been clearly done for cytochrome c between Complexes III and IV, at least in mammalian mitochondria. This review also discusses the implications concerning the amount of respiratory complexes organized in supercomplexes and the possibility that they represent associations in dynamic equilibrium with the individual complexes.
Book
This book focuses on the use of various molecules with antioxidant properties in the treatment of major male genital tract disorders, especially male infertility, erectile dysfunction, and accessory gland infection. The coverage also includes discussion of pathophysiology, the molecular basis of male infertility, and the rationale for use of antioxidants, with particular attention to coenzyme Q10 and carnitine. Oxidative stress occurs when the production of reactive oxygen species, including free radicals, exceeds the body’s natural antioxidant defences, leading to cellular damage. Oxidative stress is present in about half of all infertile men, and reactive oxygen species can produce infertility both by damaging the sperm membrane, with consequences for sperm motility, and by altering the sperm DNA. There is consequently a clear rationale for the use of antioxidant treatments within andrology, and various in vitro and in vivo studies have indicated that many antioxidants indeed have beneficial impacts. In providing a detailed and up-to-date overview of the subject, this book will be of interest to both practitioners and researchers in andrology, endocrinology, and urology.
Chapter
Human coenzyme Q (CoQ10) or ubiquinone is mainly known for its bioenergetic role as a proton and electron carrier in the inner mitochondrial membrane and is also an endogenous lipophilic antioxidant, ubiquitous in biological membranes. It is also present in plasma lipoproteins, where it plays a well-recognized antioxidant role. More recently coenzyme Q10 was also shown to affect gene expression by modulating the intracellular redox status. Its involvement in many cellular and extracellular functions suggests that its use as a food supplement could be beneficial in conditions associated with increased oxidative stress underlying different pathological conditions. In reproductive biology, CoQ10 has been shown to play a role in fertility of both males and females.
Chapter
Evidence from several investigations demonstrates the existence of supramolecular units of Complex I, Complex III, and multiple copies of Complex IV in mitochondria and indicates that specific respiratory complexes may preferentially associate to form cytochrome-containing supercomplexes in the native membrane. There are now indications thatcardiolipin,a distinctive mitochondrial lipid, stabilizes the respiratory assemblies. The isolated supercomplexes are active with respect to both their component individual complexes and the entire respiratory function that relies on Coenzyme Q and cytochrome c as intermediate substrates. The latter finding argues against previous models of a random distribution of the respiratory complexes in mitochondria. The supercomplex organization is compatible with electron transfer, but experimental evidence is scant for an effective mechanism via substrate channeling compared to free diffusion of substrates in accordance with the random collision model. The finding that Complex I is almost totally associated in a supercomplex with Complex III seems to exclude a role for the ubiquinone pool in physiological electron transfer between these two complexes, whereas it is certainly required for electron transfer from Complex II or from other dehydrogenases to Complex III; likely, only a small fraction of Complex IV forms a functional supercomplex with channeling of cytochrome c. Nevertheless, the supercomplexes may physiologically exist in equilibrium with free complexes (plasticity model). The supercomplex organization appears to prevent excessive generation of reactive oxygen species from the respiratory chain; accordingly, many pathological conditions and the mitochondrial aging phenotype characterized by a loss of supercomplex assembly correlate with mitochondrial dysfunction and increased oxidative stress. Specific metabolic signals may also arise in the cell in response to a tuned production of reactive oxygen species as a consequence of the controlled dynamics of supercomplex assembling/disassembling at different physio-pathological conditions. The present review paper provides an updated and extensive discussion on the subject.
Article
The notion of a mobile pool of coenzyme Q (CoQ) in the lipid bilayer has changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising complexes I and III; in this model, the electron transfer is thought to be mediated by tunneling or microdiffusion, with a clear kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound quinone, besides being required for electron transfer from other dehydrogenases to complex III. The mechanism of CoQ reduction by complex I is analyzed regarding recent developments on the crystallographic structure of the enzyme, also in relation to the capacity of complex I to generate superoxide. Although the mechanism of the Q-cycle is well established for complex III, involvement of CoQ in proton translocation by complex I is still debated. Some additional roles of CoQ are also examined, such as the antioxidant effect of its reduced form and the capacity to bind the permeability transition pore and the mitochondrial uncoupling proteins. Finally, a working hypothesis is advanced on the establishment of a vicious circle of oxidative stress and supercomplex disorganization in pathological states, as in neurodegeneration and cancer.
Article
Full-text available
The consequence of blocking the de novo synthesis of ubiquinone (coenzyme Q) on mitochondrial ubiquinone content and respiratory function was studied in cultured C1300 (Neuro 2A) murine neuroblastoma cells. Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was used to suppress the synthesis of mevalonate, an essential precursor for the isoprenoid side chain of ubiquinone. At a concentration of 25 microM, mevinolin completely inhibited the incorporation of [3H]acetate into ubiquinone, isolated from cell extracts by two-dimensional thin-layer chromatography. Similar results were obtained when [14C]tyrosine was used as a precursor for the quinone ring. Through the use of reverse-phase thin-layer chromatography, it was established that the principal product of the ubiquinone pathway in murine neuroblastoma cells was ubiquinone-9. Inhibition of ubiquinone synthesis for 24h in cells cultured in the presence of 10% fetal calf serum (which contains 0.14 nmol of ubiquinone/ml of serum) resulted in a 40-57% decline in the concentration of ubiquinone in the mitochondria. However, the activities of succinate-cytochrome c reductase and succinate dehydrogenase in whole-cell homogenates or mitochondria were not inhibited. The state 3 and uncoupled rates of respiration, determined by polarographic measurements of oxygen consumption in homogenates and mitochondria, were elevated slightly in the mevinolin-treated cells. The data demonstrate that, although mevalonate synthesis is important for the maintenance of the intramitochondrial ubiquinone pool in cultured cells, major changes in the ubiquinone content of the mitochondria can occur in intact cells without perturbation of respiratory function. However, the coincidence of decreased mitochondrial ubiquinone concentration and the inhibition of cell cycling previously observed in mevinolin-treated cells (Maltese, W.A. (1984) Biochem. Biophys. Res. Commun. 120, 454-460) suggests that the availability of ubiquinone may play a role in the regulation of mitochondrial and cellular proliferation.
Chapter
This chapter investigates the preparation, properties, and conditions for assay of mitochondria. Three procedures are described for the isolation of mitochondria from slaughterhouse material. These procedures for the isolation of beef heart mitochondria differ only in the manner in which the heart mince is homogenized. The fractionation of the mitochondria and washing steps are the same in the three procedures. Procedure 1 dislodges the loosely packed damaged mitochondria, and the mixture is decanted and discarded. A portion of the light beef heart mitochondria (LBHM) adheres to the wall of the centrifuge tube, and it can be removed with the aid of a glass-stirring rod. Procedure 2 involves the use of a proteolytic enzyme to aid in the homogenization of the heart mince, and has the advantage of producing a higher yield of mitochondria than the first procedure. Mitochondria prepared by this procedure manifest higher respiratory control ratios. Procedure 3 leads to the formation of a large proportion of damaged mitochondria (LBHM) but has the advantage that large amounts of material can be worked up at one time. This method uses a Waring blendor to homogenize the heart mince. Mitochondria that are prepared and assayed with distilled water that had been passed through two ion exchange beds consistently exhibit P : O ratios greater than 3 for pyruvate, β-hydroxybutarate, α-ketoglutarate and greater than 2 for succinate.
Chapter
This chapter discusses the mitochondrial respiratory control and the polarographic measurement of ADP : O ratios. The polarographic oxygen electrode technique is used for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems. Although the polarographic method measures changes in oxygen concentration of photosynthetic systems, yeast cells, and nerve, but the oxygen electrode technique is applied to a study the mitochondrial respiration and oxidative phosphorytation. The principle of the oxygen electrode has been summarized, and the design of the vibrating oxygen electrode for use with speetrophotometric studies is illustrated. The oxygen electrode apparatus can be calibrated in a number of ways. A more accurate calibration of oxygen content can be obtained by gas equilibration with various nitrogen-oxygen mixtures. When tightly coupled mitochondria are suspended in an isotonic buffer, a slow rate of oxygen uptake is measured in the presence of substrate and absence of ADP. Addition of ADP causes an immediate increase in the rate of oxygen utilization. The concentration of oxygen utilized is proportional to the amount of ADP phosphorylated to ATP. The type of oxygen electrode tracings is presented from which an ADP : O ratio (equivalent to a P : O ratio) can be directly calculated.
Article
In succinoxidase, CoQ1 had low activity, and CoQ2 through CoQ10 had comparable activities in both the acetone- and pentane-extracted systems. In the DPNH-oxidase system, CoQ1 had low activity; CoQ2, CoQ3, and CoQ4 had about one-fourth the activity; and CoQ5 and CoQ6 had about one-half the activity of CoQ10. CoQ7, CoQ8, and CoQ9 had activity comparable with that of CoQ10.Hexahydro- and octahydrocoenzyme Q4 had activities comparable with that of CoQ4 and CoQ10 for succinoxidase, but about 10–15% that of CoQ10 for DPNH-oxidase. The double bond in the isoprenoid unit adjacent to the nucleus, and possible cyclization, are not necessary for activity. 2,3-Dimethoxy-5-methyl-6-heptadecyl-l,4-benzoquinone shows succinoxidase and DPNH-oxidase activities comparable with that of hexahydrocoeuzyme Q4. The 5-methyl group is not essential for succinoxidase activity, but has some significance for DPNH-oxidase. A tertiary hydroxyl group in the isoprenoid unit adjacent to the nucleus abolishes activity for succinoxidase but not completely for DPNH-oxidase. Rhodoquinone had no activity in the succinoxidase system and 25% that of CoQ10 in the DPNH-oxidase system.Representatives of biosynthetic precursors, 2-methoxy-5-methyl-6-phytyl-1,4-benzoquinone and 2-methoxy-3-hydroxy-5-methyl-6-phytyl-1,4-benzoquinone, had no activity in either the succinoxidase or DPNH-oxidase systems. Four compounds having a mechanistic relationship between CoQ and vitamin E had no activity. These data support the concept of two sites for the electron transfer of CoQ. The site for CoQ in succinoxidase is not very specific in the structural and steric requirement for the isoprenoid side chain, and has an electron potentiality that is not met by rhodoquinone. The site for CoQ in DPNH-oxidase has a rather specific requirement for a given length of side chain, and an electron potentiality that is significantly met by rhodoquinone.
Article
We have combined a rapid-quenching protocol with HPLC analysis to measure the kinetics of reduction of coenzyme Q in a mitochondrial enzyme complex. The method has a time resolution of several milliseconds and will readily measure 1–20 nmol of the Q derivatives under investigation. By changing the HPLC solvent, either Q6 or Q10 can be studied.
Article
1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358--368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.
Article
The Coenzyme Q homologs having short isoprenoid chains are much less efficient than the higher homologs in restoring NADH oxidation in pentane-extracted lyophilized beef heart mitochondria; they have however high restoring activity for succinate oxidation. The same pattern is observed in pentane extracted submitochondrial particles ETP only if the quinones are added to detergent-treated membranes, showing that in ETP there is a decreased accessibility of the long chain quinones in comparison with the lower homologs. In intact mitochondria and ETP, CoQ3 inhibits NADH oxidation while leaving succinate oxidation unaffected; the inhibition of NADH oxidation by CoQ3 is not reversed by serum albumin but is reversed by CoQ7, particularly when the membrane has been previously “opened” with deoxycholate. CoQ3 may accept electrons from NADH in cyanide-inhibited ETP, allowing coupling at the first phosphorylation site as shown by the quenching of the fluorescence of atebrine. The mechanism of CoQ3 inhibition is probably related to its insufficient rate of reoxidation by the following segment of the respiratory chain when it has been reduced by NADH dehydrogenase.
Article
Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ9/CoQ10 molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt. c reductase, NADH-cyt. c reductase, succinate-DCIP, ubiquinol2-cyt. c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ2H2-cyt. c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23-24 degrees C.
Article
The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 109 M−1 sec−1 corresponding to a diffusion coefficient in the range of 10−6 cm2/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10−9 cm2/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.
Article
The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.
Article
The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The incorporation of small amounts of ubiquinone (Q) into pentane extracted submitochondrial particles has been studied. A procedure is described which allows nearly quantitative incorporation of as little as 0.2 to 0.3 nmol Q/mg protein, i.e., less than 10% of the Q present in the lyophilized particles prior to extraction. It is shown that nicotinamide adenine nucleotide (hydrolized) (NADH) oxidase and succinate oxidase activities can be restored to 100% of that in lyophilized particles by incorporation of 6 to 8 nmol Q/mg protein. The relative activities of both oxidases show a parallel response to an increase of the Q content of the particles between 0.2 and 22 nmol Q/mg protein, 50% reactivation being obtained at about 2 nmol Q/mg protein. 50 to 60% of the incorporated Q can be reduced by NADH or succinate, independent of the total concentration of Q in the membrane. Similar reduction values were obtained for lyophilized particles prior to extraction, suggesting that incorporated Q functions similarly to that originally present in the particles. Succinate dehydrogenase activity, which is decreased 505 by extraction of Q, can be completely restored upon incorporation of only 1.5 nmol Q/mg protein. Extraction of lyophilized particles with pentane + 10% acetone results in a more effective removal of Q, but also in a differential Q requirement for NADH oxidase and succinate oxidase. Thin layer chromatography shows that extraction with pentane + acetone removes an additional, unidentified, nonpolar lipid. The present data do not support the belief that succinate oxidase and NADH oxidase communicate with separate pools of Q.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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