The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one-electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe-S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Decreased Pasteur effect in platelets of aged individuals.

We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

Effect of the oxidative stress induced by adriamycin on rat hepatocyte bioenergetics during ageing.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on mitochondrial functions. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor's age, whilst the hydrogen peroxide is significantly higher in aged than in adult rat hepatocytes. Kinetic studies in isolated mitochondria show that the mitochondrial respiratory chain activity (NADH --> O2) of 50 microM adriamycin-treated hepatocytes is lowered both in adult and aged rats. The same adriamycin concentration induces a slight decrease of the maximal rate of ATP hydrolysis in both young and aged rats, without affecting the Km for the substrate. However, at drug concentrations lower than 50 microM, both ATPase and NADH oxidation activities decrease significantly in aged rats only. The results suggest that free radicals increase during ageing in rat hepatocytes but are unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that some effects of the drug may be due to other reasons besides oxidative stress.

Mitochondria, oxidative stress, and antioxidant defences.

Mitochondria are strongly involved in production of reactive oxygen species, considered today as the main pathogenic agent of many diseases. A vicious circle of oxidative stress and damage to cellular structures can lead to either cell death by apoptosis or to a cellular energetic decline and ageing. The early involvement of mitochondria in apoptosis includes expression of pro-apoptotic factors, release of cytochrome c from the inter-membrane space and opening of the permeability transition pore: cytochrome c release appears to precede pore opening. The mitochondrial theory of ageing considers somatic mutations (deletions) of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, Complex I appears to be mostly affected. We have developed the Pasteur effect (enhancement of lactate production by mitochondrial inhibition) as a bio-marker of mitochondrial bioenergetics in human platelets, and found it to be decreased in aged individuals. Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants coenzyme Q is the only one of endogenous biosynthesis; exogenous coenzyme Q, however, may protect cells from oxidative stress in vivo.

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.

Virgin olive oil and coenzyme Q10 protect heart mitochondria from peroxidative damage during aging.

The mitochondrial theory of aging suggests that this phenomenon is the consequence of random somatic mutations in mitochondrial DNA, induced by long-term exposure to free radical attack. There are two potential dietary means of delaying the effects of free radicals on cellular aging, i.e., enrichment of mitochondrial membranes with monounsaturated fatty acids and supplementation with antioxidants. We have performed a preliminary study on male rats, 6 or 12 month old, fed with diets differing in the nature of the fat (virgin olive oil or sunflower oil) and/or with antioxidant supplementation (coenzyme Q10), analysing hydroperoxide and coenzyme Q9 and Q10 in heart mitochondria. Preliminary results allow us to conclude that the CoQ10 dietetic supplementation as well as the enrichment of the cellular membranes with monounsaturated fatty acids, successfully protect mitochondrial membranes from aged rats against the free radical insult.

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 microM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Oxidative stress, antioxidant defences and aging.

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.

Mitochondrial complex I defects in aging.

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.

Coenzyme Q deficiency in mitochondria: kinetic saturation versus physical saturation.

The coenzyme Q (CoQ) concentration in the inner membrane of beef heart mitochondria is not kinetically saturating for NADH oxidation inasmuch as the K(m) of NADH oxidation for endogenous CoQ10 is in the mM range in membrane lipids. Using CoQ1 as an electron acceptor from complex I, we have found additional evidence that the high Km of NADH oxidase for CoQ is not an artifact due to the use of organic solvents in reconstitution studies. We have also obtained experimental evidence that CoQ concentration may be rendered more rate-limiting for NADH oxidation either by a decrease of CoQ content (as in liver regeneration or under an acute oxidative stress), or by a possible increase of the Km for CoQ, as in some mitochondrial diseases and ageing. The possibility of enhancing the rate of NADH oxidation by CoQ therapy is hindered by the fact that the CoQ concentration in mitochondria appears to be regulated by its mixability with the membrane phospholipids. Nevertheless CoQ10 incorporated into heart submitochondrial particles by sonication enhances NADH oxidation (but not succinate oxidation) up to twofold. Nontoxic CoQ homologs and analogs having shorter side-chains with respect to CoQ10 can be incorporated in the mitochondrial membrane without sonication, supporting an enhancement of NADH oxidation rate above 'physiological' values. It is worth investigating whether this approach can have a therapeutical value in vivo in mitochondrial bioenergetic disorders.

Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles.

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kröger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.

Inhibitor sensitivity of respiratory complex I in human platelets: a possible biomarker of ageing.

NADH-Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19-30 years) individuals and 18 pools from aged ones (66-107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Major changes in complex I activity in mitochondria from aged rats may not be detected by direct assay of NADH:coenzyme Q reductase.

We have investigated the respiratory activities and the concentrations of respiratory chain components of mitochondria isolated from the livers and hearts of two groups of rats aged 6 and 24 months respectively. In comparison with the adult controls (6 months), in aged rats there was a decline in total aerobic NADH oxidation in both tissues; only minor (non-significant) changes, however, were found in NADH:coenzyme Q reductase and cytochrome oxidase activities, and there was no change in ubiquinol-cytochrome c reductase activity. The coenzyme Q levels were slightly decreased in mitochondria from both organs of aged rats. The lowered NADH oxidase activity is not due to the slight decrease observed in the coenzyme Q levels, but is the result of decreased Complex I activity. Since the assay of NADH:coenzyme Q reductase requires quinone analogues, none of which can evoke its maximal turnover [Estornell, Fato, Pallotti and Lenaz (1993) FEBS Lett. 332, 127-131], its activity has been calculated indirectly by taking advantage of the relationship that exists between NADH oxidation and ubiquinol oxidation through the coenzyme Q pool. The results, expressed in this way, show a drastic loss of activity of Complex I in both the heart and the liver of aged animals in comparison with adult controls.

Underevaluation of complex I activity by the direct assay of NADH-coenzyme Q reductase in rat liver mitochondria.

We have shown that the rate of NADH-coenzyme Q reductase in rat liver mitochondria, assayed using the decyl-ubiquinone analog DB, is underevaluated, probably as a result of its low water solubility. In view of drawbacks encountered using other more soluble acceptors in this system, we demonstrate that the most reliable assay of the physiological rate of CoQ reduction by Complex I is the indirect calculation from the total rate of NADH oxidation and the rate of ubiquinol oxidation, using the pool equation of Kröger and Klingenberg [(1973) Eur. J. Biochem. 34, 358-368].

Mitochondrial activities of rat heart during ageing.

Some analytical and functional parameters of rat heart mitochondrial have been investigated at six different periods of ageing from 2 to 26 months. The fatty acid composition of the mitochondrial membranes reveals a percentage increase of polyunsaturated fatty acids (20:4 n-6, 22:6 n-3) up to 12 months, followed by a decrease; however, fluorescence polarization of the membrane probe diphenylhexatriene is not changed, revealing that membrane fluidity is not significantly affected. No major change in ubiquinone-9 and in cytochrome content is apparent, indicating that the relative ratio of the respiratory chain components is unmodified. Nevertheless, significant changes in enzyme specific activities are detected: NADH cytochrome c reductase and cytochrome oxidase activities increase up to 12 months, then decrease at 18-26 months; ubiquinol cytochrome c reductase exhibits a peak at 18 months, followed by a decrease. All these activities follow a similar trend during the whole life span of the rat, even though the 'maximum' is different. No significant changes have been found in ATP synthase. Succinate-cytochrome c reductase steadily increases over the whole life span. The results, showing activity decreases in the respiratory enzymes having subunits encoded by mitochondrial DNA, are compatible with the 'mitochondrial' theory of ageing.

Protective effect of exogenous coenzyme Q against damage by adriamycin in perfused rat liver.

We have investigated the effect of rat liver perfusion with adriamycin on mitochondrial activities. Although the perfusion treatment per se induces some decline of respiratory activities, adriamycin strongly potentiates this effect; moreover the coenzyme Q9 content of the mitochondrial membrane is significantly lowered by the antibiotic. Coaddition of coenzyme Q10 in the perfusate significantly protects the mitochondria, not only from loss of respiratory activities but also of the endogenous CoQ9 content. Exogenous CoQ10 fails to enhance respiratory activities in control rats, not treated with adriamycin, even though CoQ concentration has been proven not to be kinetically saturating in the respiratory chain under physiological conditions. Thus, the beneficial effect of CoQ10 in the perfusate does not appear to be the result of its role in the respiratory chain but is a consequence of its antioxidant action.

An updating of the biochemical function of coenzyme Q in mitochondria.

The apparent Km for coenzyme Q10 in NADH oxidation by coenzyme Q (CoQ)-extracted beef heart mitochondria is close to their CoQ content, whereas both succinate and glycerol-3-phosphate oxidation (the latter measured in hamster brown adipose tissue mitochondria) are almost saturated at physiological CoQ concentration. Attempts to enhance NADH oxidation rate by excess CoQ incorporation in vitro were only partially successful: the reason is in the limited amount of CoQ10 that can be incorporated in monomeric form, as shown by lack of fluorescence quenching of membrane fluorescent probes; at difference with CoQ10, CoQ5 quenches probe fluorescence and likewise enhances NADH oxidation rate above normal. Attempts to enhance the CoQ content in perfused rat liver and in isolated hepatocytes failed to show uptake in the purified mitochondrial fraction. Nevertheless CoQ cellular uptake is able to protect mitochondrial activities. Incubation of hepatocytes with adriamycin induces loss of respiration and mitochondrial potential measured in whole cells by flow cytometry using rhodamine 123 as a probe: concomitant incubation with CoQ10 completely protects both respiration and potential. An experimental study of aging in the rat has shown some decrease of mitochondrial CoQ content in heart, and less in liver and skeletal muscle. In spite of the little change observed, it is reasoned that CoQ administration may be beneficial in the elderly, owing to the increased demand for antioxidants.

Tryptophan phosphorescence as a structural probe of mitochondrial F1-ATPase epsilon-subunit.

We report the detection of tryptophan phosphorescence emission from the sole residue in the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the epsilon-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the epsilon-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the epsilon-subunit, whereas the opposite occurs when Mg-ATP is added to nucleotide-depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the epsilon-subunit.