Dihydrolipoic acid maintains ubiquinone in the antioxidant active form by two-electron reduction of ubiquinone and one-electron reduction of ubisemiquinone

Dihydrolipoic acid (DHLA) is a constituent of cellular energy metabolism, where it cycles between the oxidized and reduced form. The two thiol residues of DHLA make this biomolecule susceptible to most radical species and prevent Fenton-type reactions by chelating free iron. In this study we present a novel mode of action by which DHLA exerts antioxidant function in combination with coenzyme Q (ubiquinone). DHLA was found to reduce ubiquinone to ubiquinol by the transfer of a pair of electrons, thereby increasing the antioxidant capacity of coenzyme Q in biomembranes. In addition, ubisemiquinone, which was earlier shown to be an active oxygen radical source when existing in the anionic form, is removed from equilibrium by the addition of a single electron from DHLA. The high reactivity of DHLA with this potentially deleterious ubisemiquinone species not only prevents the formation of prooxidants, it also keeps ubiquinone in its antioxidant active form. Experimental data of this study demonstrate a superadditive effect of ubiquinone in combination with DHLA in preventing peroxidation of biomembranes.     

α-Tocopherol is an effective Phase II enzyme inducer: protective effects on acrolein-induced oxidative stress and mitochondrial dysfunction in human retinal pigment epithelial cells

Vitamin E has long been identified as a major lipid-soluble chain-breaking antioxidant in mammals. α-Tocopherol is a vitamin E component and the major form in the human body. We propose that, besides its direct chain-breaking antioxidant activity, α-tocopherol may exert an indirect antioxidant activity by enhancing the cell's antioxidant system as a Phase II enzyme inducer. We investigated α-tocopherol's inducing effect on Phase II enzymes and its protective effect on acrolein-induced toxicity in a human retinal pigment epithelial (RPE) cell line, ARPE-19. Acrolein, a major component of cigarette smoke and also a product of lipid peroxidation, at 75 μmol/L over 24 h, caused significant loss of ARPE-19 cell viability, increased oxidative damage, decreased antioxidant defense, inactivation of the Keap1/Nrf2 pathway, and mitochondrial dysfunction. ARPE-19 cells have been used as a model of smoking- and age-related macular degeneration. Pretreatment with α-tocopherol activated the Keap1/Nrf2 pathway by increasing Nrf2 expression and inducing its translocation to the nucleus. Consequently, the expression and/or activity of the following Phase II enzymes increased: glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase 1, heme-oxygenase 1, glutathione S-transferase and superoxide dismutase; total antioxidant capacity and glutathione also increased. This antioxidant defense enhancement protected ARPE-19 cells from an acrolein-induced decrease in cell viability, lowered reactive oxygen species and protein oxidation levels, and improved mitochondrial function. These results suggest that α-tocopherol protects ARPE-19 cells from acrolein-induced cellular toxicity, not only as a chain-breaking antioxidant, but also as a Phase II enzyme inducer.     

Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties

With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-alpha. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-alpha. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.     

Effect of exercise training on tissue vitamin E and ubiquinone content

Endurance exercise training led to an adaptive increase in the ubiquinone content and cytochrome c reductase activity of red quadriceps and soleus muscles and adipose tissues, but not of cardiac or white quadriceps muscle. These findings are consistent with the well-known positive adaptation of skeletal muscle mitochondria to endurance training. However, there was no concomitant increase in the vitamin E content of tissues, which showed an increase in mitochondrial content. Since ubiquinone is located in the mitochondrial inner membrane and the major pool of vitamin E is also associated with mitochondrial membranes, the results suggest that training causes a substantial decrease in vitamin E concentration in the proliferating muscle mitochondrial membranes, thus depleting muscle mitochondria of their major lipid antioxidant. Since vitamin E is the major cellular, lipid-soluble, chain-breaking antioxidant, these findings indicate increased free radical reactions in the tissues of exercising animals.     

Proton-coupled electron-transfer mechanism for the radical scavenging activity of cardiovascular drug dipyridamole

Dipyridamole (DIP) is a well-known pharmaceutical drug used as a coronary vasodilator and anti-platelet agent in clinics for treating several cardiovascular diseases. Primarily, the therapeutic effects of the drug are attributed to its antioxidant potency. In this research, we aim to declare the unknown antioxidant mechanism of DIP as well as its potent chain-breaking antioxidant activity in polar aqueous medium inside the cells, using different experimental methods and theoretical quantum calculations. Data demonstrated the higher antioxidant capacity of DIP against ROS and free radicals in polar cell's interior. DIP is capable of generating long living and noninvasive DIP(?) radicals in oxidant condition that leads to an effective "chain-breaking antioxidant" activity. Quantum computational data indicated that DIP antioxidant has more favorable ionization potential than trolox which means DIP has higher antioxidant activity. Also, data showed that the direct hydrogen-transfer is not a favorable process to construct DIP(?) because of high barrier energy, though electron-transfer process can more easily to produce DIP(?+) with the lowest barrier energy. Altogether, the electron donating potency of DIP to reduce ferric ion, having the low anodic oxidation peak potential, producing long lived stable DIP(?) radicals and protecting myoblast cells from oxidation, proposed the excellent "chain-breaking antioxidant" potency via electron-transfer mechanism of this vasodilator DIP drug in polar aqueous medium.     

The multiple functions of coenzyme Q

The coenzyme function of ubiquinone was subject of extensive studies in mitochondria since more than 40 years. The catalytic activity of ubiquinone (UQ) in electron transfer and proton translocation in cooperation with mitochondrial dehydrogenases and cytochromes contributes essentially to the bioenergetic activity of ATP synthesis. In the past two decades UQ was recognized to exert activities which differ from coenzyme functions in mitochondria. From extraction/reincorporation experiments B. Chance has drawn the conclusion that redox-cycling of mitochondrial ubiquinone supplies electrons for univalent reduction of dioxygen. The likelihood of O2(.-) release as normal byproduct of respiration was based on the existence of mitochondrial SOD and the fact that mitochondrial oxygen turnover accounts for more than 90% of total cellular oxygen consumption. Arguments disproving this concept are based on results obtained from a novel noninvasive, more sensitive detection method of activated oxygen species and novel experimental approaches, which threw light into the underlying mechanism of UQ-mediated oxygen activation. Single electrons for O2(.-) formation are exclusively provided by deprotonated ubisemiquinones. Impediment of redox-interaction with the bc1 complex in mitochondria or the lack of stabilizing interactions with redox-partners are promotors of autoxidation. The latter accounts for autoxidation of antioxidant-derived ubisemiquinones in biomembranes, which do not recycle oxidized ubiquinols. Also O2(.-)-derived H2O2 was found to interact with ubisemiquinones both in mitochondria and nonrecycling biomembranes when ubiquinol was active as antioxidant. The catalysis of reductive homolytic cleavage of H2O2, which contributes to HO. formation in biological systems was confirmed under defined chemical conditions in a homogenous reduction system. Apart from dioxygen and hydrogen peroxide we will provide evidence that also nitrite may chemically interact with the ubiquinol/bc1 redox couple in mitochondria. The reaction product NO was reported elsewhere to be a significant bioregulator of the mitochondrial respiration and O2 activation. Another novel finding documents the bioenergetic role of UQ in lysosomal proton intransport. A lysosomal chain of redox couples will be presented, which includes UQ and which requires oxygen as the terminal electron acceptor.     

Lysosomal ROS formation

Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone has bioenergetic and pathophysiological functions, unusually high levels of ubiquinone have also been reported in Golgi vesicles and lysosomes. In lysosomes, the interior differs from other organelles in its low pH value which is important to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of an FAD-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced giving rise to the release of HO radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated.     

Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency

The chain-breaking (peroxyl radical-trapping) antioxidant activity of plasma obtained from several patients with a very severe vitamin E deficiency has been measured. The total chain-breaking antioxidant activity in lipid extracts has been shown to be approximately equal to the concentration of vitamin E. For whole plasma there is no significant difference in the concentrations of water-soluble, chain-breaking antioxidants between the E-deficient patients and healthy adults. It is concluded that even in cases of very severe vitamin E deficiency the requirement for this vitamin is not met by some other exogenous or endogenous antioxidant.     

Tracing the tail of ubiquinone in mitochondrial complex I

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron–sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30 Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.     

The existence and significance of redox-cycling ubiquinone in lysosomes

Summary Ubiquinone is inhomogeneously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone was demonstrated to exert bioenergetic and pathophysiological functions, unusually high levels of ubiquinone were also reported to exist in Golgi vesicles and lysosomes. In lysosomes the interior differs from other organelles by the low pH value which is important not only to arrest proteins but also to ensure optimal activity of proteases. Since redox cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is a part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier reported to exist in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of a flavin adenine dinucleotide-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of ab-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor thereby also regulating acidification of the lysosomal matrix. The role of the proton-pumping redox chain has to be elucidated.Abbreviations DMPO 5,5-dimethyl-1-pyrroline N-oxide - ESR electron spin resonance - FAD flavin adenine dinucleotide - UQ ubiquinone     

Tightly-bound ubiquinone in the Escherichia coli respiratory Complex I

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.     

A dual role for lecithin:cholesterol acyltransferase (EC 2.3.1.43) in lipoprotein oxidation

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis.     

Proton Pumping of Mitochondrial Complex I: Differential Activation by Analogs of Ubiquinone

As part of the ongoing studies aimed at elucidating the mechanism of the energy conserving function of mitochondrial complex I, NADH: ubiquinone (Q) reductase, we have investigated how short-chain Q analogs activate the proton pumping function of this complex. Using a pH-sensitive fluorescent dye we have monitored both the extent and initial velocity of proton pumping of complex I in submitochondrial particles. The results are consistent with two sites of interaction of Q analogs with complex I, each having different proton pumping capacity. One is the physiological site which leads to a rapid proton pumping and a stoichiometric consumption of NADH associated with the reduction of the most hydrophobic Q analogs. Of these, heptyl-Q appears to be the most efficient substrate in the assay of proton pumping. Q analogs with a short-chain of less than six carbons interact with a second site which drives a slow proton pumping activity associated with NADH oxidation that is overstoichiometric to the reduced quinone acceptor. This activity is also nonphysiological, since hydrophilic Q analogs show little or no respiratory control ratio of their NADH:Q reductase activity, contrary to hydrophobic Q analogs.     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.     

Ubiquinone is reduced by lipoamide dehydrogenase and this reaction is potently stimulated by zinc

Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 microM in the presence and 4 microM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.     

Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol.

d-Alpha-tocopherol (2R,4'R,8'R-Alpha-tocopherol) and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but differing in their hydrocarbon "tail": tocopherol with a saturated and toctrienol with an unsaturated isoprenoid chain. d-Alpha-tocopherol has the highest vitamin E activity, while d-alpha-tocotrienol manifests only about 30% of this activity. Since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes, we studied alpha-tocotrienol as compared to alpha-tocopherol under conditions which are important for their antioxidant function. d-Alpha-tocotrienol possesses 40-60 times higher antioxidant activity against (Fe2+ + ascorbate)- and (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomal membranes and 6.5 times better protection of cytochrome P-450 against oxidative damage than d-alpha-tocopherol. To clarify the mechanisms responsible for the much higher antioxidant potency of d-alpha-tocotrienol compared to d-alpha-tocopherol, ESR studies were performed of recycling efficiency of the chromanols from their chromanoxyl radicals. 1H-NMR measurements of lipid molecular mobility in liposomes containing chromanols, and fluorescence measurements which reveal the uniformity of distribution (clusterizations) of chromanols in the lipid bilayer. From the results, we concluded that this higher antioxidant potency of d-alpha-tocotrienol is due to the combined effects of three properties exhibited by d-alpha-tocotrienol as compared to d-alpha-tocopherol: (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. The data presented show that there is a considerable discrepancy between the relative in vitro antioxidant activity of d-alpha-tocopherol and d-alpha-tocotrienol with the conventional bioassays of their vitamin activity.     

Toward a Characterization of the Connecting Module of Complex I

Complex I [NADH–ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB–NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.     

Retinal coenzyme Q in the bovine eye

Coenzyme Q plays an integral role in oxygen metabolism and management, and there is a positive correlation between low tissue coenzyme Q concentrations and the progression of many degenerative diseases. Retinal oxidative damage plays a role in the pathogenesis of many degenerative eye diseases; nevertheless, despite the retina's high rate of oxygen metabolism, there is little data relating to retinal coenzyme Q concentrations. In this study, we quantified coenzyme Q in the model bovine eye and determined whether it could function as a retinal lipid antioxidant. We found that the neural retina's ubiquinone concentration exceeded those of the vitreous humor, lens, choroid, and extraocular muscle, but it was lower than those measured in heart, kidney, liver, and brain tissues. Ubiquinol was found to be as effective as vitamin E as a retinal lipid antioxidant. The overall relatively low levels of ubiquinone found in the retina, coupled with the retina's need for lipid antioxidants and oxidative metabolism, suggests that retinal function might be sensitive to changes in ubiquinone concentrations.     

Human cultured skin fibroblasts survive profound inherited ubiquinone depletion

Beside its role in electron transfer in the mitochondrial respiratory chain, ubiquinone is known to prevent lipid peroxidation and DNA damage by trapping cellular free radicals. Thanks to its antioxidant properties, ubiquinone may represent an important factor controlling both necrotic and apoptotic processes. We have investigated the consequences of a profound inherited ubiquinone depletion on cultured skin fibroblasts of a patient presenting with encephalomyopathy. Interestingly, cell respiration, mitochondrial oxidation of various substrates, and cell growth of ubiquinone-deficient fibroblasts were only partially decreased. Moreover, these cells did not apparently overproduce superoxide anions or lipoperoxides. Finally, apoptosis did not increase as compared to control, even after serum deprivation. These observations suggest that ubiquinone may not play a major role in the antioxidant defenses of cultured fibroblasts and that its role in controlling oxidative stress and apoptosis may greatly vary across cell types, especially as not all tissues were equally affected in the patient despite the widespread ubiquinone depletion in vivo.