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HEME OXYGENASE

Mitochondrial Production of Oxygen Radical Species and the Role of Coenzyme Q as an Antioxidant

Dipartimento di Biochimica "G. Moruzzi", University of Bologna, 40126 Bologna, Italy; and
^* Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università di Parma, 43100 Parma, Italy

Abstract

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe–S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.

Key Words: oxidative stress • mitochondria • Complex I • Coenzyme Q

Reactive oxygen species (ROS) are considered as a major etiological and/or pathogenic agent of most diseases (1); moreover, ROS are involved in the progressive deterioration of cell structures accompanying aging (2–4). The free radical theory of aging (5, 6) is based on the idea that cells, continuously exposed to ROS, are progressively damaged in their most vital macromolecules. The implication of mitochondria both as producers and as targets of ROS (4, 7, 8) has been the basis for the mitochondrial theory of aging (9, 10). The theory postulates that random alterations of mitochondrial DNA (mtDNA) in somatic cells are responsible for the energetic decline accompanying senescence. It was proposed that accumulation of somatic mutations of mtDNA, induced by exposure to ROS, leads to errors in the mtDNA-encoded polypeptides (5). These errors are stochastic and randomly transmitted during mitochondrial division and cell division. The consequence of these alterations, which affect exclusively the four mitochondrial complexes involved in energy conservation, would be defective electron transfer and oxidative phosphorylation. Respiratory chain defects may become associated with increased ROS production, thus establishing a vicious circle (11).

If the energetic impairment derives from a stochastic damage to the mitochondrial genes, then it is important to select the mitochondrial activity that is most likely to be affected. Because seven of the 13 structural genes in mtDNA encode for polypeptides in Complex I, then it is Complex I that is most likely to undergo functional alterations (12).

A decrease in individual enzyme activity in a metabolic pathway is meaningful only if it is able to affect the rate of the whole pathway, and this will depend on the degree of flux control exerted by the individual step (13). In the respiratory chain, Complex I is present in the lowest amounts (14) and it is presumably the rate-limiting step of aerobic NADH oxidation. However, this is not true for the oxidation of NAD-linked substrates in phosphorylating mitochondria (15, 16). In mitochondrial diseases, the flux control coefficient at site I in permeabilized cells was found to dramatically increase (17). We have addressed this point in respiration of liver mitochondria from young and old rats and found that the threshold for decrease of NAD-linked state 3 respiration by Complex I inhibition by rotenone was dramatically increased in the older animals (18), indicating that complex I becomes strongly rate-limiting in the old.

Another approach used in our laboratory for recognition of possible early changes not only in postmitotic cells but also in short-living cells, such as blood platelets, has been looking for specific changes linked to subunits encoded for by mtDNA. Similarly to Leber’s hereditary optic neuropathy (LHON) (19), it was found that rotenone sensitivity of NADH CoQ reductase was significantly decreased in platelets from old individuals (20). The same change was exhibited by nonsynaptic mitochondria from rat brain cortex (21) and in rat liver mitochondria (18).

The primary role of ROS in the stochastic theories of aging elicits, on one hand, a better understanding of the cellular sources of these damaging species and of the factors modulating their production, and, on the other, a search for agents capable of controlling excessive ROS production and propagation of ROS-induced damages.

Mitochondrial Complex I as a Source of Superoxide Radical

The mitochondrial respiratory chain is a powerful source of ROS, primarily the superoxide radical and consequently hydrogen peroxide, either as a product of mitochondrial superoxide dismutase (22) or by spontaneous disproportionation. It was calculated that 1–4% of oxygen reacting with the respiratory chain is incompletely reduced to ROS (23). Their production may increase in state 4 with respect to state 3 (24) because oxygen concentration increases and the level of reduced one-electron donors in the respiratory chain is concomitantly increased (25).

There are two major respiratory chain regions where ROS are recognized to be produced (26), one being Complex I (NADH Coenzyme Q reductase) (27–29) and the other complex III (ubiquinol cytochrome c reductase; cf. 4).

In Complex III, antimycin is known not to completely inhibit electron flow from ubiquinol to cytochrome c. The antimycin insensitive reduction of cytochrome c is mediated by superoxide radicals. The source of superoxide in the enzyme may be either cytochrome b₅₆₆ (26), or ubisemiquinone (27), or Rieske’s iron-sulfur center (28). Ubisemiquinone is relatively stable only when protein bound (29); therefore, the CoQ pool in the lipid bilayer is no source of ROS. The role of ubiquinone within ROS production deserves some comments because it has been described both as a pro-oxidant (24, 27, 30) and as a powerful antioxidant (31, 32; see the text below).

Early experiments proved the involvement of Complex I in ROS production (33). The addition of either NADH at low concentration or of NADPH, which feeds the electrons at decreased rate into the complex, led to copious ROS production detected by lipid peroxidation. However, the addition of NADH at high concentrations, but in presence of rotenone, also induced peroxidation. In another study (34), water-soluble CoQ homologs used as electron acceptors from isolated Complex I stimulated H₂O₂ production in the order CoQ₁ > CoQ₀ > CoQ₂, whereas CoQ₆ and CoQ₁₀ were inactive; the rate of H₂O₂ production was partly inhibited by rotenone, indicating that water-soluble quinones may react with oxygen when reduced at sites both prior and subsequent to the rotenone block. There is evidence that the one-electron donor to oxygen in Complex I is a nonphysiological quinone reduction site different from the physiological site(s) (35, 36); the former hydrophilic site reduces several quinones to the corresponding semiquinone forms, which are unstable and can reduce oxygen to superoxide. This mechanism is shared by several quinones, including such drugs as anthracyclines (37) and the clinically used CoQ analog, idebenone (38). However, auto-oxidation of fully reduced quinones (39), such as those formed by NADH CoQ reductase past the rotenone inhibition site, is also a source of ROS, but this effect exclusively pertains to hydrophilic quinones and not to the physiological hydrophobic ubiquinone. Finally, in view of the experiments of Takeshige et al. (33), the hydrophilic, rotenone-insensitive site can apparently reduce oxygen to superoxide in the absence of intermediate acceptors. More recent studies confirmed that Complex I is a major source of superoxide production in several types of mitochondria (40) and localized the oxygen reducing site between the ferricyanide and the quinone reduction sites (41).

Structural changes in Complex I may also enhance ROS production. This has been ascertained in some Complex I defects induced by nuclear DNA mutations (42, 43), but there are also reasons to believe that mitochondrial DNA defects affecting Complex I, such as three mutations causing LHON (44), are accompanied by increased ROS production. Recently, Barrientos and Moraes (45) developed a cellular model of a partial Complex I defect that closely resembles LHON. In this model, besides the respiratory defect, they described increased ROS production and induction of apoptotic cell death. Moreover, a single study showed that viability of LHON cybrids, carrying the common 11778 mutation, had an increased sensitivity to oxidative stress compared to the wild-type parental cell line (46).

We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles (SMP). Complex I of the mitochondrial respiratory chain represents the first site of oxidative phosphorylation. The enzyme is still scarcely understood because of its utter complexity (47): 43 subunits in mammals, seven of which are encoded by mitochondrial DNA, and several prosthetic groups including flavin mononucleotide, at least seven iron-sulfur clusters, and a few molecules of protein-bound CoQ. The natural acceptor is CoQ dissolved in the lipid bilayer (2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone); however, the enzyme assay requires using artificial acceptors, including homologs and analogs of the natural CoQ (mostly CoQ₁₀), binding both the physiological site and additional non-physiological site(s) upstream (48).

Complex I is inhibited by several compounds, almost all binding the hydrophobic subunits of which three classes have been distinguished on the basis of the interaction sites (49). Their use allows to functionally dissect the enzyme in relation to its multiple sites of interaction. Among the inhibitors, there are some short-chain homologs of the natural CoQ, such as CoQ₂ and CoQ₃ (but not CoQ₁) acting both as (poor) acceptors and as inhibitors (48). Present knowledge, in fact, indicates that the features of the side chain in position 6 are critical for CoQ function as electron acceptor (50). The mechanism by which these homologs inhibit is not understood; however, the active form appears to be the reduced one. It is puzzling that analogs having a side chain of the same length but saturated and linear, such as decyl-ubiquinone (DB) are inactive as inhibitors, but very active as acceptors.

We have exploited the effects of different Complex I inhibitors and quinone acceptors to dissect the sites and mechanism of one-electron transfer to oxygen with superoxide formation, investigated by the method of superoxide dismutase-sensitive epinephrine oxidation to adrenochrome (51). To functionally isolate superoxide production by Complex I only, its formation by Complex III was prevented using mucidin, an inhibitor of center o. We avoided using antimycin A because this center i inhibitor is known to enhance superoxide formation (4), and myxothiazol, another center o inhibitor that also inhibits Complex I (52).

The addition of NADH to mucidin-inhibited SMP promotes superoxide formation, which is enhanced to a similar extent by Complex I inhibitors belonging to all three classes and by combinations thereof (Fig. 1A). In contrast to the findings of Barja (51), we observed very low superoxide production in noninhibited SMP. The addition of short-chain analogs and homologs of CoQ enhances superoxide formation of noninhibited Complex I, and this enhancement is further stimulated by Complex I inhibitors (Fig. 1B). Among the quinones tested, idebenone is particularly effective in inducing a dramatic increase of superoxide production (Fig. 1C). Because idebenone is clinically used in mitochondrial cytopathies and neurodegenerative diseases (53), its strong pro-oxidant effect raises doubts on its safety as a drug. However, Complex I inhibitors, acting at the level of iron-sulfur clusters, such as p-hydroxymercuribenzoate (51), inhibited superoxide production. Similar results were obtained with SMP obtained from rat heart and rat liver, although the latter were significantly less active in superoxide formation (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Figure 1. Superoxide radical production by Complex I in bovine SMP. (A) In the presence of 125 µM NADH and no Complex I acceptor; mucidin (1.8 µM) was added to inhibit Complex III in all samples except where indicated (SMP). Inhibitor concentrations: p-hydroxymercuribenzoate (pHMB, FeS cluster inhibitor), 59 µM; rolliniastatin-2 (ROL, center A inhibitor), 0.2 nmol/mg protein; rotenone (ROT, center B inhibitor), 0.2 nmol/mg protein; capsaicin (CAP, center C inhibitor), 4 µmol/mg protein. (B) In the presence of 125 µM NADH, 1.8 µM mucidin and (when indicated) 60 µM decyl-ubiquinone (DB) as acceptor. Inhibitor concentrations: rotenone 0.2 nmol/mg protein; rolliniastatin-2 0.2 nmol/mg protein; myxothiazol (MYX, center C inhibitor), 230 nmol/mg protein. Inhibitor classes are according to the nomenclature of Degli Esposti (49). (C) In the presence of 1.8 µM mucidin and idebenone (IDE, 2 µmol/mg protein).

View larger version (35K): [in this window] [in a new window]   Figure 2. Superoxide production in rat heart (left) and liver (right) submitochondrial particles (SMP) (for abbreviations, see legend of Fig. 1).

View larger version (77K): [in this window] [in a new window]   Figure 3. A model of electron transfer and the site of superoxide production by Complex I. The scheme follows the model of Degli Esposti (49) and depicts FeS cluster N2 as the source of electrons to bound ubiquinone (center B) and to the ubiquinone molecule deriving from the pool (center A). The two deriving semiquinones dismutate so that center B contains oxidized ubiquinone, whereas the reduced ubiquinone (ubiquinol) moves to center C, where it is released to the pool. The effect of different inhibitors and acceptors (see text) is compatible with FeS-cluster N2 as the source of one electron to oxygen or to exogenous quinones (in place of the endogenous bound CoQ), which, in turn, reduce oxygen monoelectronically. Idebenone behaves both as an acceptor and as a type A inhibitor.

View larger version (42K): [in this window] [in a new window]   Figure 4. Effect of CoQ10 extraction and reconstitution on superoxide production by bovine heart SMP. CoQ10 content was checked by high-performance liquid chromatography analysis: CoQ-depleted particles contained a residual amount of 83 pmol/mg protein while the quinone content in the reconstituted particles was 15.7 nmol/mg protein (i.e., almost five times higher than the physiological content).

Cells contain enzymatic systems that are capable of converting ROS into less toxic or nontoxic species (1). The co-ordinate action of superoxide dismutase and peroxidases (glutathione peroxidase being of particular importance) detoxifies superoxide to water. If the action of superoxide dismutase is, however, not accompanied by that of peroxidases, the accumulation of hydrogen peroxide would rather induce production of the damaging hydroxyl radical by means of the Fenton reaction (56).

Other defense systems include metal-binding proteins, which prevent the pro-oxidant action of heavy metals, metabolic intermediates acting as free radical scavengers, and antioxidants, largely taken in by the organism through nutrition, such as ascorbic acid, vitamin E, carotenes, polyphenols, and flavonoids (1).

Among antioxidants, a special position is held by CoQ (CoQ₁₀ in humans), which is the only lipid-soluble antioxidant that is normally synthesized by the organism (57). Its strong hydrophobicity because of the long isoprenoid chain at the 6-position, allows the insertion of the molecule in the membrane phospholipid bilayer.

The biosynthesis of CoQ is particularly complex (58). The benzoquinone ring is synthesized from the essential amino acid phenylalanine up to 4-hydroxy-benzoate, whereas the isoprenoid chain is formed by a pathway common to cholesterol and dolichol biosynthesis. CoQ biosynthesis requires the dietary intake of several vitamin cofactors. It is, therefore, conceivable that one or more such factors may become limiting under physiological or pathological conditions (57), thus, slowing down ubiquinone biosynthesis and inducing a ubiquinone deficiency state.

Besides its bioenergetic role, as a component of the mitochondrial respiratory chain, CoQ is also a component of extra-mitochondrial redox chains (59), whose function, among others, would be to remove excess reducing power formed by glycolysis when mitochondrial respiration is decreased (60, 61).

As an antioxidant, the reduced form of CoQ is exploited either directly upon superoxide or indirectly on lipid radicals (31, 32); ubiquinol can also act together with vitamin E (-tocopherol) by re-generating the active form from the tocopheroxyl radical (62, 63).

The antioxidant action of ubiquinol yields the ubisemiquinone radical. This species is converted back to its antioxidant form by re-reduction, which occurs either through the electron transfer chain in mitochondria, or through various quinone reductases present in different cell fractions (64–66).

Studies in perfused rat liver (67) and in isolated rat hepatocytes (65) clearly show the antioxidant effect of exogenous added CoQ₁₀. The anticancer quinone glycoside, adriamycin, induces oxidative stress by enhancing ROS production in mitochondria and endoplasmic reticulum. In hepatocytes, adriamycin enhances ROS production. Concomitantly endogenous CoQ is re-oxidized and the mitochondrial membrane potential falls. Incubation of the cells with exogenous CoQ₁₀ prevents ROS formation and protects both reduced CoQ and the _mit. The cytosolic enzyme DT-diaphorase seems to be responsible for reduction of both endogenous and exogenous CoQ, as shown by the effect of dicoumarol, an inhibitor of DT-diaphorase, preventing the protective action of exogenous CoQ addition (65). Studies in platelets from transfusional buffy coats also showed protection by added CoQ of the mitochondrial function, investigated by way of the Pasteur effect (68). The protection was enhanced by the addition of an exogenous source of CoQ reduction, such as purified DT-diaphorase in presence of NADPH (Fig. 5).

View larger version (49K): [in this window] [in a new window]   Figure 5. -lactate values in platelets subjected to an oxidative stress and effect of oxidized CoQ in presence and absence of a reducing system. Oxidative stress was accomplished in washed platelets by incubation with 50 mM 2,2'-azobis-2-amidinopropane (AAPH) for 3 hr at 37°C during the incubation for -lactate determination. The samples had been preincubated for 1 hr with or without addition of different CoQ suspensions at room temperature with stirring. The CoQ sample (20 µM) was a soluble formulation, guttaQuinonTM, kindly donated by Dr. F. Enzmann of MSE Pharmazeutika GmbH, Bad Homburg, Germany. The reducing system consisted of 150 µM ß-NADPH and 1000 units of DT-diaphorase (a kind gift from Dr. J. Segura-Aguilar).

If aging is the result of prolonged oxidative stress, an adequate antioxidant supply might contrast the process. The content of vitamin antioxidants depends on dietary intake, and may be subjected to decreases due to deficient intestinal absorption defects and bad dietary habits of the aged. CoQ, being synthesized, is a special case. Some studies have shown a CoQ decrease with age (71, 72); however, this is not true for the brain, where high levels are maintained throughout aging (73, 74) in accordance with the steady level of nonaprenyl-4-hydroxybenzoate transferase (75). Nevertheless, even if the levels of CoQ and other antioxidants do not dramatically fall with aging, we must consider that the antioxidant defenses should actually strongly increase to cope with enhanced oxidative stress.

In some instances of acute or subacute stress, this is actually the case. The CoQ plasma level in the rat doubles by simple sham operation (76), indicating that surgical stress can induce increased ubiquinone release from tissues and/or increased biosynthesis. Conversely, a stronger metabolic stress, such as liver resection, followed by regeneration (76), can exhaust the CoQ biosynthesis capability, yielding lowered plasma CoQ levels.

Senescence is associated with increased incidence of degenerative diseases, such as Parkinson’s and Alzheimer’s diseases and age-linked macular degeneration. These diseases often have a strong genetic component that is, however, associated with exogenous factors, among which oxidative stress and mitochondrial involvement may be major triggering factors (4, 77–79). A decrease of CoQ₁₀ and of antioxidant defenses in plasma was found in our laboratory in patients affected by age-related macular degeneration of the retina (80).

The proposal of antioxidant therapy, as an attempt to reduce or retard the damaging effect of ROS in aging and age-related degenerative diseases, is strongly supported by the excellent results obtained in Alzheimer’s disease and Huntington’s disease (77, 81). The use of CoQ as a dietary supplement requires its distribution to deficient tissues. Dietary CoQ is quickly taken into blood, where it is mainly transported by LDL (82), and is rapidly incorporated in liver and reticulo-endothelial cells. The uptake of CoQ₁₀ by other tissues has not been shown in nutritional experiments in the rat (83), although there are indirect observations suggesting its incorporation in tissues when the endogenous levels are lowered (81). Despite the lack of uptake of dietary CoQ₁₀ by rat heart mitochondria, the exogenous quinone was found to strongly protect mitochondria from oxidative changes (84).

Footnotes

This work was supported in part by PRIN "Longevity Determinants in Humans," MURST, Rome, and by a grant from the International Coenzyme Q10 Association.

¹ To whom requests for reprints should be addressed at Dipartimento di Biochimica "G. Moruzzi", Via Irnerio 48, 40126 Bologna, Italy. E-mail: lenaz{at}biocfarm.unibo.it

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