Superoxide Production by the Mitochondrial Respiratory Chain

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1 Bioscience Reports, Vol. 17, No. 1, 1997 REVIEW Superoxide Production by the Mitochondrial Respiratory Chain Received October 25, 1996 Julio F. Turrens This mini-review describes the role of different mitochondrial components in the formation of reactive oxygen species under normal and pathological conditions and the effect of inhibitors and uncouplers on superoxide formation. KEY WORDS: Mitochondria; reactive oxygen species; superoxide; hydrogen peroxide; superoxide dismutase; catalase; lipid peroxidation. SUPEROXIDE FORMATION IN THE RESPIRATORY CHAIN Most of the oxygen consumed by aerobic organisms is reduced to water by the enzyme cytochrome c oxidase in the terminal reaction of the mitochondrial respiratory chain. Since oxygen in the ground state is in a triplet configuration (with two unpaired electrons in the outer shell) its reduction to water must occur in four consecutive one-electron steps. Some of the partially reduced oxygen intermediates generated in this process are very stable but the enzyme cytochrome c oxidase is able to retain them until all the electrons are transferred. However, a small proportion of the oxygen molecules (1-2% (Boveris et al., 1973)) are converted to superoxide anion radical (Of) by other respiratory components. There are two main sites in the respiratory chain where these reactions occur: Complex I [NADH dehydrogenase, (Turrens et al., 1980)] and Complex III [ubisemiquinone, (Boveris et al., 1976; Cadenas et al., 1977; Turrens et al., 1985)]. Several studies have shown that both sites generate only Of as opposed to a mixture of partially reduced oxygen species, suggesting that the electron donors are either stable radicals or one-electron donors. Hydrogen peroxide is produced as a secondary product (via Of dismutation), and therefore the stoichiometry between Of and H2O2 is close to 2.0 (Cadenas et al., 1977). Department of Biomedical Sciences, University of South Alabama, Mobile, Alabama 36688, USA /97/ $12.50/ Plenum Publishing Corporation

2 4 Turrens EFFECT OF MITOCHONDRIAL INHIBITORS ON SUPEROXIDE FORMATION Superoxide formation by Complexes I and III in the respiratory chain is a non-enzymatic process (equation 1). Thus, this activity should increase by mass action when the electron carriers are highly reduced or in the presence of mitochondrial inhibitors. Superoxide production by mitochondrial electron carriers should also increase with oxygen concentration (equation 1) since cytochrome c oxidase, with a Km for oxygen in the submicromolar range, is practically saturated under physiological conditions and will not compete for the extra oxygen. While Oi formation increases with oxygen concentration [both under normobaric or hyperbaric conditions (Boveris et al., 1973; Chance et al., 1978; Turrens et al., 1982b; Turrens et al., 1982a)], changes in the steady state reduction of mitochondrial respiratory components affect O^ formation in different ways, depending on which carriers are reduced and whether respiratory inhibitors are present. For example, in the absence of inhibitors and under State III conditions (in the presence of oxygen, ADP to phosphorylate and reduction equivalents) the electron carriers are more oxidized and O^ formation is minimal (Boveris et al., 1973). When ASP is exhausted the respiratory chain becomes more reduced (State IV) and O^ formation concomitantly increases (Boveris et al., 1973). In the presence of inhibitors the picture becomes more complicated. For example, rotenone, an inhibitor of Complex I, stimulates O^ formation by the enzyme NADH dehydrogenase (Turrens et al., 1980; Turrens et al., 1982b). Antimycin, an inhibitor of the b - c\ complex, also stimulates OJ formation by both Complex I and III (Boveris et al., 1973, Turrens et al., 1980; Turrens et al., 1982b). However, inhibition of electron flow at any point between the Rieske iron-sulfur protein and oxygen (i.e., by cyanide, myxothiazol or by cytochrome c extraction) stimulates Oj formation by Complex I but blocks this activity in Complex III (Turrens et al., 1982a; Turrens et al., 1982b; Turrens et al., 1985). The inhibitory effect of myxothiazol and cyanide on O^ formation by Complex III suggests that the species responsible for superoxide formation must be one of the intermediates in the Q-cycle (i.e., ubisemiquinone or cytochrome b, Fig. 1). Moreover, when myxothiazol is added to antimycin-blocked mitochondria, cytochrome b remains reduced but O^ formation is blocked (Turrens et al., 1985). This suggests that cytochrome b is not responsible for O^ production since, otherwise, it would become fully oxidized by molecular oxygen before Oj production stops. Thus, the oxidizable species responsible for O^ formation is likely to be ubisemiquinone (Turrens et al., 1985). According to the traditional models of the Q-cycle (Trumpower, 1990), Qo (the ubisemiquinone located on the outer side of the inner membrane) could be the autoxidizable species, with its more negative reduction potential. However, several pieces of evidence suggest that O^ is formed in the inner side of the inner mitochondrial membrane. For example, NADH-dependent OJ formation is

3 Superoxide production by the mitochondrial respiratory chain 5 Fig. 1. Scheme of the electron transport chain and the Q-cycle. The names of several respiratory inhibitors are shown in italics next to the site where they block electron flow. undetectable in intact mitochondria but easily measured in submitochondrial particles (Turrens et al., 1980; Turrens et al., 1982b). Yet, H2O2 formation (the product of Oj" dismutation) is detectable in intact mitochondria as it freely diffuses through the mitochondrial membranes. If OJ were produced on the outer side of the inner mitochondrial membrane, it would not dismute but rather react with cytochrome c regenerating oxygen (k = 107M~' sec"1 (McCord et al., 1969)) and therefore H2O2 could never be detected. UNCOUPLERS MAY STIMULATE OR INHIBIT SUPEROXIDE FORMATION The effect of uncouplers on mitochondrial production of reactive oxygen species also varies depending on whether inhibitors are present in the preparation. Since uncouplers increase the rate of electron transfer in the absence of inhibitors (thus decreasing the steady state reduction of electron carriers), they inhibit OJ formation by mitochondria in State IV (Boveris et al., 1973). Recently Skulachev has proposed that certain hormones (i.e., thyroid hormone) may in vivo uncouple mitochondria reversibly, thus preventing Oj formation occurring under State IV conditions (Skulachev, 1955,1996). The effect of uncouplers is the opposite if the respiratory chain is inhibited. In the presence of antimycin, the autoxidation of ubisemiquinone drives a continuous electron flow to cytochrome c oxidase which slowly create a proton gradient. Thus, uncouplers stimulate Oj" formation in the presence of antimycin (Boveris et al., 1973; Cadenas et al., 1980).

4 6 Turrens ENZYMATIC DEFENSES AGAINST OXIDATIVE STRESS The production of O^ and its dismutation to H2O2 provides the substrates for hydroxyl radical formation, particularly in the presence of transition metals (Halliwell, 1978; Halliwell et al, 1986). In the case of mitochondria, this species may be responsible for the higher (104-fold) proportion of oxidized bases in mitochondrial DNA compared to nuclear DNA (Richter et al., 1988). Nevertheless, mitochondria have several defenses against oxidative stress to maintain a low steady state formation of oxidants. Superoxide anion is eliminated by a specific Mn-containing superoxide dismutase, similar to the bacterial forms but different from the Cu, Zn isozyme found in the cytoplasm (Weisiger et al., 1973; Chance et al., 1979). While the cytosolic form is a constitutive enzyme, the mitochondrial superoxide dismutase may be induced under a variety of circumstances, including hyperoxia, radiation or as a response to cytokines (Stevens et al., 1977; Oberley et al., 1987; Masuda et al., 1988; Wong et al., 1988). Mitochondrial H2O2 is eliminated by glutathione peroxidase (Chance et al., 1979). Heart mitochondria also contain catalase (Radi et al., 1991; Radi et al., 1993), an enzyme that in most other organs is confined to peroxisomes (Chance et al., 1979). In addition, several groups have described a membrane-linked succinatedependent reductase that could be responsible for the detoxification of several radicals in vivo, as well as for the regeneration of reduced vitamin E (Maguire et al, 1989; Packer et al, 1989; Maguire et al., 1992). MITOCHONDRIAL GENERATION OF REACTIVE OXYGEN SPECIES UNDER PATHOLOGICAL CONDITIONS The mitochondrial formation of reactive oxygen species has been implicated in a variety of pathological scenarios. Not only mitochondria are able to generate these species but also, when subjected to oxidative stress, they become uncoupled or inhibited affecting energy production. For example, mitochondria are in part responsible for the cardiotoxic effect of the antitumor agent adriamycin. This drug redox cycle generating Of at the level of Complex I (Marcillat et al., 1989) and the resulting oxidative insult cause heart failure. Under hyperoxic conditions, mitochondrial Oj generation augments linearly with oxygen concentration (Turrens et al., 1982b). This is probably the cause of the initial morphological changes occurring in the lung under hyperoxic conditions which include alterations of mitochondrial shape with appearance of structures resembling lamellar bodies (Crapo et al., 1980). Mitochondria may buffer intracellular Ca2+, particularly at high concentrations since the endoplasmic reticulum has a much lower Km for Ca2+ than mitochondria (Sheu et al., 1994). However, oxidative stress alters mitochondrial Ca2+ homeostasis, particularly affecting the oxidation of specific thiol groups in proteins (Valle et al., 1993). This effect is potentiated by Ca2+. A specific scenario in which this insult may be particularly relevant is after reperfusion of ischemic tissue, in which Ca2+ accumulates and precipates in the matrix (Malis et al., 1988;

5 Superoxide production by the mitochondrial respiratory chain 7 Nomoto et al., 1989; Hearse et al., 1978). Results from our laboratory showed that both mitochondria exposed to high Ca2+ and mitochondria isolated from ischemic tissues show inhibition of electron flow accompanied by an increased generation of H2O2 (Turrens et al., 1991). The mitochondrial production of reactive oxygen species has also been implicated in many other processes including aging (Sohal et al., 1995), apoptosis (Kroemer et al., 1995), alcoholism-related tissue damage (Cardellach et al., 1992). CONCLUSIONS In summary, a small proportion of the oxygen utilized by aerobic cells undergoes partial reduction by respiratory chain components but the toxic effects of these reactive species is prevented by antioxidant defenses. Under pathologic conditions, however, these defenses become overwhelmed, causing oxidative stress and eventually damaging mitochondria and other cell compartments, leading to cell death. REFERENCES Boveris, A. and Chance, B. (1973) Biochem. J. 134: Boveris, A., Cadenas, E. and Stoppani, A. O. M. (1976) Biochem. J. 156: Cadenas, E., Boveris, A., Ragan, C. I. and Stoppani, A. O. M. (1977) Arch. Biochem. Biophys. 180: Cadenas, E. and Boveris, A. (1980) Biochem. J. 188: Cardellach, F., Galofr6, J., Grau, J. M., Casademont, J., Hoek, J. B., Rubin, E. and Urbano-Maquez, A. (1992) Ann. Neural. 31: Chance, B. and Boveris, A. (1978) in: Extrapulmonary Manifestations of Respiratory Diseases (Robin, E. D. ed.), Hyperoxia and Hydroperoxide Metabolism. Marcel Dekker. pp Chance, B., Sies, H. and Boveris, A. (1979) Physiol. Rev. 59: Crapo, J. D., Barry, B. E., Foscue, H. A. and Shelburne, J. (1980) Am. Rev. Respir. Dis. 122: Halliwell, B. (1978) FEBS Lett. 96: Halliwell, B. and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246: Hearse, D. J., Humphrey, S. M. and Bullock, G. R. (1978) J. Mol. Cell. Cardiol. 10: Kroemer, G., Petit, P., Zamzami, N., Vayssiere, J. L. and Mignotte, B. (1995) FASEB J. 9: Maguire, J. J., Wilson, D. L. and Packer, L. (1989) J. Biol. Chem. 264: Maguire, J. J., Kagan, V., Ackrell, B. A. C., Serbinova, E. and Packer, L. (1992) Arch. Biochem. Biophys. 292: Malis, C. D. and Bonventre, J. V. (1988) Prog. Clin. Biol. Res. 282: Marcillat, O., Zhang, Y. and Davies, K. J. A. (1989) Biochem. J. 259: Masuda, A., Longo, D. L., Kobayashi, Y., Appella, E., Oppenheim, J. J. and Matsushima, K. (1988) FASEB J. 2: McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244: Nomoto, K.-I., Mori, N., Miyamoto, J., Shoji, T. and Nakamura, K. (1989) Exp. Mol. Pathol. 51: Oberley, L. W., St. Clair, D. K., Autor, A. P. and Oberley, T. D. (1987) Arch. Biochem. Biophys. 254: Packer, L., Maguire, J. J., Mehlhom, R. J., Serbinova, E. and Kagan, V. E. (1989) Biochem. Biophys. Res. Commun. 159: Radi, R., Turrens, J. F., Chang, L. Y., Bush, K. M., Crapo, J. D. and Freeman, B. A. (1991) J. Biol. Chem. 266: Radi, R., Sims, S., Cassina, A. and Turrens, J. F. (1993) Free Radic. Biol. Med. 15: Richter, C., Park, J. W. and Ames, B. N. (1988) Proc. Natl. Acad. Sci. USA 85:

6 8 Turrens Sheu, S.-S. and Jou, M.-J. (1994) J. Bioenerg. Biomembr. 26: Skulachev, V. P. (1995) Molec. Biol. (Moscow) 29: Skulachev, V. P. (1996) Quart. Rev. Biophys. 29: Sohal, R. S., Sohal, B. H. and Orr, W. C. (1995) Free Radio. Biol. Med. 19: Stevens, J. B. and Autor, A. P. (1977) J. Biol. Chem. 252: Trumpower, B. L. (1990) J. Biol. Chem. 265: Turrens, J. F. and Boveris, A. (1980) Biochem. J. 191: Turrens, J. F., Freeman, B. A. and Crapo, J. D. (1982a) Arch. Biochem. Biophys. 217: Turrens, J. F., Freeman, B. A., Levitt, J. G. and Crapo, J. D. (1982b) Arch. Biochem. Biophys. 217: Turrens, J. F., Alexandre, A. and Lehninger, A. L. (1985) Arch. Biochem. Biophys. 237: Turrens, J. F., Beconi, M., Barilla, J., Chavez, V. B. and McCord, J. M. (1991) Free Rod. Res. Comms : Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R. and Vercesi, A. E. (1993) Arch. Biochem. Biophys. 307:1-7. Weisiger, R. A. and Fridovich, I. (1973) J. Biol. Chem. 248: Wong, G. H. W. and Goeddel, D. V. (1988) Science 242:

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