Mitochondria, Oxidants, and Aging

The free radical theory of aging postulates that the production of intracellular reactive oxygen species is the major determinant of life span. Numerous cell culture, invertebrate, and mammalian models exist that lend support to this half-century-old hypothesis. Here we review the evidence that both supports and conflicts with the free radical theory and examine the growing link between mitochondrial metabolism, oxidant formation, and the biology of aging. The free radical theory of aging postulates that the production of intracellular reactive oxygen species is the major determinant of life span. Numerous cell culture, invertebrate, and mammalian models exist that lend support to this half-century-old hypothesis. Here we review the evidence that both supports and conflicts with the free radical theory and examine the growing link between mitochondrial metabolism, oxidant formation, and the biology of aging. Many believe that the seeds of aging can be traced back to a chance encounter that occurred sometime between one and two billion years ago. The event of note involved the presumed incorporation of an uninvited eubacteria into an Archea-type host. This was presumably not the first such encounter between a host cell and invading bacteria. Nonetheless, it is generally presumed that previous interactions had led to the death of the invader, the host, or more likely both organisms. However, on this day, rather then a case of mutual destruction, the results of this incursion led to an agreement between host and bacteria that has persisted more or less intact until this day. Although most of the initial details are unknown, over time, the two initial antagonists began to rely on each other in ever more intricate and interconnected ways. The eubacteria is believed to have evolved into the mitochondria, able to safely replicate within limits inside its host. Slowly, as it became more comfortable in its new environment, more and more responsibility for its own replication and maintenance was shifted to the cell. Indeed, today, of the thousand or so proteins that make up the mitochondria, only a handful are still encoded by mitochondrial DNA. The host, too, began to rely more heavily on its onetime invader. It began to see the advantage of specialization and compartmentalization. Although the cell still retained the capacity to produce energy independent of the mitochondria, more and more of the day-to-day responsibility was turned over to this new organelle. Suddenly, the ability of these onetime unwanted invaders to efficiently produce chemical energy seemed to allow the possibility of powerful muscles or prodigiously beating hearts. Our story might have ended there, a heart-warming tale of two potential enemies that joined forces to work together for the common good. Yet old habits are indeed hard to break. And although it is true that, on that day, the invading eubacteria did not immediately kill its host, it has also become increasingly clear that it may not have entered the agreement with full disclosure. For, although mitochondria are marvels of energy production, they also have another, less beneficial legacy in the cell. Increasingly, this other property, the continuous production of potentially harmful reactive oxygen species (ROS), has become a central focus of aging research. The purpose of this review is therefore to reexamine the terms of a now nearly two billion-year-old agreement and in particular to evaluate to what degree the mitochondrial production of ROS and the cellular response to oxidative stress may be a major determinant of how we age. It has been nearly 50 years since Harman proposed the “free radical theory” of aging (Harman, 1956Harman D. Aging: a theory based on free radical and radiation chemistry.J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (5753) Google Scholar). The initial theory suggested that aging, as well as the associated degenerative diseases, could be attributed to deleterious effects of free radicals on various cell components. When originally proposed, the notion that cells actually produced free radicals remained unproven and hotly debated. Even after Harman’s proposal, this controversy raged for the next decade or so, until it was ultimately settled with the discovery of the enzyme superoxide dismutase (McCord and Fridovich, 1969McCord J.M. Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). The existence of an intracellular enzyme whose sole function was to scavenge superoxide seemed indirect but powerful evidence that cells must continuously produce their own free radicals. Nonetheless, with relatively little direct experimental support, Harman initially speculated that the source of age-inducing free radicals was most likely “the interaction of the respiratory enzymes involved in the direct utilization of molecular oxygen.” Such a hypothesis was also generally consistent with early theories suggesting a correlation between overall metabolic rate and life span. In the 1920s, Pearl proposed the “rate of living” hypothesis that directly linked metabolic rate with the longevity of an organism (Pearl, 1928Pearl R. The Rate of Living. University of London Press, London1928Google Scholar). Pearl was unclear what the precise mechanism was that linked metabolism with life span and therefore suggested that some vital cellular element was somehow consumed in proportion to overall metabolic rate. His concept was that, when this unknown but vital element was exhausted, death occurred. Since Harman’s initial formulation, ensuing experimentation has solidified but not proven his underlying theory. Today, although aerobic metabolism and the corresponding generation of ROS remain the most widely accepted cause of aging, substantial gaps and unknowns persist. Indeed, fundamental questions regarding what governs the relationship between overall metabolic rate and the production of ROS remain unclear. Perhaps more important, relatively little is known about what are the relevant intracellular targets for ROS and how oxidative modification of these targets might influence life span. Fifty years after its formulation, we now know that cells make free radicals on a continuous basis, yet we are still uncertain as to whether they cause or merely correlate with aging. ROS are generated in multiple compartments and by multiple enzymes within the cell. Important contributions include proteins within the plasma membrane, such as the growing family of NADPH oxidases (Lambeth, 2004Lambeth J.D. NOX enzymes and the biology of reactive oxygen.Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2188) Google Scholar); lipid metabolism within the peroxisomes; as well as the activity of various cytosolic enzymes such as cyclooxygenases. Although all these sources contribute to the overall oxidative burden, the vast majority of cellular ROS (estimated at approximately 90%) can be traced back to the mitochondria. The generation of mitochondrial ROS is a consequence of oxidative phosphorylation, a process that uses the controlled oxidation of NADH or FADH to generate a potential energy for protons (ΔΨ) across the mitochondrial inner membrane. This potential energy is in turn used to phosphorylate ADP via the F1-F0 ATPase. At several sites along the cytochrome chain, electrons derived from NADH or FADH can directly react with oxygen or other electron acceptors and generate free radicals. In the past, the generation of ROS or other free radicals was thought of as “slippage” or an unproductive side reaction. More recently, as will be discussed later in more detail, it has been proposed that mitochondrial ROS may actually be important in various redox-dependent signaling processes (Nemoto et al., 2000Nemoto S. Takeda K. Yu Z.X. Ferrans V.J. Finkel T. Role for mitochondrial oxidants as regulators of cellular metabolism.Mol. Cell. Biol. 2000; 20: 7311-7318Crossref PubMed Scopus (307) Google Scholar, Werner and Werb, 2002Werner E. Werb Z. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases.J. Cell Biol. 2002; 158: 357-368Crossref PubMed Scopus (181) Google Scholar, Dada et al., 2003Dada L.A. Chandel N.S. Ridge K.M. Pedemonte C. Bertorello A.M. Sznajder J.I. Hypoxia-induced endocytosis of Na, K-ATPase in aveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta.J. Clin. Invest. 2003; 111: 1057-1064Crossref PubMed Scopus (204) Google Scholar) as well as in the aging clock. A schematic diagram of the flow of electrons through the cytochrome chain is presented in Figure 1. NADH, generated by associated Krebs cycle dehydrogenases (DH) (Robinson and Srere, 1985Robinson Jr., J.B. Srere P.A. Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria.J. Biol. Chem. 1985; 260: 10800-10805Abstract Full Text PDF PubMed Google Scholar), is initially oxidized at site I. As the electrons from NADH are passed to the first mobile electron acceptor, oxidized coenzyme Q, the energy is dissipated by the ejection of protons. Coenzyme Q can also accept electrons from the site II complex donated by FADH, thereby bypassing site I and one proton ejection site. Coenzyme Q next donates electrons to cytochrome b in site III in a near potential energy neutral process. In site III, the electrons are passed to cytochrome c1 with the dissipative ejection of protons. Cytochrome c1 transfers its electrons to the second mobile element in the cytochrome chain, cytochrome c. Cytochrome c in turn reduces cytochrome a,a3 (i.e., cytochrome oxidase [COX] referring to the terminal electron acceptor) in site IV, which ultimately reduces molecular oxygen to form water. This final dissipation of the redox energy in NADH/FADH at site IV is also associated with a final ejection of protons. In this manner, the cytochrome chain transforms the redox energy of the rather stable molecules NADH and FADH into a ΔΨ across the inner mitochondrial. In this complex reaction sequence, several important questions naturally arise: Where are ROS generated? What is the basal rate of mitochondrial ROS generation? What regulates mitochondria ROS generation? How are ROS eliminated? The two major sites for ROS generation are believed to be at sites I and III where large changes in the potential energy of the electrons, relative to the reduction of oxygen, occur (see Figure 1). Experimental manipulations that increase the redox potential of site I (Kushnareva et al., 2002Kushnareva Y. Murphy A.N. Andreyev A. Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.Biochem. J. 2002; 368: 545-553Crossref PubMed Scopus (481) Google Scholar) or site III (Chen et al., 2003Chen Q. Vazquez E.J. Moghaddas S. Hoppel C.L. Lesnefsky E.J. Production of reactive oxygen species by mitochondria: central role of complex III.J. Biol. Chem. 2003; 278: 36027-36031Crossref PubMed Scopus (1118) Google Scholar) generally increase the rate of ROS generation, supporting the notion that the redox potential of these reactive sites is important in free radical formation. Site I remains the least understood of the cytochrome chain elements (Yagi and Matsuno-Yagi, 2003Yagi T. Matsuno-Yagi A. The proton-translocating NADH-quinone oxidoreductase in the respiratory chain: the secret unlocked.Biochemistry. 2003; 42: 2266-2274Crossref PubMed Scopus (253) Google Scholar). This multisubunit complex is believed to be composed of ∼46 proteins with a combined molecular weight exceeding 1 MDa and is thought to contain at least one bound flavin mononucleotide (FMN) and eight iron sulfur groups. Both the iron sulfur groups (Genova et al., 2001Genova M.L. Ventura B. Giuliano G. Bovina C. Formiggini G. Parenti C.G. Lenaz G. The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2.FEBS Lett. 2001; 505: 364-368Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) and FMN sites have been implicated in ROS generation (Liu et al., 2002Liu Y. Fiskum G. Schubert D. Generation of reactive oxygen species by the mitochondrial electron transport chain.J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (848) Google Scholar). At site III, an even more complicated story is found with the Q cycle contributing to the generation of O2− through reduced ubisemiquinone either on the inner or outer membrane surfaces (Turrens et al., 1985Turrens J.F. Alexandre A. Lehninger A.L. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria.Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1004) Google Scholar, Aguilaniu et al., 2003Aguilaniu H. Gustafsson L. Rigoulet M. Nystrom T. Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.Science. 2003; 299: 1751-1753Crossref PubMed Scopus (475) Google Scholar ). Surprisingly, the terminal oxidation step at cytochrome oxidase is not believed to be a significant source of ROS in intact systems, despite the fact that this site is capable of in vitro ROS generation (Fridovich and Handler, 1961Fridovich I. Handler P. Detection of free radicals generated during enzymic oxidations by the initiation of sulfite oxidation.J. Biol. Chem. 1961; 236: 1836-1840Abstract Full Text PDF PubMed Google Scholar). Since the precise mechanisms of ROS generation are unknown, we can construct a simple relationship that might globally describe the generation of ROS in the mitochondria. First, we will define the term Eox as the net driving force for the reduction of oxygen. Eox can be estimated as the difference between the redox potential for donating a single electron to oxygen (Eo = −160 mV, Wood, 1988Wood P.M. The potential diagram for oxygen at pH 7.Biochem. J. 1988; 253: 287-289Crossref PubMed Scopus (377) Google Scholar) and the redox potential of a particular electron donor at a given reaction site. Also important is the oxygen tension, PO2, and a hypothetical first order reaction constant, Kox, resulting in the following simple equation for total net ROS generation (QROS): QROS≈∑site+nsite(KOXsiteEOXsite[site])×PO2(1) In the equation, “site” represents all of the mitochondrial ROS-generating sites in a given cell. Since the concentration of aqueous O2 is significantly higher than that of O2− or H2O2 (Cadenas and Davies, 2000Cadenas E. Davies K.J. Mitochondrial free radical generation, oxidative stress, and aging.Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2055) Google Scholar), under most conditions, the reverse reaction rate can be ignored. Based on this simplistic approach, any perturbation to oxidative phosphorylation that changes these terms, including the number of mitochondria or cytochrome chain equivalents within a cell, would increase the production of ROS. The regulation of QROS can be evaluated for each one of these elements. For instance, putting the mitochondria in a reduced state without ADP or Pi for oxidative phosphorylation (state 4) (Loschen et al., 1971Loschen G. Flohe L. Chance B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria.FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (406) Google Scholar) or the addition of electron transport inhibitors that secondarily increase the Eox of site I or site III all tend to increase QROS (Staniek and Nohl, 2000Staniek K. Nohl H. Are mitochondria a permanent source of reactive oxygen species?.Biochim. Biophys. Acta. 2000; 1460: 268-275Crossref PubMed Scopus (123) Google Scholar, Aguilaniu et al., 2003Aguilaniu H. Gustafsson L. Rigoulet M. Nystrom T. Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.Science. 2003; 299: 1751-1753Crossref PubMed Scopus (475) Google Scholar, Loschen et al., 1971Loschen G. Flohe L. Chance B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria.FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (406) Google Scholar ). Experimentally, a large increase in ROS formation is often seen in the condition known as reverse electron flow. This is usually achieved when a site II substrate, succinate, is added in the presence of a site III inhibitor, thereby generating a reverse flow of electrons from site II to site I (Loschen et al., 1971Loschen G. Flohe L. Chance B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria.FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (406) Google Scholar, Aguilaniu et al., 2003Aguilaniu H. Gustafsson L. Rigoulet M. Nystrom T. Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.Science. 2003; 299: 1751-1753Crossref PubMed Scopus (475) Google Scholar ). Reverse electron flow might also be responsible for the high ROS generation occurring with fatty acid oxidation that also generates electrons for site II via FADH (Boveris et al., 1972Boveris A. Oshino N. Chance B. The cellular production of hydrogen peroxide.Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1127) Google Scholar, Boveris and Chance, 1973Boveris A. Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen.Biochem. J. 1973; 134: 707-716Crossref PubMed Scopus (1930) Google Scholar). Another “endogenous” example in which Eox and ROS are increased is with the release of cytochrome c during apoptosis. In this condition, the absence of cytochrome c results in a block in electron flow and a rise in the Eox of site I (Kushnareva et al., 2002Kushnareva Y. Murphy A.N. Andreyev A. Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.Biochem. J. 2002; 368: 545-553Crossref PubMed Scopus (481) Google Scholar) with a subsequent rise in ROS produced at this site (Kushnareva et al., 2002Kushnareva Y. Murphy A.N. Andreyev A. Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.Biochem. J. 2002; 368: 545-553Crossref PubMed Scopus (481) Google Scholar). This does not appear to happen at site III since in the absence of electron transport there is also an inhibition of Q cycle turnover that is required for generating the active ubisemiquinone (Turrens et al., 1985Turrens J.F. Alexandre A. Lehninger A.L. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria.Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1004) Google Scholar). Finally, another important physiological regulator of Eox is the family of uncoupling proteins (UCPs) (Echtay et al., 2002Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. et al.Superoxide activates mitochondrial uncoupling proteins.Nature. 2002; 415: 96-99Crossref PubMed Scopus (1044) Google Scholar, Casteilla et al., 2001Casteilla L. Rigoulet M. Penicaud L. Mitochondrial ROS metabolism: modulation by uncoupling proteins.IUBMB Life. 2001; 52: 181-188Crossref PubMed Scopus (137) Google Scholar). Since Eox and ΔΨ are coupled through the proton ejection process, these parameters are proportional under most conditions. If ΔΨ is reduced by the action of uncoupling proteins “leaking” protons across the inner membrane (see Figure 1), then predictably Eox and therefore QROS would be decreased. Thus, uncoupling has been proposed as an important mechanism to reduce ROS levels (Casteilla et al., 2001Casteilla L. Rigoulet M. Penicaud L. Mitochondrial ROS metabolism: modulation by uncoupling proteins.IUBMB Life. 2001; 52: 181-188Crossref PubMed Scopus (137) Google Scholar, Brand et al., 2004Brand M.D. Affourtit C. Esteves T.C. Green K. Lambert A.J. Miwa S. Pakay J.L. Parker N. Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins.Free Radic. Biol. Med. 2004; 37: 755-767Crossref PubMed Scopus (741) Google Scholar). Interestingly, UCPs might also be directly activated by superoxide anions, thereby providing an overall feedback circuit for ROS production (Echtay et al., 2002Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. et al.Superoxide activates mitochondrial uncoupling proteins.Nature. 2002; 415: 96-99Crossref PubMed Scopus (1044) Google Scholar). An additional recent proposal for ROS regulation is that the entry of electrons into and through the cytochrome chain, especially at the level of the DH-site I complex, is highly regulated. This electron gate would presumably only permit oxidative phosphorylation to occur when it was required by cellular energetic needs (Bose et al., 2003Bose S. French S. Evans F.J. Joubert F. Balaban R.S. Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate.J. Biol. Chem. 2003; 278: 39155-39165Crossref PubMed Scopus (157) Google Scholar, Joubert et al., 2004Joubert F. Fales H.M. Wen H. Combs C.A. Balaban R.S. NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP): applications to enzyme and mitochondrial reaction kinetics, in vitro.Biophys. J. 2004; 86: 629-645Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This mechanism potentially permits the modulation of oxidant formation without the requirement to dissipate ΔΨ through the energetically nonproductive action of UCPs. There is also growing appreciation that the activity of components of the electron transport chain can also be altered by covalent modification and that these modifications may be important in ROS formation (Ludwig et al., 2001Ludwig B. Bender E. Arnold S. Huttemann M. Lee I. Kadenbach B. Cytochrome C oxidase and the regulation of oxidative phosphorylation.ChemBioChem. 2001; 2: 392-403Crossref PubMed Scopus (163) Google Scholar). Finally, oxygen tension represents another variable known to regulate the rate of mitochondrial ROS production (Turrens et al., 1982Turrens J.F. Freeman B.A. Crapo J.D. Hyperoxia increases H2O2 release by lung mitochondria and microsomes.Arch. Biochem. Biophys. 1982; 217: 411-421Crossref PubMed Scopus (277) Google Scholar). The mechanisms underlying the matching of tissue oxygen level to metabolic demand are poorly understood. Yet it is clear that tissue pO2 can be dynamically regulated, and, indeed, the increase in venous oxygen content observed during brain activation is the physiological parameter that forms the basis for fMRI detection (Ogawa et al., 1992Ogawa S. Tank D.W. Menon R. Ellermann J.M. Kim S.G. Merkle H. Ugurbil K. Intrinsic signal changes accompanying sensory stimulation: functional brain mapping with magnetic resonance imaging.Proc. Natl. Acad. Sci. USA. 1992; 89: 5951-5955Crossref PubMed Scopus (2648) Google Scholar). From this brief review, it is clear that there are indeed many factors that can regulate mitochondria ROS generation. Presumably, this complexity contributes in part to the numerous conflicting reports in the literature regarding the nature, control, and degree of mitochondrial ROS generation. The above discussion does, however, raise an important caveat concerning the relationship between oxidant formation and metabolic rate (i.e., oxygen consumption). In general, the higher the rate of oxidative phosphorylation and oxygen consumption, the lower the overall value of cytochrome chain Eox. Consistent with this notion was the early observation obtained from isolated mitochondria that augmenting oxidative phosphorylation resulted in a reduction, not an augmentation, of ROS generation (Loschen et al., 1971Loschen G. Flohe L. Chance B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria.FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (406) Google Scholar). Although it is commonly assumed that an increase in oxygen consumption produces an increase in ROS production, we would argue that this positive correlation is only true if the increase in oxygen consumption was secondary to a higher tissue pO2 or an increase in the number of “sites”, i.e., functional mitochondria. In contrast, an increase in oxygen consumption in the setting of constant tissue oxygen concentrations and a fixed number of mitochondria would favor a decrease in ROS levels. There is a wide variance in the literature regarding what percentage of basal mitochondrial oxygen consumption ultimately leads to ROS generation. This is not surprising, since most of these conclusions are based on isolated mitochondria studies in which the Eox of many of the redox elements were held at very unphysiological states or under conditions in which reverse electron flow was possible. Based on these initial observations (for review, see Chance et al., 1979Chance B. Sies H. Boveris A. Hydroperoxide metabolism in mammalian organs.Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4530) Google Scholar), it was suggested that ∼2% of the total oxygen consumption was funneled to ROS generation. This number has been widely cited despite the fact that, even in these early studies, it was appreciated that the ROS measurements were made under artificial conditions. Subsequent studies, under more physiological conditions, have reduced this basal value to ∼0.2% (Staniek and Nohl, 2000Staniek K. Nohl H. Are mitochondria a permanent source of reactive oxygen species?.Biochim. Biophys. Acta. 2000; 1460: 268-275Crossref PubMed Scopus (123) Google Scholar, Aguilaniu et al., 2003Aguilaniu H. Gustafsson L. Rigoulet M. Nystrom T. Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.Science. 2003; 299: 1751-1753Crossref PubMed Scopus (475) Google Scholar ). This lower value does not imply that the baseline production is unimportant but does suggest that the mitochondrial ROS load on most tissues may not be as severe as once thought. The discovery of superoxide dismutase (SOD), as discussed previously, was a major step in establishing the generation of ROS or H2O2 in the mitochondria. Two intracellular SOD enzymes exist within the cell: SOD2, a manganese-dependent enzyme in the matrix, and SOD1, a copper-containing enzyme primarily in the cytosol. Both of these enzymes convert O2− into H2O2 that is then further deactivated by catalase (Radi et al., 1991Radi R. Turrens J.F. Chang L.Y. Bush K.M. Crapo J.D. Freeman B.A. Detection of catalase in rat heart mitochondria.J. Biol. Chem. 1991; 266: 22028-22034Abstract Full Text PDF PubMed Google Scholar) to water and oxygen or by the various glutathione peroxidases to reduced glutathione and water. The discovery of the peroxiredoxins (Chang et al., 2004Chang T.S. Cho C.S. Park S. Yu S. Kang S.W. Rhee S.G. Peroxiredoxin III, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria.J. Biol. Chem. 2004; 279: 41975-41984https://doi.org/10.1074/jbc.M407707200.Abstract Full Text Full Text PDF PubMed Google Scholar) represents yet another family of important scavenging enzymes in the mitochondria (Taylor et al., 2003Taylor S.W. Fahy E. Zhang B. Glenn G.M. Warnock D.E. Wiley S. Murphy A.N. Gaucher S.P. Capaldi R.A. Gibson B.W. Ghosh S.S. Characterization of the human heart mitochondrial proteome.Nat. Biotechnol. 2003; 21: 281-286Crossref PubMed Scopus (566) Google Scholar). Superoxide that is not immediately scavenged can directly react with oxidized cytochrome c (Joubert et al., 2004Joubert F. Fales H.M. Wen H. Combs C.A. Balaban R.S. NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP): applications to enzyme and mitochondrial reaction kinetics, in vitro.Biophys. 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USA. 1988; 85: 6465-6467Crossref PubMed Scopus (1388) Google Scholar). Reasons for these differences are thought to include the proximity of mitochondrial DNA to the source of oxidants and the lack of any protective histone covering. This postulated and observed increased sensitivity o