Mitochondria and reactive oxygen species

Free Radical Biology & Medicine 47 (2009) 333–343

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Review Article

Mitochondria and reactive oxygen species Alicia J. Kowaltowski a, Nadja C. de Souza-Pinto a, Roger F. Castilho b, Anibal E. Vercesi b,⁎ a b

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, 13083-887 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history: Received 19 January 2009 Revised 29 April 2009 Accepted 6 May 2009 Available online 8 May 2009 Keywords: Mitochondria Oxidative stress Free radicals Respiration Uncoupling Electron transport chain Energy metabolism

a b s t r a c t Mitochondria are a quantitatively relevant source of reactive oxygen species (ROS) in the majority of cell types. Here we review the sources and metabolism of ROS in this organelle, including the conditions that regulate the production of these species, such as mild uncoupling, oxygen tension, respiratory inhibition, Ca2+ and K+ transport, and mitochondrial content and morphology. We discuss substrate-, tissue-, and organism-specific characteristics of mitochondrial oxidant generation. Several aspects of the physiological and pathological roles of mitochondrial ROS production are also addressed. © 2009 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . ROS metabolism in mitochondria . . . . . . . . . Sources of mitochondrial ROS . . . . . . . . . . . Tissue-, substrate-, and organism-specific properties Mechanisms controlling ROS release . . . . . . . . Mild mitochondrial uncoupling . . . . . . . . . . Respiratory inhibition . . . . . . . . . . . . . Changes in oxygen tension . . . . . . . . . . Calcium . . . . . . . . . . . . . . . . . . . Mitochondrial content and morphology . . . . Summary and perspectives . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Introduction

Abbreviations: mitoKATP, ATP-sensitive K+ channels; FCCP, carbonyl cyanide p(trifluoromethoxy)phenylhydrazone; COX, cytochrome c oxidase; ETC, electron transport chain; FMN, flavin mononucleotide; LDL, low-density lipoprotein; mtDNA, mitochondrial DNA; ΔΨ, mitochondrial inner membrane potential; MPT, mitochondrial permeability transition; NOS, nitric oxide synthase; UQ, oxidized coenzyme Q; ROS, U reactive oxygen species; UQH2, reduced coenzyme Q; UQ −, semiquinone radical; SOD, superoxide dismutase. ⁎ Corresponding author. Fax: +55 19 3521 9434. E-mail address: [email protected] (A.E. Vercesi). 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.05.004

It has now been over 40 years since H2O2 production from mitochondria was first recorded [1–3], followed quickly by the detection of mitochondrially generated superoxide radical anions U (O2−) [4–6]. These findings, together with the uncovering of a specific, Mn-containing isoform of superoxide dismutase (SOD) in the mitochondrial matrix [7,8], brought attention to this organelle as an important intracellular source of reactive oxygen species (ROS). Indeed, mitochondrial energy metabolism is recognized today as the most quantitatively important source of ROS in the majority of eukaryotic cell types. Mitochondrial ROS are known to be important

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determinants in cell function, participating in many signaling networks and also in a variety of degenerative processes. Here, we critically review aspects concerning mitochondrial ROS, including the types of ROS present in the mitochondrial microenvironment, how they are generated, how levels are controlled, and the conditions that promote changes in the production of these species. ROS metabolism in mitochondria U The primary ROS generated by mitochondria is O2−, as a result of monoelectronic reduction of O2 (see Scheme 1). The mitochondrial electron transport chain continuously reduces the bulk of O2 consumed to water, a four-electron reduction, but a small quantity U U of O2− is also generated. O2−, a reasonably reactive ROS [9,10], is transformed into more stable H2O2 in mitochondria through the activity of matrix Mn-SOD, as well as Cu,Zn-SOD in the intermembrane space [7,8,11,12]. The abundance of this enzyme, as well as its presence in both mitochondrial compartments, attests to the U importance of removing mitochondrially generated O2−. Indeed, knockout animals for Mn-SOD present perinatal lethality unless treated with a cell-permeative SOD mimetic [13–15]. H2O2 generated in mitochondria has many possible fates. Because H2O2 is relatively stable and membrane-permeative (and transported by aquaporins [16] present in the inner mitochondrial membrane [17]), it can diffuse within the cell and be removed by cytosolic antioxidant systems such as catalase, glutathione peroxidase, and thioredoxin peroxidase (reviewed in [18,19]). Mitochondrially generated H2O2 can also act as a signaling molecule in the cytosol, affecting multiple networks that control, for example, cell cycle, stress response, energy metabolism, and redox balance (reviewed in [9,20]). Within mitochondria, many new signaling effects of H2O2 have been uncovered over the past few years, including an important

role in the activation of mild mitochondrial uncoupling pathways, which are themselves key regulators of mitochondrial ROS generation ([21–24], see discussion below). H2O2 can be eliminated by mitochondrial enzymes (see Scheme 1). At least in heart [25] and liver [26], catalase is present in mitochondria. Interestingly, in yeast, catalase is targeted to the mitochondrial matrix under conditions that favor ROS accumulation in this organelle [27]. Mitochondria also present efficient peroxiredoxins and associated reductases to remove H2O2. These include the mitochondrial glutathione peroxidase/glutathione reductase system, which removes H2O2 using reduced glutathione as an electron source, and the mitochondrial thioredoxin peroxidase/thioredoxin reductase system, which uses electrons from thioredoxin (reviewed in [28,29]). Interestingly, 1-Cys peroxiredoxins (which are located in mitochondria in yeast [30]) can also use ascorbate as a reducing agent [31], a property that may be highly relevant physiologically owing to the abundance of intracellular ascorbate. As occurs with the cytosolic counterparts of these systems, both mitochondrial thioredoxin and glutathione are reduced by NADPH. Thus, the levels of NADPH are closely related to the mitochondrial antioxidant capacity. In mitochondria, NADP is reduced, in part, by the activity of the NADH/NADP transhydrogenase (reviewed in [32]), which functions as a proton pump, using the respiration-generated electrochemical H+ gradient to displace the reaction toward NADPH formation (see Scheme 1). This characteristic links mitochondrial coupling and the membrane potential to the redox potential. Therefore, if mitochondria are not fully coupled or if the membrane potential decreases, the energy-linked transhydrogenase may be unable to respond readily to high levels of NADPH oxidation, and oxidative damage may occur owing to the lower rate of hydroperoxide removal. Indeed, molecules that oxidize pyridine nucleotides increase oxidative damage in Ca2+loaded mitochondria [33–38]. This was evidenced by early studies

U U Scheme 1. Mitochondrial ROS metabolism. Superoxide radical anions (O2−) are formed by monoelectronic reduction of O2, mainly at Complexes I and III of the respiratory chain. O2− is dismutated to H2O2 by Cu,Zn-SOD in the intermembrane space and Mn-SOD in the matrix. H2O2 can be removed by mitochondrial catalase or thiol peroxidases such as glutathione and thioredoxin peroxidase, using reduced glutathione (GSH) and thioredoxin (TrxSH) as substrate, respectively. Oxidized glutathione (GSSG) and thioredoxin (TrxS−) are reduced by their respective reductases, using NADPH as an electron source. NADP can be kept reduced by the activity of the NAD/NADP transhydrogenase, with proton transport into the matrix, providing a link between the inner membrane potential and the mitochondrial redox capacity. Alternatively, NADP+ is reduced by isocitrate dehydrogenase.

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showing that mitochondrial Ca2+ accumulation and retention was favored by reduced pyridine nucleotides, whereas oxidation of these nucleotides was followed by release of accumulated Ca2+ [35]. This Ca2+ release was later associated with the mitochondrial permeability transition (MPT), a nonselective form of inner-mitochondrial membrane permeabilization induced by several conditions, including oxidative stress (see [34] for review). The mitochondrial NADP+ pool can also be maintained in the reduced state through the activity of isocitrate dehydrogenase [39], a system that may be of high physiological relevance [40]. Our group recently uncovered the importance of this system in a condition of mitochondrial redox imbalance in hypercholesterolemic LDL receptor knockout mice [41,42]. This is associated with low mitochondrial antioxidant capacity and increased levels of mitochondrial markers of oxidative damage, due to a large consumption of reducing equivalents from NADPH to sustain high rates of de novo cholesterol synthesis. The elevated production of H2O2 in mitochondria isolated from these mice was decreased by the addition of exogenous isocitrate, which keeps NADP fully reduced [41]. We hypothesize that high rates of lipogenesis in the hypercholesterolemic mice decrease mitochondrial NADPH/NADP+ ratios owing to enhanced NADPH consumption rates. This is supported by experiments demonstrating the recovery of respiratory rates in the hypercholesterolemic mice by isocitrate supplementation and the finding that in vivo treatment of these mice with citrate partially restored both the rate of oxygen consumption and the capacity to sustain NADPH levels. Thus, mitochondrial oxidative imbalance in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates that can be, at least in part, replenished by oral administration of citrate [41]. When not metabolized by mitochondrial antioxidant systems, U H2O2 may generate the hydroxyl radical (HO ), through the metalU catalyzed Fenton reaction [43]. HO is highly reactive and generally believed to act essentially as a damaging molecule. For this reason, mitochondria are believed to have developed efficient H2O2 removal systems, as well as metal-chelating mechanisms, preventing the formation of this radical. Experiments using iron chelation to prevent mitochondrial damage and loss of integrity due to enhanced ROS U generation demonstrate the importance of HO as a damaging species in mitochondria [33,44]. Mitochondria have also been credited as a source of reactive U U nitrogen species derived from nitric oxide (NO ). NO is generated enzymatically by a family of nitric oxide synthases (NOS), which includes neuronal NOS (nNOS), endothelial NOS, and inducible NOS. U These enzymes synthesize NO using L-arginine as a substrate and NADPH as an electron source, in a manner favored by the presence of Ca2+ ions and reduced thiols. Indeed, NO⋅ is a well-established signaling molecule in biological systems, and many of its known targets, which include heme and thiol groups [45,46], are abundantly present in mitochondria. Studies suggesting that a NOS isoform is present in mitochondria initially appeared in the early 1990s [47–49], mostly through experiments showing staining against NOS near to or within U mitochondria. Subsequent work measured NO production by a putative NOS in isolated mitochondria from different tissues [50,51]. The main function of this enzyme is believed to be regulation of respiratory rates due to reversible inhibition of cytochrome c oxidase U by NO (see [52] for review). Yet, despite a large number of publications that have accumulated over the years, the existence of a mitochondrial NOS is still quite controversial. Different groups have suggested distinct molecular identities for the enzyme (although U more recent data suggest it is a nNOS [52]); a very wide range of NO production rates has been recorded, and the activity measured has been suggested to be related to contaminant enzymes from other intracellular compartments (see contrasting views in [53,54]). U Furthermore, alternative sources of NO have been described in

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mitochondria, including respiratory chain-dependent nitrite reductase activities [55–57]. U Irrespective of where and how NO is produced, mitochondria are certainly a target for this species, which is stable enough, as well as diffusible enough, to act upon targets distant from its production site. Mitochondria are rich in both proteins and lipids and, as a result, U substantial targets for NO . Heme- and metal-containing enzymes of U the electron transport chain can be nitrosylated by NO . Indeed, reversible nitrosylation of cytochrome c oxidase may constitute an important form of respiratory chain regulation [58,59]. Higher levels of U NO are associated with the nitrosation of oxygen-, nitrogen-, or sulfurcontaining amino acid side chains, resulting in functional changes that may be involved in protection against ischemia (reviewed in [60,61]). U U Furthermore, the reaction of NO with O2−, generating the highly reactive nitrogen species peroxynitrite (ONOO−), can lead to oxidation and protein nitration [62]. Whereas peroxynitrite-induced protein oxidation can be partially repaired by the thioredoxin reductase system, tyrosine nitration seems to be irreversible [63]. A major protein target for nitrosation and nitration in mitochondria is respiratory Complex I [64], whereas large ONOO− concentrations can promote more generalized mitochondrial protein and lipid modifications, leading to nonselective inner membrane permeabilization and loss of function [65]. Another highly reactive oxidant that has gained attention in the U U past few years is the carbonate radical (CO3−). CO3− formation has been directly demonstrated to occur only in mixtures of ONOO− with bicarbonate [66], but its formation has been proposed to occur under many biologically relevant conditions (see [67] for review). Indeed, because biological systems present millimolar concentrations of U bicarbonate, it is reasonable to propose that CO3− can have important biological implications, including in mitochondria. Singlet oxygen (1O2) can also have a role in the mitochondrial redox state. This ROS may be important in mitochondrial DNA (mtDNA) damage promoted by a solar radiation mimetic, such as sublethal UVA irradiation [68], although other reactive species may be involved too. Singlet oxygen induces the mitochondrial permeability transition [69] and may cause mitochondrial damage when generated purposefully with the aim of promoting cell death, such as in photodynamic therapy, which often focuses on mitochondria as primary targets for photosensitizer action [70–74] to activate intrinsic apoptotic pathways by releasing proapoptotic mitochondrial proteins (reviewed in [75]). Although the production of singlet oxygen by mitochondria has not yet been directly detected, indirect evidence suggests that this species may be generated in mitochondria via cytochrome c-catalyzed peroxidation of carbonyl substrates, such as diphenylacetaldehyde and 3-methylacetone [76]. The oxidation of such aldehydes by peroxidases generates carbonyl compounds in the electronically excited triplet state [77,78]. Nantes et al. [79] detected a red emission from rat liver mitochondria incubated with diphenylacetaldehyde, which was tentatively assigned to singlet oxygen. Thus, mitochondria produce and are in contact with a very diverse group of oxidants, each with different strengths, reactive properties, and ability to diffuse and to be removed by specific antioxidants. These oxidants are also generated in different environments within the organelle or cell. In this manner, mitochondrial ROS present the same diversity of characteristics as ROS in other biological systems: they encompass a widely varied group of chemical species that cannot be considered a single entity. As a result, responses to their presence depend on the species, rate, quantity, accumulation, and microenvironment of generation [80]. Sources of mitochondrial ROS The bulk of mitochondrial ROS generation occurs at the electron transport chain (ETC), as a by-product of respiration [81–83]. As a result, this generation can occur at relatively high rates compared to

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cytosolic ROS production and is primarily determined by metabolic conditions. The result of generating ROS at potentially high rates and in a manner not primarily determined by signaling networks is that mitochondrially-generated ROS are associated mostly with damaging processes, although signaling roles for these species are now being uncovered (see discussion below). Interestingly, new evidence suggests that one of these signaling roles involves cross-talk with another cellular oxidant generating system—the NADPH oxidase [84]. The ETC, in which numerous one-electron transfer reactions occur, has been recognized as an important intracellular source of ROS for approximately 40 years [1,3–5,85–87]. Cytochrome c oxidase (Complex IV) is the terminal component of the ETC and reduces one O2 molecule to two H2O, in a process involving four one-electron reduction reactions [88]. Although reactive intermediates are produced within the enzyme, it is highly adapted and does not seem to release these intermediates in measurable quantities. Cytochrome c, the electron donor to cytochrome c oxidase, is not a documented ROS source in biologically relevant situations [89–91]. Indeed, some experimental evidence indicates that cytochrome c may act as a U mitochondrial antioxidant, oxidizing O2− back to O2 (for further discussion, see [92]). On the other hand, Q-cytochrome c oxireductase (Complex III) is a well-documented source of mitochondrial ROS [85–87,93–95]. This respiratory complex receives electrons from reduced coenzyme Q (UQH2) and donates them to cytochrome c. UQH2 is oxidized to UQ in a complex set of reactions that involves first the formation of the U semiquinone radical (UQ −) at the Qp site of Complex III, which faces the intermembrane space, by donation of an electron from UQH2 to the U Reiske protein and then to cytochrome c. An electron from the UQ − formed at Qp is then transferred to the Qn site (which faces the U U mitochondrial matrix), where UQ is reduced to UQ −. The Qn UQ − is U reduced to UQH2 by an electron provided by a second UQ − formed at U the Qp site [88]. The result of this cycle is that UQ − is formed at both the U− U Qp and the Qn site. Because the UQ /UQ pair is highly reducing, O2− may be formed by electron donation from it, as long as O2 has access to any of these sites within the complex. Although the Qp site is generally believed to be more accessible to O2, rendering it a more significant U source of O2− [96,97], there is also evidence of superoxide formation at the Qn site [95]. The effect of respiratory inhibitors confirms that UQ⋅− is the source of mitochondrial ROS generated by Complex III. Mixothiazol, U which prevents UQ − formation at the Qp site, prevents mitochondrial ROS generation at Complex III (although it may increase this release at Complex I). On the other hand, antimycin, which inhibits electron transfer from the Qp to the Qn site, thus leading to an accumulation of U UQ − at Qp, enhances ROS release by Complex III [4,87,94,95,97,98] (but see [93]). U Ubiquinone cycling may also be responsible for O2− formation within NADH-Q oxireductase (Complex I). Indeed, mixothiazol, which keeps the ubiquinone pool highly reduced, enhances ROS formation by Complex I more significantly than the Complex I inhibitor rotenone U [99]. This suggests that Complex I has at least two O2− formation sites: one upstream and one downstream of the rotenone inhibitory site. The downstream site is most probably the ubiquinone binding site, whereas upstream sites may be the FMN group or iron–sulfur centers. U Mixothiazol reduces and favors O2− formation at all of these sites, U whereas rotenone reduces FMN and iron–sulfur centers, facilitating O2− formation at these levels. Although succinate dehydrogenase (Complex II) is a flavoprotein, and can theoretically generate one-electron O2 reductions, U significant O2− formation from this enzyme has not been measured. This may be because the enzyme’s structure does not allow O2 access to the intrinsic FAD [100]. Despite the lack of ROS formation by Complex II itself, succinate is an important source of ROS in many tissues. This is due to reverse electron transfer from succinate to ubiquinone (via Complex II) and back to Complex I [101]. ROS release by reverse electron transfer occurs primarily at sites

upstream of the rotenone inhibition site, as indicated by the prevention of this formation by rotenone. Furthermore, reverse electron transfer is strongly stimulated by high ΔΨ, which thermodynamically allows electron donation from Complex II to Complex I. Other mitochondrial enzymes are documented ROS sources, although pinpointing their specific role within the mitochondrial microenvironment, rich in redox activity, is often a difficult task. The flavoproteins acyl-CoA dehydrogenase and glycerol phosphate dehydrogenase generate ROS and may account for enhanced levels of ROS release seen in some tissues when oxidizing lipid-derived substrates [94,97,102–104]. Monoamine oxidase and dihydroorotate dehydrogenase are also documented mitochondrial ROS sources [81,105]. Furthermore, pyruvate and α-ketoglutarate dehydrogenase both contain flavoenzyme dihydrolipoyl dehydrogenase subunits, which are very important ROS sources in brain [106,107] and may be involved in aging, at least in model organisms [108]. Tissue-, substrate-, and organism-specific properties of mitochondrial ROS formation Given the various mitochondrial enzymes capable of promoting ROS formation, it is expected that intact mitochondria will present changes in redox state when in the presence of various classes of respiratory substrates. Indeed, in vitro mitochondrial ROS release is strongly altered by the respiratory substrate used, with higher rates often observed in the presence of succinate and acyl-CoA [94,97]. Furthermore, not all mitochondria are alike, and organelles from various tissues and organisms present quite diverse ROS release patterns, owing both to changes in ROS production and to differing levels of mitochondrial antioxidant systems [109–111]. Examples of organelle-specific characteristics of mitochondrial ROS release include: (i) a strong importance of reverse electron transfer in brain, skeletal muscle, and heart [101,112–115], but not liver mitochondria [115], and (ii) palmitate is a substantial ROS source in heart and skeletal muscle [97], whereas (iii) glycerol phosphate may be an important ROS source in brain when higher intracellular Ca2+ levels are present [103,104]. Further studies are necessary to understand how these different responses to substrates, observed in isolated mitochondria, relate to ROS release rates in vivo in cells using different substrates. At least in skeletal muscle cells, the oxidation of fatty acids enhances mitochondrially generated oxidants [102]. Many fewer studies have focused on mitochondrial ROS formation in nonmammalian tissues, despite evidence of interesting and diverse properties. In the yeast Saccharomyces cerevisiae, mitochondrial ROS release is substantial [108] and occurs at the level of Complex III and NADH dehydrogenase, as well as at the level of dihydrolipoyl dehydrogenase [90,108]. Interestingly, ROS release in yeast is largely stimulated by growth under fermentative conditions, rather than under conditions that stimulate mitochondrial respiration [108,116,117]. Some pathological fungi, such as the Candida genus, present complex branched respiratory chains, as well as uncoupling pathways [118–120], which may prevent electron accumulation at specific sites, decreasing ROS release. In C. parapsilosis, ubiquinone is the main source of intracellular ROS, and promoting its reduction by inhibiting downstream electron transport pathways leads to cellular oxidative stress and lack of growth [121]. Plant mitochondria also present branched respiratory chains and uncoupling pathways, which are involved in tolerance against oxidative stress [122–130]. Mechanisms controlling ROS release The existence of branched respiratory chains and mechanisms decreasing oxidative phosphorylation efficiency in fungi, amoeba, and plants [118,119,122–125,127,129–131] suggests that enhancing

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Scheme 2. Mechanisms leading to ΔΨ and ROS generation control in mitochondria. ATP synthase activity decreases ΔΨ and ROS release and can be stimulated by increased ADP formation in mitochondria owing to the activity of mitochondrially bound hexokinase (HXK) or creatine kinase (not depicted). The adenine nucleotide translocator (ANT), in addition to transporting ADP and ATP across the inner membrane, transports anionic fatty acids from the matrix to the inner membrane space, where they are protonated, resulting in an uncharged molecule that can flip-flop across the membrane, releasing the proton in the matrix. Uncoupling proteins (UCP) similarly transport anionic fatty acids across the membrane, promoting uncoupling, which can also be stimulated by agents (such as dinitrophenol and FCCP) or conditions (such as changes in membrane structure) that increase the proton leak. Mitochondrial ATP-sensitive K+ channels (mitoKATP) promote K+ transport into the mitochondrial matrix and, in concert with the K+/H+ exchanger, lead to mild uncoupling. Ca2+ uniporters allow uptake of the ion, stimulated by the electrochemical gradient.

respiratory rates without ATP synthesis is advantageous under some conditions. Among the many proposed functions for these inefficient respiratory chains, one of the best demonstrated is their role in preventing mitochondrial ROS formation. Indeed, a variety of conditions that increase electron transport in different tissues, including oxidative phosphorylation, uncoupling, and activation of alternative respiratory pathways, prevent mitochondrial ROS release [132–137] (see Scheme 2). Mild mitochondrial uncoupling We have recently demonstrated that systemically increasing respiratory rates in vivo by using low doses of uncoupler is a highly effective antioxidant strategy, preventing even low levels of oxidative damage to biomolecules observed in healthy mice [133]. This evidence adds to significant in vitro data demonstrating prevention of ROS formation associated with mitochondrial uncoupling and associated increases in respiratory rates [3,22,83,101,112,132,134–140]. Increasing electron transport prevents ROS release by mitochonU dria through many mechanisms involving the prevention of O2− formation (reviewed in [82,137]). By increasing O2 consumption, enhanced electron transport may prevent ROS formation by decreasing O2 tension in the mitochondrial microenvironment. A second effect of enhanced electron transport is to favor more oxidized levels of respiratory chain intermediates, in particular at initial steps of the electron transport chain such as Complexes I and III, known to be substantial ROS sources, as described above. Enhanced electron transport also keeps NADH levels lower, which prevents ROS formation by mitochondrial matrix flavoenzymes [106–108]. Finally, increased electron transport rates are often accompanied by lower ΔΨ, a condition that thermodynamically disfavors reverse electron transfer from Complex II to Complex I [83]. A recent review [82] comprehensively and quantitatively discusses these effects. Indeed, mitochondrial pathways that decrease oxidative phosphorylation efficiency and increase electron transport (see Scheme 2), including uncoupling proteins [135,140], ATP-sensitive K+ channels (mitoKATP; [22,113,139,141,142]), and the alternative oxidase [136,138],

have been widely reported to prevent ROS release by these organelles. Interestingly, uncoupling proteins [24,143–145] and mitoKATP channels [22,113,146,147] are activated by oxidants, resulting in an elegant negative feedback system controlling mitochondrial ROS formation by promoting mild uncoupling. The alternative oxidase is also redox sensitive, although, intriguingly, it is inhibited by thiol oxidation [148]. In addition to being redox sensitive, these energy-dissipating pathways are also regulated by metabolic conditions. As examples, the alternative oxidase is activated by α-ketoacids [148], uncoupling proteins depend on free fatty acids to increase mitochondrial transmembrane proton transport (reviewed in [149]), and mitoKATP channels are activated by long-chain acyl-CoA esters [150,151]. Thus, mitochondrial energy metabolism and redox state are interrelated at many levels. In fact the activity of mitochondrially bound hexokinase or creatine kinase [152,153] can decrease ROS production, by lowering ΔΨ (Scheme 2). Respiratory inhibition Whereas enhancing mitochondrial respiratory rates generally prevents ROS release, pathological conditions that lead to lower respiratory rates are often accompanied by enhanced ROS release, as is the case in respiratory deficiency associated with neurodegenerative diseases and mitochondriopathies [154–159]. Interestingly, whereas Complex IV inhibition has been linked to increased ROS release in vitro [83,141,145,160], in vivo experiments revealed that cells from COX-deficient animals did not show elevated oxidative stress, possibly because of a lack of assembly of respiratory chain supercomplexes [157]. Several groups have reported increased ROS production in cell lines and tissue from transgenic animals and patients carrying mutant mtDNA [161–164] (for review, see [165,166]), suggesting that impaired expression of mitochondrially encoded subunits leads to increased electron leak at the ETC. In fact, Ishikawa et al. [167] have recently shown that cybrid cell lines carrying specific mtDNA mutations were more metastatic than their nonmutant counterparts, an effect they correlated to increased ROS production at Complex I.

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Although the specificity of their ROS measurements has been questioned [168], these results lend strong support to the hypothesis that mtDNA mutations may lead to conditions that are associated with oxidative stress. On the other hand, results obtained with transgenic mice expressing a proofreading-deficient mitochondrial DNA polymerase γ [169] cast some doubt on this direct relationship. Although these mice display a pronounced accelerated aging phenotype and accumulate high levels of mtDNA point mutations and deletions [169–171], they show no obvious signs of oxidative stress [169,170]. These results suggest that the simple accumulation of random mtDNA mutations may not be sufficient to drive enhanced ROS production and aging. Together with new evidence showing that some forms of mtDNA deletions do indeed drive aging [172], these findings raise the possibility that selected alterations in mtDNA, and not the overall mutational load, are relevant to the phenotypic expression of mitochondrial dysfunction. Changes in oxygen tension Variations in oxygen tension are also expected to have an important impact on mitochondrial ROS formation and release. It is of note that most isolated mitochondrial and cell culture studies are conducted with oxygen concentrations around 200 μM, the solubility of O2 at 37°C, or even higher when lower experimental temperatures are used. On the other hand, oxygen tension within tissues can be as low as 5 μM in working skeletal muscle [173,174]. Mitochondrially generated NO⋅ has been suggested to be a regulator of tissue oxygen tension by inhibiting respiratory activity at higher tension sites and allowing diffusion toward more distant locations [175,176]. Although lowering oxygen tensions does not substantially change mitochondrial respiration, owing to the high affinity of cytochrome c oxidase, the results of changes in oxygen tension on ROS formation in vivo still remain to be uncovered. Early work demonstrated that the oxygen tension to which an animal is subjected changes ROS release in its mitochondria. Namely, hypobaric oxygen conditions decreased ROS release in many tissues, whereas hyperbaric oxygen led to increased mitochondrial ROS release [177,178]. Indeed, primary cells growing under low oxygen tension (3%) show senescent phenotypes later than cells growing under normoxic oxygen levels, which was associated with lower oxidative damage [179,180]. Moreover, reoxygenation protocols in normoxia promote more favorable outcomes than those involving oxygen supplementation [181,182]. However, the relationship between oxygen tension and ROS release is not always so straightforward: although lower oxygen tension would be expected to prevent the formation of these species, many conditions that involve hypoxia are associated with enhanced ROS levels [183,184]. An interesting example regarding the complexity of the effects of changes in oxygen tension on mitochondrial ROS release is pathologies involving tissue ischemia. Mitochondrial oxidative stress is widely demonstrated to be involved in postischemic damage. The postischemic reperfusion period is accompanied by increases in intracellular Ca2+ levels, low ATP and high phosphate concentrations, enhanced pH (relative to ischemia), as well as enhanced ROS formation, ideal conditions for MPT [34,38,160]. Indeed, MPT has been extensively demonstrated to be causative of mitochondrial damage leading to limitation of postischemic tissue recovery in brain, heart, and liver and is widely viewed as a target for tissue protection under these conditions (reviewed in [185–189]). Interestingly, mitochondrially generated ROS are not involved only in tissue damage during reperfusion but also in signaling events leading to cardiac protection after ischemic preconditioning [190]. Ischemic preconditioning is the phenomenon in which brief, nondamaging, ischemic periods are promoted to protect against subsequent longer ischemic periods. This process depends on moderate

increases in mitochondrial ROS generation that occur during the brief ischemic periods [190] and prevent oxidative stress during reperfusion [142]. The increments in ROS release are concomitant with the brief ischemic periods, and ischemic preconditioning is thus an example of a condition in which lowering of oxygen tensions increases mitochondrial ROS. The mechanisms through which preconditioning induces ROS release, and how these ROS mediate cardioprotection, are the subject of many studies. Unfortunately, contrasting theories have been put forward, and the sequence of events is still a matter of debate. We favor the idea that preconditioning leads to enhanced mitochondrial ROS release resulting from partial inhibition of the electron transport chain [190,191], perhaps due to Complex I Snitrosation caused by the activation of nitric oxide synthases, which is associated with preconditioning [60,61,192]. This moderate increase in mitochondrial ROS levels activates uncoupling pathways, including uncoupling proteins, fatty acid cycling by the adenine nucleotide transporter [21,193,194], and mitoKATP [22,141,191]. The result of mild mitochondrial uncoupling, as discussed above, is the prevention of mitochondrial ROS formation during reperfusion [113,141,142,195]. Furthermore, activation of mitoKATP channels prevents the loss of cellular ATP and mitochondrial Ca2+ accumulation during ischemia. Consequently, conditions favoring MPT during reperfusion are attenuated, and the tissue is preserved [196–200] (but see a contrasting view in [201]). Calcium Mitochondrial bioenergetics and redox state are also determined by intracellular Ca2+ levels. Mitochondria present Ca2+ uniporters capable of rapidly promoting Ca2+ uptake into the matrix, driven by ΔΨ, most notably when Ca2+ is released by the endoplasmic reticulum within the mitochondrial microenvironment [202]. Because of the larger volume of the mitochondrial matrix relative to the endoplasmic reticulum, Ca2+ can be accumulated in vast quantities in mitochondria, although the affinity of mitochondrial Ca2+ transporters tends to be lower than that of those found in the reticulum and plasma membrane. Within the mitochondrial matrix, enhanced Ca2+ levels act as important activators of pyruvate dehydrogenase, enzymes from the citric acid cycle, and respiratory chain components (reviewed in [203,204]). Ca2+ also affects mitochondrial ROS release. Uptake of the cation leads to a transitory decrease in ΔΨ and enhanced electron transport, which can, under some conditions, decrease ROS formation [205,206]. On the other hand, excessive Ca2+ accumulation has been extensively associated with mitochondrial oxidative stress [33,206–213]. Ca2+ may increase mitochondrial ROS formation by many mechanisms, including enhancing citric acid cycle activity and NADH formation [207], activating ROS-generating enzymes such as glycerol phosphate and U α-ketoglutarate dehydrogenase [104], enhancing NO generation and consequent respiratory inhibition [214], and promoting the loss of cytochrome c due to the mitochondrial permeability transition, which is promoted by excessive Ca2+ accumulation (revised in [34,38,188]). In addition, MPT can also result in increased mitochondrial ROS release [215–218] (but see [205]). An early event in Ca2+induced mitochondrial ROS generation may be an increase in lipid packing in the mitochondrial inner membrane. Grijalba et al. [219] demonstrated that Ca2+ binding to cardiolipin induced lipid domain formation and cardiolipin-enriched immobilized lipid clusters in submitochondrial particles. These led to lipid/protein alterations, which could in turn promote increased ROS production at the ETC. Moreover, in in vitro model systems consisting of phosphatidylcholine/diethyl phosphate liposomes, phosphate and Ca2+ cooperate to facilitate the propagation of radical reactions initiated by a triplet acetone-generating system [212]. Interestingly, not only are changes in mitochondrial redox state a consequence of Ca2+ accumulation: it is now clear that redox state is an important determinant of Ca2+

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signaling responses in other intracellular environments (reviewed in [220,221]). Mitochondrial content and morphology Another determinant of the impact of mitochondrial ROS formation is the quantity of mitochondria present. Mitochondrial biogenesis is controlled by Ca2+ levels, NO⋅, oxygen tension, and energy metabolism, all conditions that are also linked to changes in redox state (see [222] for review). However, increased mitochondrial content as a result of enhanced biogenesis is not necessarily accompanied by increased levels of mitochondrial ROS generation. For example, caloric restriction promotes mitochondrial biogenesis through a pathway signaled by lower insulin levels, enhanced NO⋅, and the activation of the transcriptional coactivator PCG-1α and is also associated with lower levels of mitochondrial ROS formation, possibly due to enhanced uncoupling promoted by this dietary regimen [223–227]. In fact, increased mitochondrial biogenesis signaled by PGC-1α may constitute a facet of a concerted stress response pathway. This program includes the up-regulation of antioxidant and repair systems, as well as a boost in energy metabolism [228] (for review, see [229]). In addition to being regulated by mitochondrial content, oxidant generation may also be determined by mitochondrial morphology. Yu and co-workers [230] demonstrated that the increase in ROS release from mitochondria induced by hyperglycemia is related to mitochondrial fission. Fission inhibition abrogated hyperglycemia-induced ROS production [231] and cell death, owing to the prevention of mitochondrial membrane permeabilization and cytochrome c release. These results suggest that the regulation of mitochondrial morphology could be a target for therapeutic interventions preventing oxidative stress under pathological conditions. Summary and perspectives Mitochondrial ROS have been extensively implicated in a large variety of human diseases and degenerative conditions, such as aging (reviewed in [81,154,157,232]). Although much is already known about mitochondrial ROS metabolism, as reviewed here, some basic mechanistic aspects still remain to be uncovered, especially in vivo. A significant aspect regards the differences between mitochondria from different tissues and under distinct metabolic conditions. A more comprehensive understanding of mitochondrial redox transformations is needed to develop new interventions capable of regulating the role of mitochondrial ROS production in human health. Acknowledgments The authors are supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Instituto Nacional de Processos Redox em Biomedicina—Redoxoma, Instituto Nacional de Obesidade e Diabetes, and Pró-Reitoria de Pesquisa, USP. The authors thank Felipe D.T. Navarete for help in preparing the schemes. References [1] Hinkle, P. C.; Butow, R. A.; Racker, E.; Chance, B. Partial resolution of the enzymes catalyzing oxidative phosphorylation. XV. Reverse electron transfer in the flavincytochrome beta region of the respiratory chain of beef heart submitochondrial particles. J. Biol. Chem. 242:5169–5173; 1967. [2] Jensen, P. K. Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles. I. pH dependency and hydrogen peroxide formation. Biochim. Biophys. Acta 122:157–166; 1966. [3] Loschen, G.; Azzi, A.; Flohé, L. Mitochondrial H2O2 formation: relationship with energy conservation. FEBS Lett. 33:84–87; 1973. [4] Boveris, A.; Cadenas, E. Mitochondrial production of superoxide anions and its relationship to the antimycin insensitive respiration. FEBS Lett. 54:311–314; 1975.

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