Disruption of mitochondrial redox circuitry in oxidative stress

Chemico-Biological Interactions 163 (2006) 38–53

Disruption of mitochondrial redox circuitry in oxidative stress Dean P. Jones ∗ Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University, Atlanta, GA 30322, USA Available online 31 July 2006

Abstract The present review and commentary considers oxidative stress as a disruption of mitochondrial redox circuitry rather than an imbalance of oxidants and antioxidants. Mitochondria contain two types of redox circuits, high-flux pathways that are central to mechanisms for ATP production and low-flux pathways that utilize sulfur switches of proteins for metabolic regulation and cell signaling. The superoxide anion radical (hereafter termed “superoxide”, O2 •− ), a well known free radical product of the high-flux mitochondrial electron transfer chain, provides a link between the high-flux and low-flux pathways. Disruption of electron flow and increased superoxide production occurs due to inhibition of electron transfer in the high-flux pathway, and this creates aberrant “short-circuit” pathways between otherwise non-interacting components. A hypothesis is presented that superoxide is not merely a byproduct of electron transfer but rather is generated by the mitochondrial respiratory apparatus to serve as a positive signal to coordinate energy metabolism. Electron mediators such as free Fe3+ and redox-cycling agents, or potentially free radical scavenging agents, could therefore cause oxidative stress by disrupting this normal superoxide signal. Methods to map the regulatory redox circuitry involving sulfur switches (e.g., redox-western blotting of thioredoxin-2, redox proteomics) are briefly presented. Use of these approaches to identify sites of disruption in the mitochondrial redox circuitry can be expected to generate new strategies to prevent toxicity and, in particular, promote efforts to re-establish proper electron flow as a means to counteract pathologic effects of oxidative stress. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Superoxide anion; Glutathione; Thioredoxin; Oxidative phosphorylation; Respiratory control; Antioxidants

1. Introduction Accumulation of data on redox signaling pathways and lack of major benefits in intervention trials with free radical scavengers [1–9] has prompted efforts to refine the definition of oxidative stress. The definition of oxidative stress as “a disturbance in the prooxidant–antioxidant balance in favor of the former” [10], implies that a disturbance due to prooxidant conditions can be corrected by addition of appropriate antiox-

∗

Present address: Whitehead Biomedical Research Center, 615 Michael Street, Suite 205P, Atlanta, GA 30322, USA. Tel.: +1 404 727 5970; fax: +1 404 712 2974. E-mail address: [email protected].

idants. However, redox mechanisms function in cell signaling, and cells are very sensitive to loss of these regulatory and control systems [11]. Consequently, an alternate definition for oxidative stress is “a disruption of redox signaling and control” [12]. These two concepts have recently been incorporated into a new definition as “an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage” [13]. However, this definition suggests that disruption of signaling and control is caused by an imbalance of prooxidants and antioxidants; from the perspective of oxidative stress in the mitochondria, this may be inverted in cause and effect. In the present review and commentary, I consider an alternative definition that may provide a better

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description of oxidative stress in mitochondria: “a disruption of electron transfer reactions leading to an oxidant/antioxidant imbalance and oxidative damage to macromolecules”. This provides the basis to view the complex issues of oxidative stress in mitochondria in terms of disruption of normal redox biology, with reactive oxygen species (ROS) such as the free radical superoxide anion (O2 •− ) and hydrogen peroxide being intermediates connecting two types of redox circuits, high-flux circuits that function in energy metabolism and low-flux circuits that function in control of metabolism and cell signaling. This focuses attention on the disruption of electron flow from the high-flux metabolic pathways to low-flux signaling and control pathways as the fundamentally important process characterizing oxidative stress in mitochondria. The first section summarizes the foundation for this view of oxidative stress derived from historical concepts of cellular respiration. These historical concepts lead to a first principle that mitochondria evolved as redox organelles containing electron transfer machinery which have very high rates of transfer compared to other oxidation–reduction processes in cells. The next section addresses a second principle, that sulfur switches are components of low-flux redox circuits which function in cell signaling and metabolic control. This section is based upon accumulating knowledge concerning thiol/disulfides in redox signaling in the cell cytoplasm and nuclei. In mitochondria, interference with high-flux electron transfer causing disruption of low-flux signaling and control mechanisms provides a simple and logical way to define oxidative stress. The following section addresses the principle that ROS are products of the high-flux metabolic pathways connecting these with the low-flux redox signaling and control pathways. This section includes the speculative hypothesis that O2 •− is generated by the electron transport chain as a signaling molecule, providing a signal that the electron transport pathway is competent for generation of the protonmotive force needed for ATP synthesis. The last section addresses practical aspects of this new “definition” of oxidative stress, namely that oxidation of specific proteins can serve as reporters of disruption of redox pathways and thereby improve mechanistic details of oxidative stress [14,15]. Use of these approaches to identify and eliminate dietary and environmental agents which disrupt mitochondrial redox circuitry, such as those implicated in Parkinson’s disease [16], and develop therapeutic interventions to re-establish proper electron flow could provide novel strategies to protect against oxidative stress.

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2. Historical precedents to the concept that mitochondrial oxidative stress represents a disruption of mitochondrial redox circuits In the 1920s, Warburg found a complex ironcontaining substance in tissue which functioned in oxidative processes and was inhibited by cyanide or sulfide. He referred to this substance as the “Atmungsferment”, meaning respiratory enzyme, a kinetically active component for reduction of molecular oxygen. About the same time, Keilin used spectral changes of cytochromes in response to oxidants and reductants to elucidate the major components of electron transfer in the respiratory chain [17]. He found that oxidation of cytochromes was also inhibited by cyanide and sulfide, and concluded that Warburg’s Atmungsferment was cytochrome oxidase. During the ensuing decades, these central oxidative processes were associated with mitochondrial ATP production. Three features of the early studies are noteworthy in terms of oxidative stress, namely, that the respiratory chain in mitochondria is the body’s most active electron transport system, that the associated electron transfer components are normally well insulated from other electron transfer reactions, and that mediators and inhibitors of electron transfer disrupt electron flow. As early as 1876–1877, Hoppe-Seyler showed that cyanide prevented tissues from using oxygen and inhibited most of the oxidation processes. The subsequent connection of the chemical-induced inhibition of O2 consumption to the mitochondrial cytochrome chain associated quantitatively important redox reactions with mitochondria. Electron transfer between the cytochromes occurred in a well defined sequence, and Keilin introduced alternative electron carriers, which he termed “mediators”, to study electron transfer at specific sites. One of these mediators was methylviologen, a chemical today known as paraquat, a potent inducer of oxidative stress. In the presence of oxygen and electron donating systems, paraquat undergoes cyclical transfer of electrons from the donor to oxygen, creating ROS that damage macromolecules in a process now termed “redox cycling” [18]. I believe that these early observations encompass the essence of mitochondrial oxidative stress: electron flow normally occurs through well defined redox circuits. Conditions that disrupt this normal electron flow create an imbalance of oxidants and antioxidants and cause macromolecular damage. In the 1950s, studies to elucidate details of mitochondrial electron transfer used the crossover theorem [19]. This theorem states that blocks in a pathway cause an increase in precursors upstream of the block and a decrease in products downstream of the block. Thus,

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Fig. 1. The crossover theorem provides a means to identify sites of disruption in electron transfer pathways. As shown in (A), changes in reduction state of cytochromes following addition of an inhibitor such as antimycin A provides a means to identify the site of inhibition. Similarly, redox changes of signaling and control pathways can be used to reveal sites of regulation of disruption of electron flow. The example shown in (B) illustrates the predicted change in the pathway from NADPH to control redox state of the mitochondrial permeability transition pore (PT pore) that would occur if an inhibitor were introduced that blocked electron flow from thioredoxin reductase-2 (TR2) to thioredoxin-2 (Trx2).

inhibitors that blocked electron transfer at specific sites could be used to order the electron transfer components (Fig. 1). With the view that oxidative stress is a disruption of redox circuits, the crossover theorem is applicable today as a general means to elucidate mechanisms determining oxidative stress. For instance, the mitochondrial permeability transition (MPT) can be activated by oxidants [20], presumably by oxidation of specific amino acid residues of component proteins in the permeability transition (PT) pore complex. Thioredoxin-2 (Trx2) is a protein that functions in reduction of disulfides and sulfoxides, and regulation of the PT pore could occur by the redox pathway consisting of NADPH → thioredoxin reductase-2 (TrxR2) → Trx2 → PT pore protein. Based upon this sequence, activation of the MPT by oxidative stress could occur by at least three mechanisms, increased rates of oxidation of the critical amino acid residues of the PT pore, inhibition of electron transfer within the pathway that supplies reducing equivalents to Trx2, or failure of NADPH supply. Application of the crossover theorem can distinguish between such mechanisms. For instance, inhibition of electron transfer from TrxR2 to Trx2 would cause TrxR2 to be more reduced and Trx2 and the PT pore protein to be more oxidized (Fig. 1). Methods are available, e.g., redox-western blot analysis [21,22] and redox proteomics [23], to use the crossover theorem to map such sites of perturbation in electron flow even in redox circuits that have relatively

low rates of electron transfer compared to the mitochondrial chain. Thus, new details of mitochondrial oxidative stress can be obtained by drawing upon the classical approaches of respiratory biology and applying modern proteomic methods. Studies in the 1970s showed that mitochondria are a source of ROS and that inhibition of electron transfer stimulates generation of ROS [24,25]. Subsequent studies showed that abnormal electron flow upon reperfusion following ischemia also generates ROS [26]. It is noteworthy that each of these prototypic conditions of mitochondrial oxidative stress is caused by a disruption of normal electron flow. 3. First principle: the mitochondrial electron transfer chain contains high-flux redox circuits that are effectively insulated from other electron transfer pathways Although discussions of mitochondrial oxidative stress often begin with consideration of ROS being produced as an unavoidable byproduct of respiration, I feel that a more appropriate starting principle concerns the nature of the mitochondrial electron transport chain. This chain is quantitatively the most significant redox system in aerobic organisms, accounting for perhaps 98–99% of all O2 consumption. The pathway can be viewed as a high-flux electron transfer pathway because

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the rates of electron transfer far exceed all but a few specialized oxidative systems (e.g., phagocyte NADPH oxidase). Because the catalytic rates for electron transfer can exceed other redox reactions by orders of magnitude, short-circuits created by electron transfer from this pathway has the potential to be highly disruptive to other redox processes. Mitochondria contain two central water-soluble electron carrier systems, NADH/NAD+ and NADPH/ NADP+ , that transfer electrons from intermediary metabolites to electron transfer chains. The NADH/ NAD+ couple transfers electrons to the mitochondrial chain and supports ATP production while the NADPH/ NADP+ couple functions principally in biosynthetic and detoxification reactions, including supply of reducing equivalents to the redox pathways containing GSH and thioredoxins. Estimates of the electron current in hepatocytes indicate that maximal flow through the NADPH/NADP+ couple is only about 12% of the rate through the NADH/NAD+ couple [27]. Thus, the NADH-utilizing mitochondrial chain can be conceptualized as a high-flux electron transfer pathway, which, if disrupted, can transfer electrons to lower flux pathways and disrupt their function.

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An overview of this concept is given in Fig. 2. On the left, electrons flow into the mitochondrial redox pathways through the NADH/NAD+ couple, then through complexes I, III and IV, ultimately being transferred to O2 . Each of the respiratory complexes contains multiple proteins with at least two redox-active centers, creating the possibility that O2 •− can be generated at multiple sites. Under aerobic conditions without inhibitors, the redox potential of NADH/NAD+ is about −300 mV [28] while downstream components are considerably more oxidized. The midpoint potential for O2 •− /O2 is −330 mV, so energetics are favorable for generation of O2 •− at complex I even without inhibition. This raises the possibility that complex I could continuously generate O2 •− , and O2 •− could serve as a signaling molecule (Fig. 2). In the presence of inhibitors of electron transport, the continued transfer of reducing equivalents from NADH results in increased reduction of the other complexes so that generation of O2 •− becomes energetically favorable at other sites. Several naturally occurring and synthetic inhibitors are known for each complex, and as illustrated in Fig. 2, rotenone (Rot) inhibition of complex I, antimycin A (AA) inhibition of complex III or cyanide (CN) inhibition of complex IV,

Fig. 2. Mitochondrial redox circuits. Two types of electron transfer circuits can be delineated in mitochondria, ones that function in metabolism and ones that function in signaling and metabolic control. The metabolic circuits typically utilize redox-active centers such as flavins and heme and catalyze high-flux electron transfers. In contrast, the signaling and control circuits typically utilize low-flux pathways involving protein cysteine or methionine residues. Efficient function of these pathways depends upon specificity of interactions and transfer of electrons, meaning that high-flux circuits are normally isolated from low-flux circuits. Mitochondrial oxidative stress occurs when there is a disruption of the normal electron flow, typically occurring from high-flux to low-flux component. Disruption of signaling and control pathways can activate the permeability transition (PT) pore and cause necrosis, or activate the apoptosis signal-regulating kinase-1 (ASK1) and cause apoptosis. Disruption of electron flow can occur by inhibitors that create “opens” in the high-flux pathways with resulting increase in electron transfer to the signaling and control circuits, or by electron mediators that accept electrons from the high-flux components and transfer these to disrupt signaling and control. These mediators include free metal ions, such as Fe3+ , as well as lipophilic redox cycling agents. For reference, approximate redox states of components are indicated relative to the scale on the left. TR2: thioredoxin reductase-2; Trx2: thioredoxin-2; Prx3: peroxiredoxin-3; MPT: redox-sensitive protein controlling the PT pore.

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results in stimulated ROS generation. This abnormal O2 •− can cause a short-circuit because the O2 •− is a diffusible electron carrier that can interact with other redox pathways. In this model, other electron mediators, such as Fe3+ and redox cycling chemicals, may disrupt redox signaling by interfering with normal O2 •− signaling. The NADPH/NADP+ couple is about −400 mV [28], a value which is considerably more reducing that the NADH/NAD+ couple. Consequently, the energetics are more favorable to generate O2 •− from enzymes using NADPH than from those using NADH. Indeed, the cytoplasmic NADPH oxidases that function in signaling catalyze O2 •− generation from NADPH [29]. However, NADPH is not known to support O2 •− generation in mitochondria, functioning instead to support GSH and thioredoxin-2 (Trx2)-dependent antioxidant systems. In Fig. 2, these NADPH-dependent pathways are identified as examples of low-flux redox signaling and control circuits. In the Trx2 pathway, Trx2 is reduced by electron transfer from NADPH, catalyzed by thioredoxin reductase-2 (TrxR2). Trx2 functions in regulation of apoptosis signal-regulating kinase-1 (Ask-1) [30], as well as control of mitochondrial permeability transition [31]. Trx2 also supplies electrons to peroxiredoxin3 (Prx3), a peroxidase which reduces H2 O2 to water. Oxidative stress occurs when short-circuits are created by disruption of electron transfer in the high-flux metabolic pathway and this causes low-flux systems to fail. Thus, the second principle of oxidative stress is that low-flux redox circuits function in mitochondrial signaling and control, and these are critical to survival.

N-terminal kinase and ERK1/2 pathways, was modulated by Ras, and was blocked by overexpression of catalase. Thus, H2 O2 signaling of these kinase pathways was shown to be mediated by discrete redox circuits rather than by global redox effects [33]. These results show that low-flux redox pathways can transmit important biologic signals despite coexistence with higher capacity redox systems that are dependent upon Trx or GSH. In other words, the fundamental concept derived from the early respiratory studies is applicable to lowflux systems, namely, that the redox signaling circuits are well insulated from other redox processes in the cell. Evidence has accumulated that many cell signaling circuits depend upon specific sulfur switches in proteins which undergo reversible oxidation–reduction [11]. The most common types of sulfur switches are cysteinyl groups that undergo S-thiylation with GSH, cysteine or other proteins; proximal cysteinyl groups (dithiols) which undergo disulfide formation within the protein; methionine residues that are oxidized to methionine sulfoxides; and cysteines that are oxidized to cysteine sulfenic acids which are stabilized by interaction with an amine. Because these redox-controlled switches can operate with negligible electron current relative to that used for metabolic purposes, minor changes in electron flow from the high-flux metabolic circuits can have major effects on the signaling and control circuits. In this way, a small fraction of the total mitochondrial electron flow to generate O2 •− can potentially have catastrophic effects by oxidizing critical protein residues.

4. Second principle: low-flux redox circuits involving sulfur switches function in cell signaling and control

4.1. Compartmentation of cellular redox and redox signaling

Early evidence for redox signaling mechanisms was obtained from studies showing that low levels of H2 O2 stimulated cell proliferation [32] and that high concentrations of thiols such as N-acetylcysteine (NAC) also alter signaling [11]. Studies to examine whether H2 O2 effects are mediated indirectly via a general redox signal or whether H2 O2 targets specific signaling pathways were performed using cells expressing NADPH oxidase-1 (Nox1), an enzyme which produces H2 O2 and induces cell growth, transformation, and tumorigenicity [29]. A luciferase reporter regulated by the antioxidant response element (ARE4 of glutamate–cysteine ligase) was increased 15-fold in Nox1 cells under conditions where there was no effect on the redox state of the major thiol antioxidants, GSH or Trx1 [33]. H2 O2 signaling was found to be mediated by activation of both the c-Jun

Three thiol/disulfide redox control nodes have been identified, i.e., GSH/GSSG, Trx1 (–SH2 /–SS–) and cysteine/cystine (Cys/CySS) [34]. Studies in several cell lines and normal human cells show that proliferating cells have GSH/GSSG redox potential in the range of −240 to −260 mV, i.e., not too different from the NADH/NAD+ couple but far more oxidized than the NADPH/NADP+ couple. Measures in HT29 cells showed that the differentiating agent sodium butyrate resulted in 60 mV oxidation (from −260 to −200 mV [35]), an oxidation sufficient for a 100-fold change in protein dithiols/disulfide ratio. Similar data was obtained with other cell types [36–38] and in vivo [37]. Thus, the GSH/GSSG system is considerably more oxidized than the NADPH/NADP+ couple and undergoes sufficient redox changes under physiologic conditions to function in redox control [35].

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Given the data cited above showing that redox signaling occurs by discrete pathways without global redox changes, a critical question is whether global changes in thiol/disulfide redox functions in signaling and control. Investigation of the role of GSH/GSSG in control of detoxification systems showed that the inducer benzyl isothiocyanate resulted in only a 12–16 mV oxidation in non-differentiated HT29 cells but a 40 mV oxidation (to −160 mV) in differentiated cells [35]. The redox state correlated with glutathione S-transferase and NADPH:quinone reductase activities, suggesting that changes in GSH redox could directly control redox signaling. In support of this conclusion, depletion of cysteine from culture media or addition of BSO to inhibit GSH synthesis resulted in substantial oxidation of cellular GSH [40,41], sufficient to activate movement of the transcription factor Nrf2 to the nuclei and increase transcription of an ARE-reporter [42]. Thus, these results indicate that both discrete redox signaling pathways and control by global changes in central redox control nodes are important in biologic control mechanisms. Studies of Trx1 and Cys/CySS show that these pools are not equilibrated with GSH/GSSG and function independently in control of redox processes [43]. Comparison of redox states of Trx1 and GSH/GSSG in Caco2 cells during progression from proliferation to growth arrest showed that the Trx1 redox state was more reduced and did not change in association with the oxidation of GSH/GSSG redox state during spontaneous differentiation [39]. Similar studies showed that Cys/CySS redox state is not equilibrated with either cellular GSH/GSSG or Trx1 [34]. This latter finding is unexpected given the well known exchange reactions of thiols and disulfides. Reed et al. [44] provided a possible explanation by showing that GSH released from cells reacted with CySS to generate Cys. While this reaction clearly occurs, calculations using rate constants for thiol/disulfide exchange and enzymatic catalysis by Grx1 and Trx1 [45–47] revealed that the exchange under physiologic conditions is slow compared with the rates of Cys metabolism in cells [34]. For instance, at physiologic concentrations of CySS and GSH, the rate of reduction is only about 7 ␮M min−1 , but higher rates of utilization of Cys are needed for protein synthesis (60 ␮M min−1 , calculated from [35]) and GSH synthesis (50 ␮M min−1 , calculated from [41]) [34]. The conclusion that the major thiol/disulfide couples are not equilibrated is reinforced by data from human plasma which also show that the redox states of GSH/GSSG and Cys/CySS are not equilibrated with each other or with the cellular pools [12,48,49] and are differentially oxidized in association with aging [50] and age-related diseases [38,48,51].

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4.2. Thiol/disulfide redox states in the mitochondrial compartment Although the GSH/GSSG system is closely linked to the cytoplasmic system, mitochondrial pH is considerably more alkaline, and mitochondria contain unique forms of thioredoxin and thioredoxin reductase. Thus, mitochondrial thiol/disulfide systems must be considered separately from the other subcellular compartments [22]. In hepatocytes, where 15% of cell volume is mitochondria, approximately 15% of the total GSH is contained in the mitochondria [52–56]. Mitochondria do not contain the enzymes needed to synthesize GSH [57], and mitochondrial GSH is maintained by transport systems that import negatively charged GSH against the electrochemical gradient [58,59]. Transport is stimulated in energized mitochondria and inhibited by glutamate or disruption of the protonmotive force [58]. Two carriers, the dicarboxylate and 2-oxoglutarate transporters [59–62], which are anion exchange systems using the energy of the mitochondrial membrane potential, drive the uptake of the anionic GSH against the prevailing membrane potential (negative inside). Early studies showed cytoplasmic GSH was selectively depleted by diethylmaleate (DEM) thereby establishing the independence of mitochondrial and cytoplasmic pools [52,63]. Selective depletion of mitochondrial GSH was shown with (R,S)-3-hydroxy-4pentenoate (3-HP), an agent activated to an electrophile within mitochondria by ␤-hydroxybutryate dehydrogenase [63]. A decrease in mitochondrial GSH plays a role in the pathogenesis of alcoholic liver disease (ADL), where mitochondrial GSH is decreased by 60% [64,65]. Radiolabeling experiments show that transport of GSH into the mitochondria is decreased by approximately 35%, an effect that mostly accounts for mitochondrial GSH depletion with ethanol exposure [66]. Acetaminophen also preferentially depleted mitochondrial GSH as compared to that in the cytosol [67], suggesting a possible role for mitochondrial GSH depletion in acetaminophen toxicity. Estimates of mitochondrial GSH/GSSG redox potential in cells are limited because of the difficulty in measuring GSH and GSSG concentrations in the mitochondrial compartment without artifacts due to fractionation. Based upon available data and assuming a pH of 7.8, the mitochondrial GSH/GSSG pool is about −280 mV and becomes oxidized to at least −235 mV during apoptosis [68,69]. Studies of the possibility that oxidation of GSH was central in apoptosis signaling showed that oxidation of mitochondrial GSH/GSSG was secondary to

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cytochrome c release in cells treated with staurosporine [69]. In addition to the mitochondrial GSH peroxidase and GSSG reductase system for elimination of peroxides [70], mitochondria also contain glutaredoxin-2 (Grx2) [71] and a fully functional thioredoxin system consisting of thioredoxin-2 (Trx2), thioredoxin reductase-2 (TR2) and peroxiredoxin-3 (Prx3). Each of these systems protects against oxidative stress and apoptosis [71,72]. Grx2 is a homolog of cytoplasmic glutaredoxin-1 and catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane proteins. Molecular studies show that Grx2 has a central role in GSH-dependent redox regulation in mitochondria [73]. Overexpression in Hela cells inhibited cardiolipin loss and prevented apoptosis induced by doxorubicin. Thus, Grx2 is a component of one of the central mitochondrial redox systems for protection against oxidative stress. Because Grx2 catalyzes reversible oxidation of proteins, which can regulate function, it appears reasonable to consider this a redox control pathway (see Fig. 2). Trx2 has a better defined regulatory function, binding to apoptosis signal-regulating kinase-1 (ASK1) and inhibiting activity [30]. Oxidation of Trx2 frees ASK1, allowing apoptosis to ensue. In addition, overexpression of Trx2 also protects against oxidative stress and apoptosis [31] probably functioning in the detoxification of H2 O2 through peroxiredoxins. Peroxiredoxin-3 (Prx3), a peroxiredoxin found exclusively in the mitochondria, was overexpressed in numerous cancer cell lines and also protected against apoptosis [74]. Although the central GSH/GSSG and Trx2 redox pathways are established, the full range of protein targets of Grx2 and Trx2 has not been identified. These targets could include proteases and other proteins that function in protein import [75], enzymes of citric acid cycle, ␤-oxidation pathway supplying reducing equivalents for NADH or NADPH, regulation of ion transport activity, mitochondrial protein synthesis or other aspects of mitochondrial metabolism and homeostasis. Consequently, efforts to map the diverse range of redoxsensitive proteins using proteomic techniques [76,77] will be important as a foundation for understanding the details of mitochondrial oxidative stress. 5. Third principle: superoxide anion produced by the high-flux pathways connects the high-flux circuits with the low-flux circuits As illustrated in Fig. 2, inhibitors or electron mediators stimulate electron flow from high-flux electron transfer pathways to O2 to generate O2 •− . With a steady-

state redox potential for the NADH/NAD+ couple in the mitochondrial matrix about −300 mV [28], the energy available for 1-electron transfer from the NAD+ /NADH couple is ample to reduce O2 to produce O2 •− . If the reaction were near equilibrium in cells, then with O2 at a concentration of 10 ␮M and the E0 for O2 /O2 •− of −330 mV, the steady-state O2 •− concentration could be as high as 10 nM. The actual concentration of O2 •− is much lower due to spontaneous and enzymatic dismutation of O2 •− to produce H2 O2 . H2 O2 is rapidly removed by mitochondrial GSH peroxidases and peroxiredoxins, and these reactions provide an obvious connection between the high-flux electron transfer pathways and low-flux thiol/disulfide pathways that is apparently present under all aerobic conditions (Fig. 2). The relationship of the O2 •− production by complex I to energetics of ATP production is interesting because, in principle, variation of O2 •− generation can occur without an obligatory failure in ATP synthesis. This can happen because the free energy available from electron transfer to drive proton pumping is proportional to the steady-state differences in the redox potentials between the donor and acceptor redox couples for each of the coupling sites (G = −nFE). As long as electron flow maintains donor/acceptor couples in appropriate redox states, sufficient energy is available from electron transfer through the coupling sites to maintain the electrochemical proton gradient for ATP synthesis. Although electron transfer from NADH to O2 exclusively through the coupling sites would be needed for maximal energy efficiency, as long as an adequate reducing force is available to maintain the electrochemical proton gradient, simultaneous electron transfer to other acceptors will have no effect on ATP synthesis. This character is important because it allows for the possibility that O2 •− can be produced by complex I as a signaling molecule without disruption of mitochondrial energetic functions. 5.1. Proposed function of mitochondrial O2 •− in energy regulation In analogy to the known functions of ROS in extramitochondrial signaling, O2 •− produced by the mitochondrial chain could serve as a signal that is sensed by proteins containing iron–sulfur centers or sulfur switches and provide a regulatory mechanism for respiration. The background for this hypothesis is that earlier research indicated the existence of signaling molecules which could provide a coordinated multisite regulation (CMR) of energy metabolism [78]. Specifically, for osmotic stability during metabolic transitions, a mechanism is needed to balance the generation of the protonmotive

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Table 1 Postulates for coordinated multisite regulation (CMR) of cellular energy metabolism by superoxide anion

Fig. 3. Coordinated multisite regulation (CMR) of cellular energy metabolism by the superoxide anion (O2 •− ). Mitochondria contain many systems coupled to p, and mitochondria are susceptible to osmotic disruption if p fluctuates. Oxygen deficiency and failure of metabolic substrate supply due to starvation are physiologic challenges which must be accommodated without collapse of p. Postulate 1: Superoxide anion production by NADH dehydrogenase has characteristics that are appropriate to function in CMR, being generated as a function of both O2 and NADH concentrations. Postulate 2: By functioning as a positive regulator of phosphate uptake by the inorganic phosphate transporter, ATP/ADP exchange by adenine nucleotide translocator, and ATP synthesis by the ATP synthase, superoxide generated in Step 1 regulates the use of p for supply of ATP to cytoplasm. Under anoxic conditions, loss of this activator would slow collapse of p. Postulate 3: O2 •− controls supply of NADH when this is already adequate for generation of p. A rheostat function for aconitase has previously been proposed because this enzyme is inhibited by O2 •− . The present concept also proposed O2 •− -dependent inhibition of Ca2+ uptake by the Ca2+ uniporter because this mechanism would provide a general inhibition of Ca2+ -activated NAD+ -linked dehydrogenases. Postulate 4: O2 •− activates uncoupling proteins (UCPs) to prevent excessive p. Evidence is available supporting a role for O2 •− in activating UCP’s, and this activity together with postulates 2 and 3 would provide an effective coordination of mechanisms generating, using and dissipating p.

force (p), the utilization of p for ATP production and the utilization of p for ion transport and other functions [78]. O2 •− produced by complex I has the appropriate characteristics to provide a signal that there is enough O2 and NADH for optimal bioenergetic functions (Fig. 3). In the following, I have developed the hypothesis that O2 •− functions in CMR in terms of four postulates (Table 1). The first postulate is that O2 •− is produced by the NADH dehydrogenase complex at rates dependent upon the O2 concentration and the redox state of complex I maintained by NADH/NAD+ . Although not specifically designed to test this concept, earlier data show that ROS increases with O2 partial pressure [24,79], and numerous studies with respiratory inhibitors show that the complex becomes more reduced under conditions which result in

1. O2 •− is a metabolite of NADH dehydrogenase produced as a signal of adequacy of O2 and NADH for generation of the protonmotive force (p) 2. O2 •− provides an “on” switch for the generation and supply of ATP to the cytoplasm by activating the adenine nucleotide transporter (ANT), ATP synthase and the inorganic phosphate carrier (PiT) 3. O2 •− is a negative regulator of the Ca2+ uniporter and aconitase to limit supply of electrons to produce NADH when there is adequate supply. Ca2+ uptake by the Ca2+ uniporter stimulates NAD+ -linked dehydrogenases and increases mitochondrial O2 consumption 4. O2 •− is an activator of uncoupling proteins to prevent osmotic instability by limiting the magnitude of the electrochemical proton gradient

increased O2 •− production. Thus, available data appear to support this postulate. The next two postulates (Table 1) are based upon the model for CMR, which was developed from studies showing that mitochondrial membrane potential, pH gradient and ion gradients were preserved in mitochondria during short-term anoxia [80–82]. These data showed that p and mitochondrial and cytoplasmic concentrations of ATP, ADP and inorganic phosphate were maintained such that there was sufficient energy available from p for ATP synthesis, yet this synthesis did not occur. The results showed that the adenine nucleotide transporter (ANT) was inhibited rather than functioning in reverse and using glycolytic ATP to maintain the p. Furthermore, a pyruvate gradient was maintained, indicating maintenance of a pH gradient, but inorganic phosphate was not accumulated in accordance with the gradient, indicating that phosphate uptake was also inhibited. The data suggested a coordinated regulation of ψ-linked and pH-linked ion transport systems to provide a mechanism by which oxidative phosphorylation could be regulated without disruption of the ionic homeostasis and osmotic stability [78]. The signaling mechanisms for CMR remain unidentified, but previous research suggested that the signal could be a lipid that is structurally similar to the prostaglandin B-like lipid, dicalciphor [83,84]. The current consideration of O2 •− as a signal would require that O2 •− have the opposing character of dicalciphor, i.e., stimulating activities of ψ- and pH-dependent processes in response to NADH (for complex I) and O2 (for complex IV) (Fig. 3). If so, O2 •− would have to function as a negative control for metabolic suppression [78,81].

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This leads to the second postulate, that O2 •− is an “on” switch for ANT, ATP synthase and the inorganic phosphate carrier (PiT). When O2 •− generation is limited, the system is throttled down, i.e., is not as active in use of the p for driving ATP/ADP exchange, phosphate uptake, or ATP synthesis, thereby preserving the p for support of other functions, including maintenance of normal ion gradients and osmotic stability. Although I am not aware of any evidence to support this postulate, iron–sulfur proteins and other mitochondrial components with un-identified functions could provide such O2 •− -sensitive regulation. A third postulate (Table 1), also based upon the CMR model, is that O2 •− should inhibit Ca2+ uptake and key components of NADH-generating mechanisms, such as aconitase. Ca2+ serves as a universal activator of cytoplasmic processes that require energy. Previous studies show that increased Ca2+ over the physiologic range found in cytoplasm results in increased mitochondrial Ca2+ uptake by the Ca2+ uniporter and stimulates NAD+ -linked dehydrogenases with increased mitochondrial O2 consumption [78]. Inhibition of the uniporter therefore provides a mechanism to block the Ca2+ uptake when sufficient reducing equivalents are already available to support generation of the p. Similarly, inhibition of aconitase could regulate electron flow to NAD+ , a concept that has previously been developed in a “rheostat” model for control of mitochondrial functions [85]. Although critical experiments are needed to test this postulate, the rheostat model is based upon evidence that supports this concept. A final postulate is that O2 •− produced by complex I provides a signal to activate uncoupling proteins (UCP), providing a mechanism to limit p [86–88]. This mechanism has been studied previously [86–88] and is important in the overall hypothesis that O2 •− is a signaling molecule integrating mitochondrial energy metabolism because it provides a feedback loop controlling p (Fig. 3). However, such a mechanism may not be universally needed because of the differences in NADH supply mechanisms in tissues. 5.2. Extramitochondrial uses of mitochondrial O2 •− In addition to the proposed functions in regulation of energy metabolism, O2 •− is an anion that is transported out of mitochondria down its electrochemical gradient, presumably by anion transporters. This could function to maintain low matrix concentrations, drive the import of other anions (e.g., in exchange for pyruvate, phosphate, etc.), provide oxidizing equivalents for protein import

[75] or provide a transmembrane redox signal to activate nuclear expression of detoxification genes, e.g., by activation of NF-␬B. While purposeful functions of such release are largely speculative, the distribution of components is also appropriate for O2 •− to deliver electrons to cytochrome c in the intermembrane space. Guidot et al. showed that mitochondria are very efficient in metabolizing O2 •− that is generated exogenously [89]. If O2 •− were efficiently delivered from the matrix to cytochrome c in the intermembrane space, such a mechanism would be functionally equivalent to an inward proton leak and could have evolved to either balance functions of complex I and complex IV, to provide a bypass protecting against complex III inhibition or to provide an alternative mechanism to superoxide dismutase-2 (SOD2, MnSOD) for protection against high O2 •− concentrations. 6. Implications resulting from the definition of mitochondrial oxidative stress as a disruption of redox circuitry Although much of what is known about mitochondrial oxidative stress is unaffected, a shift in definition of oxidative stress would have important implications concerning analytic methods for measurement, mechanisms of injury and strategies for intervention. In the following brief discussion, most of the implications apply regardless of whether O2 •− is a toxic byproduct of respiration or a purposefully generated signaling molecule. Important differences, particularly concerning interventional strategies, are noted. 6.1. Analysis of mitochondrial oxidative stress A redefinition of oxidative stress requires consideration of how this would impact measurement of oxidative stress. My impression is that it would have no impact on use of increased ROS, decreased antioxidants (e.g., GSH), or oxidation of macromolecules (e.g., cardiolipin, DNA) because these are consequences of oxidative stress regardless of whether this is caused by an “imbalance” or a “disruption of redox circuitry”. On the other hand, if oxidative stress is a disruption of redox circuitry, then efforts to detect perturbations in specific redox pathway components could yield more sensitive and specific indicators of oxidative stress. For thiol/disulfide systems, methods are available to measure oxidation of specific amino acids in specific proteins. Although not widely used for mitochondria, these methods provide the theoretical capability to identify specific sites of disruption of low-flux redox pathways as well as high-flux pathways.

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Trx2 is specific to the mitochondria and is well suited to determine oxidative stress within the mitochondria. The use of antibodies specific to Trx2 allows measurement of mitochondrial redox state by the redox-western blot method without a need for subcellular fractionation [14]. The redox-western blot technique for Trx2 utilizes a thiol-reactive reagent, 4-acetamido-4 -maleimidylstilbene-2,2 -disulfonic acid (AMS), to derivitize reduced Trx2 [14,15]. Samples are separated via non-reducing SDS-PAGE. Derivatized Trx2 with AMS is larger (approximately 1 kDa) and migrates through the gel more slowly as compared to oxidized/non-derivitized Trx2. Utilizing this method, results show that H2 O2 or tert-butylhydroperoxide preferentially oxidizes Trx2 as compared to Trx1 (Chen and Jones, unpublished). Similarly, tumor necrosis factor-␣ (TNF␣) caused preferential oxidation of Trx2 in HeLa cells without oxidation of cytoplasmic Trx1 [90]. This preferential Trx2 oxidation occurred within 10 min of TNF␣ treatment, and overexpression of Trx2, but not Trx1, decreased TNF␣-induced ROS generation. Thus, the results show a mitochondrial compartmentation of ROS production and specific detoxification by Trx2, not Trx1 [90]. Inhibition of NF-␬B nuclear translocation following TNF␣ treatment was also inhibited by Trx2 overexpression but not with a dominant negative activesite mutant, C93S Trx2. These results show that specific oxidations can be discerned by analysis of redox states of specific proteins, providing a way to elucidate disruption of redox circuitry [90]. A similar application of this approach showed that treatment of keratinocytes with EGF-induced ROS production and cytoplasmic Trx1 oxidation without oxidation of mitochondrial Trx2 [14]. Thus, the approach can also provide useful exclusion of mitochondrial mechanisms in studies of oxidative stress and redox signaling in other subcellular compartments. An alternate method to measure changes in redox circuits has been applied to mitochondrial thioredoxin reductase-2 (TrxR2). Thiol-modified affinity separation followed by western blotting was used to measure oxidation of TrxR2 in Hela cells in response to tumor necrosis factor-␣ (TNF␣) [91]. In this approach, cell extracts were treated with biotin-conjugated iodoacetamide, immunoprecipitated with an antibody to TrxR1 (cytoplasmic TrxR1) or TrxR2, and probed by western blotting with HRP-conjugated streptavidin. The results showed a rapid oxidation of TrxR2 in response to TNF␣, with changes apparent at 10 min. Together with the above studies, the results show that detailed mapping of redox circuits is possible with existing methods. A study of metal toxicity using redox-western blot further illustrates the utility of this approach for deter-

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mining specificity of redox processes in oxidative stress [92]. Results comparing oxidation of Trx2, Trx1 and GSH/GSSG showed that metals have different effects on the major thiol antioxidant systems. Copper, iron and nickel oxidized GSH/GSSG but caused little oxidation of either Trx1 or Trx2. Arsenic, cadmium and mercury showed little oxidation of GSH/GSSG but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium and mercury were greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20–40 mV). Apoptosis signal-regulating kinase 1 (ASK1) activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. Thus, metals have differential oxidative effects on the major thiol antioxidant systems, with Trx2 showing the greatest sensitivity [92]. Such comparisons of sensitivities of GSH, Trx and other proteins can have great utility in identifying specific sites that are sensitive to different agents that cause oxidative stress. Redox-sensitive GFPs (roGFP) also provide a useful approach for determining mitochondrial redox states. Useful forms show different fluorescence in the reduced state as compared to the oxidized state, allowing ratiometric methods for quantification [93,94]. Using constructs with different Eh values, a relatively wide range of redox potentials can be measured. A mitochondrially targeted roGFP1 with a midpoint potential of approximately −280 to −291 mV [94] indicated that mitochondria have a redox potential of about −360 mV, but it is not clear which components are being reported by this redox indicator. However, the method is remarkable in that redox effects can be measured in single mitochondria. Identification of how such indicators interact with endogenous redox circuits will be needed to enhance utility and improve understanding of mitochondrial oxidative stress. 6.2. Mitochondrial targets of oxidative stress and mechanisms of injury Three mitochondrial targets have emerged as possible common elements in toxicity, chronic disease and functional declines in aging, namely, the permeability transition pore complex [95], the inner membrane cardiolipin [96] and the mitochondrial DNA [97]. Shifting the definition of oxidative stress to disruption of redox circuitry would have no impact upon concepts related to these targets of injury because they are downstream consequences. On the other hand, there are important implications concerning how one interprets the conditions and sequence of events that cause these outcomes.

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A key question is whether variation in concentration of an oxidant, per se, is critical for damage or whether the pathway for electron transfer is really the critical parameter. NADH dehydrogenase reduces O2 to form O2 •− , but O2 •− is not a very potent oxidant (see [98] for additional information on chemistry of O2 •− ). Macromolecular damage from O2 •− is considerably enhanced by more powerful oxidants generated by Fenton chemistry in the presence of H2 O2 and a transition metal ion. In eliminating O2 •− , SOD2 produces the other reactant (H2 O2 ) that creates conditions primed for oxidative damage. But the rate-limiting component of the Fenton reaction is free Fe3+ , a catalyst for formation of the highly reactive hydroxyl radical. In the absence of free Fe3+ , O2 •− and H2 O2 have limited reactivities. Thus, it would appear that the pathway for electron transfer, i.e., the short-circuit created by Fe3+ , is the main determinant of injury. If the CMR hypothesis is correct that O2 •− and/or H2 O2 are normal products of mitochondria, then the redefinition of oxidative stress would shift the focus from the balance of O2 •− and free radical scavengers to the sources of free Fe3+ , Cu2+ and other agents that disrupt normal electron flow. Agents that disrupt FeS centers, releasing Fe3+ , may be especially worthy of investigation. Release of H2 S from these centers could also inhibit cytochrome oxidase, further contributing to disruption of electron flow. Application of the disruption of redox circuitry concept to targets of toxicity reveals that disruption of different redox circuits could converge with the same biologic outcome. The mitochondrial permeability transition (MPT) pore is an inner membrane/outer membrane protein complex that opens in response to Ca2+ and causes the mitochondrial matrix to undergo high amplitude swelling. Possible functions of the MPT in normal cell physiology have been discussed [99,100]. Earlier studies implicated ANT as a key component of the pore complex, but studies with double knockout of liver forms of ANT in mice showed that ANT is a regulatory component but not an integral component of the pore [101]. Studies using cyclophilin D (CyD) knockout mice show that the MPT is a key event in necrotic cell death but not in apoptosis. The MPT is activated by oxidants, and multiple thiols have been implicated. ANT has thiols that are subject to oxidation [102,103], and recent evidence also indicates redox sensitivity of CyD [99,100]. Thus, evaluation of redox circuits controlling these different sites could reveal that oxidation of thiols in either one or both of these proteins could trigger the MPT. The growing recognition that redox regulation can occur by different mechanisms, i.e., S-glutathionylation versus

disulfide crosslinking within or between proteins, underscores the importance of understanding such mechanistic details. While activation of the MPT has been associated with necrosis, oxidation of cardiolipin causes cytochrome c release and activates apoptosis. There is a possibility that lipoxidases catalyze cardiolipin oxidation, but it would appear more likely that free radical mechanisms are involved. In comparison to the activation of the MPT, in which thiols could be oxidized by thiol–disulfide exchange catalyzed by enzymes such as Grx2, oxidation of cardiolipin is likely to involve ROS and transition metals. Thus, depending upon the prevailing redox pathways, oxidative stress could differentially activate the MPT and necrosis or cardiolipin oxidation and apoptosis. This leads to the conclusion that the disruption of redox circuitry provides a better way to think about the mechanisms of oxidative stress. 6.3. Implications for interventional strategies for oxidative stress in humans Circumstantial evidence indicates that oxidative reactions contribute to many human disease processes including cardiovascular disease [104], pulmonary diseases [105], diabetes [106], neurodegenerative diseases [107] and cancer [108]. Plausible free radical mechanisms are known, including those described above that could affect mitochondrial function, and supportive data are available. However, large-scale interventional studies with free radical scavenging antioxidants have not clearly demonstrated health benefits in terms of quantitative measures of disease outcome [1–9]. Consideration of oxidative stress as a disruption of redox circuitry implies that approaches are needed to assess disruption in redox circuitry and strategies are needed to restore proper electron flow. The newly developing fields of redox proteomics and redox metabolomics provide the opportunity to develop comprehensive approaches to evaluate redox circuitry in individuals. Such methods can address function of electron transfer systems in terms of redox states of individual protein thiols and patterns of intermediary metabolites. Thus, in contrast to the common efforts to evaluate oxidative stress in terms of free radical antioxidant capacity and global changes in oxidation end products, oxidative stress could be evaluated using a systems biologic approach to assess disruption of oxidative functions. The need for such approaches can be illustrated by a speculative example involving SOD2-dependent prolongation of lifespan. Lifespan is increased in

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transgenic animals with increased SOD2 expression [109]. This association implies that the steady-state concentration of mitochondrial O2 •− and associated oxidative stress are determinants of survival. However, as indicated in the preceding sections, high-flux redox pathways of energy metabolism are inextricably linked to ROS generation and redox control. Because caloric restriction also prolongs lifespan, the prolongation of lifespan with SOD2 could be related to O2 •− signaling and energetic changes, being mechanistically similar to caloric restriction rather than due to a change in free radical-induced injury, per se. Thus, the elucidation of how SOD2 alters redox pathways is critical to understanding this instructive experimental model. The view that oxidative stress is a disruption of redox circuitry has important implications concerning approaches to prevent oxidative stress. Unlike the most common approaches to improve the prooxidant/antioxidant balance by providing better free radical scavenging activity, this view implies that other fruitful approaches might include methods to avoid disruption of the redox circuitry or strategies to restore proper electron flow. As indicated above, trace metals create deleterious electron transfer pathways, and improved control of trace metal ions may be a more effective approach to prevent oxidative stress than increased free radical scavengers. Redox cycling agents that interact with high-flux electron transfer pathways are also well known to cause catastrophic oxidative stress. Efforts to identify and eliminate chemicals with redox cycling properties from food sources, environmental and occupational exposures and therapeutic use, could provide health benefits by preventing disruption of redox circuits. It should also be noted that chemicals with effective in vitro antioxidant properties could also disrupt normal redox signaling by inappropriate interaction with redox pathways in vivo. Efforts to identify and eliminate inhibitors of electron transfer pathways, such as those that inhibit complex I and are implicated in Parkinson’s disease, could also be effective in protection against oxidative stress. Many naturally occurring inhibitors of the respiratory chain occur in bacterial and fungal species [110], and the low-level toxicity of rotenone [16] suggests that dietary and environmental exposures to low levels of such mitochondrial toxicants could cause oxidative stress by disrupting normal electron transfer pathways. Finally, it would appear that if sites of disruption of electron transfer could be identified, new therapeutic approaches could be developed to restore proper electron flow. This approach would be analogous to the use of electron mediators to bypass a genetic block in the mitochondrial chain in mitochondrial myopathy. Devel-

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opment of improved means to therapeutically regulate mitochondrial electron transfer pathways could have considerable applicability to controlling oxidative stress as well as energy metabolism. 7. Summary and conclusion The present review considers oxidative stress in mitochondria as a disruption of redox circuits rather than as an imbalance of oxidants and antioxidants. Two types of redox circuits are conceptually distinguished, those with metabolic functions and those with functions in metabolic control and cell signaling. The former circuits have high electron transfer rates relative to the latter. Oxidative stress occurs when electron transfer through these high-flux circuits is disrupted by conditions that block electron transfer or facilitate electron transfer between components and create “shortcircuits”. Reactive oxygen species are viewed as normal products of the high-flux pathways, with O2 •− and H2 O2 being relatively inert in the absence of Fe3+ or another electron transfer catalyst and providing a redox connection between the high-flux and low-flux pathways. Four postulates are presented for the hypothesis that O2 •− generated by the electron transfer pathway functions as a signaling molecule in coordinated multisite regulation (CMR) of cellular energy metabolism: (1) O2 •− is a metabolic product of the electron transfer chain that is produced to signal that NADH and O2 are available for generation of the protonmotive force (p), (2) that O2 •− functions as an activating signal for ion transport systems that use the p for generation and supply of ATP to the cytoplasm, (3) that O2 •− functions as a negative regulator for supply of reducing equivalents to NAD+ , and (4) that O2 •− activates uncoupling proteins to avoid excessive p. Although speculative, this hypothesis focuses attention to the need to address specific mechanisms of disruption of redox circuitry in oxidative stress. Finally, the view of oxidative stress as a disruption of redox circuitry has important implications concerning assays, mechanisms and interventions for oxidative stress. Importantly, application of redox-western blot analysis and redox proteomics provides the opportunity to map mitochondrial redox circuits and identify sites of disruption during oxidative stress. Such approaches can provide a new level of detail in mechanistic studies of oxidative stress, allowing distinction of sources and targets of redox change. The focus on disruption of redox circuitry rather than balance between prooxidants and antioxidants suggests that efforts are needed to identify and eliminate exposures to agents that disrupt

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mitochondrial redox circuitry by releasing Fe3+ from endogenous sources, inhibiting electron transfer or catalyzing redox cycling reactions. This view also suggests that novel therapeutic approaches to controlling oxidative stress could be obtained by developing means to restore proper electron transfer reactions.

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