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. 2017 Aug 24;292(41):16804–16809. doi: 10.1074/jbc.R117.789271

Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions

Hoi-Shan Wong 1,1, Pratiksha A Dighe 1, Vojtech Mezera 1, Pierre-Axel Monternier 1, Martin D Brand 1
PMCID: PMC5641882  PMID: 28842493

Abstract

Mitochondrial production of superoxide and hydrogen peroxide is potentially important in cell signaling and disease. Eleven distinct mitochondrial sites that differ markedly in capacity are known to leak electrons to oxygen to produce O2˙̄ and/or H2O2. We discuss their contributions to O2˙̄/H2O2 production under native conditions in mitochondria oxidizing different substrates and in conditions mimicking physical exercise and the changes in their capacities after caloric restriction. We review the use of S1QELs and S3QELs, suppressors of mitochondrial O2˙̄/H2O2 generation that do not inhibit oxidative phosphorylation, as tools to characterize the contributions of specific sites in situ and in vivo.

Keywords: Complex I, hydrogen peroxide, mitochondria, reactive oxygen species (ROS), superoxide ion, complex III

Introduction

Superoxide (O2˙̄)2 and hydrogen peroxide (H2O2) are produced by leaks of electrons from donor redox centers of the mitochondrial electron transport chain and associated metabolic enzymes to cause either one-electron or two-electron reduction of oxygen (1, 2). To date, at least 11 sites that produce O2˙̄ and/or H2O2 (O2˙̄/H2O2), namely sites OF, BF, AF, PF, IF, IQ, IIF, IIIQo, GQ, EF, and DQ, have been identified in mammalian mitochondria (Fig. 1), with each of them exhibiting distinct properties (3–33). The majority of the work on site-specific mitochondrial O2˙̄/H2O2 production has focused on measurement of the maximum capacities of these sites under conditions in which concentrations of substrates were optimal (usually saturating) and inhibitors of electron transport were used to induce optimal (usually maximal) reduction of redox centers (16, 34, 35). However, maximum capacities, which are mainly dependent on the abundance of the relevant proteins or complexes in the mitochondria, are not necessarily related to the importance of particular mitochondrial sites in physiological or pathological O2˙̄/H2O2 production in vivo. The measurement of “native” rates of O2˙̄/H2O2 production from mitochondrial sites under physiological conditions in the absence of inhibitors of electron transport is required to answer such questions. Rates of mitochondrial O2˙̄/H2O2 production from sites IF and IIIQo in any condition can be assessed indirectly by measuring endogenous reporters (36). Such endogenous reporters allow assessment of native rates following specific inhibition of the site of interest by measurement of the decrease in overall rate after correction for the secondary changes in the rates from other sites (assessed using the endogenous reporters) (36–38).

Figure 1.

Figure 1.

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Mitochondrial sites of O2˙̄/H2O2 production. Eleven distinct mitochondrial sites that leak electrons to oxygen to produce superoxide and/or hydrogen peroxide during substrate oxidation have been identified (21, 25–28, 30–33). Yellow circles indicate flavin groups, and orange circles indicate quinone-binding sites of enzymes and complexes. ETF, electron-transferring flavoprotein; ETF:QOR, electron-transferring flavoprotein-ubiquinone oxidoreductase; QH2, ubiquinol; Q, ubiquinone. Sites OF, BF, AF, PF, IF, IQ, IIF, IIIQo, GQ, EF, and DQ refer to the flavin (subscript F) or ubiquinone-binding sites (subscript Q) of the 2-oxoglutarate (O), branched chain 2-oxoacid (B), 2-oxoadipate (A), and pyruvate (P) dehydrogenase complexes, of complex I, complex II, and the outer ubiquinone site of complex III (IIIQo), and of mitochondrial glycerol-3-phosphate dehydrogenase (G), the ETF/ETF:QOR system (E), and dihydroorotate dehydrogenase (D).

Although excessive production of reactive oxygen species (ROS) from mitochondria is associated with a broad spectrum of pathologies (39–41), there is growing evidence supporting a role of mitochondrial ROS as signaling molecules in physiological pathways (42–45). In this context, mitochondrial ROS-mediated signaling cascades have been proposed to be involved in the response or adaptation to bioenergetic changes (46, 47). Therefore, the characterization of mitochondrial O2˙̄/H2O2 production in response to changes in bioenergetic states allows a better understanding of the physiological roles of O2˙̄/H2O2 as signaling molecules and may illuminate potential therapeutic target(s) for related pathologies.

In the following sections we discuss the contributions of different mitochondrial sites to physiological O2˙̄/H2O2 production under different bioenergetic conditions. We also discuss the use of S1QELs and S3QELs, site-specific suppressors of mitochondrial O2˙̄/H2O2 generation that have no effect on oxidative phosphorylation, as tools for such characterization in situ and in vivo.

Why native rates?

Understanding of the origins and rates of mitochondrial O2˙̄/H2O2 production under physiological conditions is important for understanding normal cellular behavior and signaling, and it is an important prerequisite for investigating the roles of mitochondrial O2˙̄/H2O2 production in pathology. Conventionally, the characterization of mitochondrial O2˙̄/H2O2 production involves the use of specific combinations of conventional substrates and electron transport inhibitors to provide information about the kinetics and the capacities of particular sites of interest. However, the impairment of electron flow by these inhibitors and the subsequent redistribution of electrons to other redox centers lead to misinterpretation when studying the contributions of particular mitochondrial sites to physiological O2˙̄/H2O2 production. Instead, the measurement of native rates of O2˙̄/H2O2 production is required when evaluating the contribution of each site under different experimental conditions.

Quinlan et al. (36) introduced measurement of the redox states of endogenous reporters as an indirect way to estimate the rates of O2˙̄/H2O2 production from mitochondrial sites. They identified unique relationships between the reduction state of the mitochondrial NAD pool and the rate of O2˙̄ or H2O2 production at the flavin site of mitochondrial complex I (site IF). The relationship between the reduction state of cytochrome b566 of mitochondrial complex III and the rate of O2˙̄ production at the Qo site of mitochondrial complex III (site IIIQo) was also elucidated. Calibration curves of measured H2O2 production arising from site IF against the mitochondrial NAD(P)H redox state and measured H2O2 production arising from site IIIQo against the cytochrome b566 redox state allow the quantification of the contribution of sites IF and IIIQo to total H2O2 production under various experimental conditions. These endogenous reporters also serve as a tool to correct for secondary changes in the redox state of the NAD pool and cytochrome b566 when specific inhibitors or omissions of particular substrates are used to shut down other sites, allowing the quantitative determination of O2˙̄/H2O2 production rates from these other mitochondrial sites as well (36–38, 48).

Mitochondrial O2˙̄/H2O2 production and substrate oxidation

Mitochondrial enzyme complexes oxidize a wide variety of substrates to support mitochondrial respiration. The origin of O2˙̄/H2O2 production when mitochondria oxidize different substrates mostly depends on the redox levels at which the substrates donate their electrons. Quinlan et al. (38) demonstrated the following contributions of different mitochondrial sites to H2O2 production during oxidization of the following: (i) complex I-linked substrates, glutamate plus malate; (ii) the complex II-linked substrate, succinate; (iii) a substrate for fatty acid oxidation, palmitoylcarnitine plus carnitine; and (iv) the substrate for mitochondrial glycerol-3-phosphate dehydrogenase (an enzyme abundantly expressed in skeletal muscle), glycerol 3-phosphate (Fig. 2). The overall rates of mitochondrial H2O2 production differed when mitochondria were oxidizing different substrates as fuel molecules. The oxidation of succinate led to the highest rate of O2˙̄/H2O2 production. Succinate is a classical substrate that can be used to study O2˙̄/H2O2 production from complex I during reverse electron transport (site IQ). Because of its high sensitivity to inhibitors of the quinone-binding site of complex I, the O2˙̄/H2O2 production of site IQ is defined as the rotenone-sensitive signal when mitochondria are oxidizing succinate. After correcting for changes in O2˙̄/H2O2 production from site IF and site IIIQo upon rotenone inhibition (using endogenous reporters), 83% of the overall rate of mitochondrial O2˙̄/H2O2 production during succinate oxidation was attributed to site IQ (38). The total rate of O2˙̄/H2O2 production with glutamate plus malate as substrate was considerably lower, only 20–25% of the rate with succinate, showing the importance of the source of reducing equivalents in determining the rate of mitochondrial O2˙̄/H2O2 production.

Figure 2.

Figure 2.

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Relative contributions of specific sites of O2˙̄/H2O2 production to total H2O2 production by isolated rat muscle mitochondria under different bioenergetic conditions. The diameters of the pie charts are proportional to the total rates of mitochondrial H2O2 production. Total rates of mitochondrial H2O2 production (pmol of H2O2 min−1 mg protein−1) were ∼890 (succinate), ∼620 (glycerol 3-phosphate), ∼200 (palmitoylcarnitine), ∼180 (glutamate plus malate), ∼340 (rest), ∼80 (mild aerobic exercise), and ∼50 (intense aerobic exercise). Data are from Refs. 38 and 48.

Importantly, the engagement of particular mitochondrial sites changes with different substrates (Fig. 2), due mostly to the different entry points of reducing equivalents during metabolism and the resulting different redox states of distinct sets of enzymes and complexes. Taking complete oxidation of fatty acids as an example, fatty acids, or more specifically palmitoylcarnitine, have three entry points into the electron transport chain as follows: complex I (via the reduction of NAD+ by hydroxyacyl-CoA dehydrogenase and the dehydrogenases of the tricarboxylic acid cycle); electron-transferring flavoprotein (ETF-ubiquinone oxidoreductase via its reduction by ETF and mitochondrial acyl-CoA dehydrogenases); and to a minor extent complex II (via the formation of succinate formed during operation of the tricarboxylic acid cycle) (37). During oxidation of palmitoylcarnitine, site IF, site IIF (the flavin site of mitochondrial complex II), and site IIIQo were the main contributors to mitochondrial O2˙̄/H2O2 production under non-phosphorylating conditions; the rate of O2˙̄/H2O2 production from the ETF system was negligible. The contribution of site IIF was caused by backflow of electrons from the ubiquinone pool into complex II rather than by forward flow from succinate formed in the tricarboxylic acid cycle. It is worth noting that sites IF and IIIQo contributed to O2˙̄/H2O2 production during oxidization of all tested substrates. This is because the redox states of the NAD and ubiquinone pools differ little between substrates; they are common entry points for most substrates to the electron transport chain, and their redox states determine the rates of O2˙̄/H2O2 production from these two sites.

Mitochondrial substrate oxidation plays an important role in determining the origin and rates of O2˙̄/H2O2 production. Changes in bioenergetic states, for example, by physical exercise or caloric restriction, influence mitochondrial substrate availability and lead to the resultant alterations in the rates and the contributions of different sites of mitochondrial O2˙̄/H2O2 production.

Mitochondrial O2˙̄/H2O2 production during physical exercise

As mentioned above, individual mitochondrial sites produce O2˙̄/H2O2 at different rates depending on the substrate being oxidized (38, 48). In vivo and in whole cells, many substrates are metabolized simultaneously, and the availability of these substrates can change. In skeletal muscle, for example, the concentrations of respiratory substrates, effectors such as Ca2+ and intracellular pH, and energy demand (reflected in the concentrations of ADP and phosphate) change dramatically between states of rest, mild exercise, and exhaustive exercise (48).

Sites IF and IIIQo, or more generally mitochondrial complexes I and III, are conventionally recognized as the major sites of mitochondrial production of O2˙̄/H2O2. However, sites IIF and IQ also have the potential to produce O2˙̄/H2O2 at high rates in isolated mitochondria oxidizing low concentrations of succinate under non-phosphorylating conditions in which protonmotive force is high and rapid reoxidation of the ubiquinone pool is prevented. Such conditions seem relevant to resting muscles (30), where the rates of mitochondrial respiration and ATP synthesis are low (49). When skeletal muscle mitochondria were incubated with physiological concentrations of substrates and effectors under conditions mimicking rest ex vivo, sites IIF and IQ were found to be major sites of mitochondrial O2˙̄/H2O2 production, each contributing a quarter of the total rate of mitochondrial O2˙̄/H2O2 production; the remainder was mostly from sites IF and IIIQo for the reason discussed previously (Fig. 2) (48).

Exercise is associated with an increase in the generation of free radicals (50, 51). With the use of electron paramagnetic resonance spectroscopy, a rise in free radicals was seen in intact muscles during exercise, and mitochondria were proposed to be the major source (52). In contrast, an increasing body of evidence suggests a decreased production rate of these reactive species from mitochondria during muscular contractile activity (53). During exercise, the concentrations of ADP and mitochondrial substrates and effectors all increase, and the mitochondria respire rapidly to produce ATP. It is not obvious whether the increased substrate supply will raise mitochondrial O2˙̄/H2O2 production or the decreased protonmotive force and reduction state of mitochondrial redox centers will lower it. When skeletal muscle mitochondria were incubated with physiological concentrations of substrates and effectors under conditions mimicking exercise ex vivo, the resultant decrease in protonmotive force and the more oxidized ubiquinone pool led to decreased rates of O2˙̄/H2O2 production despite the increased substrate availability (48). This decrease was particularly marked for O2˙̄/H2O2 production from site IQ, which is sensitive to the pH gradient (ΔpH) across the mitochondrial inner membrane, and for site IIIQo, which responds to a decreased steady-state concentration of semiquinone in the electron transport chain (21, 26, 48, 54). Under ex vivo conditions that mimic mild aerobic exercise in skeletal muscles, the overall rate of mitochondrial O2˙̄/H2O2 production was found to be ∼50% lower than in the resting state, whereas the low-capacity site IF remained as the major contributor. Sites IQ, IIIQo, and probably EF and GQ also generated some O2˙̄/H2O2 during mild exercise (48). With an increase in the intensity of aerobic exercise, the protonmotive force (and more importantly ΔpH) is lower and the ubiquinone pool is further oxidized, which leads to further decreases in O2˙̄/H2O2 production from sites IQ and IIIQo. As a consequence, site IF contributed most of the overall O2˙̄/H2O2 production under conditions mimicking intense exercise (Fig. 2) (48).

Despite the lack of in vivo evidence, the ex vivo findings demonstrate the heterogeneity of the sources of mitochondrial O2˙̄/H2O2 in skeletal muscle during rest and mild and intense aerobic exercise. An overall decrease in O2˙̄/H2O2 production in response to conditions mimicking increasing intensities of physical exercise was also observed in skeletal muscle mitochondria.

Mitochondrial O2˙̄/H2O2 production during caloric restriction

In parallel to physical exercise, caloric restriction is considered to be a health-promoting intervention. Caloric restriction is defined as a reduction of food intake with adequate intake of protein and micronutrients to avoid malnutrition (55–57). Although caloric restriction was found to extend the life span of several animal species (58), its effect on mitochondrial O2˙̄/H2O2 production is still controversial. Caloric restriction decreased the maximum capacity of O2˙̄/H2O2 production when mitochondria isolated from brain, heart, kidney, liver, and skeletal muscle of rodents oxidized pyruvate and malate in the presence of rotenone (59–61). A similar trend in rotenone-sensitive H2O2 production from liver mitochondria energized with succinate was also observed in mice subjected to a 2-month course of caloric restriction (60). These findings suggest a decreased capacity for O2˙̄/H2O2 production from site IF and site IQ of the mitochondrial electron transport chain as well as from the upstream dehydrogenases, particularly pyruvate dehydrogenase, after caloric restriction. However, none of these studies included the crucial measurement of native rates.

As reviewed by Walsh et al. (62), the beneficial effects of caloric restriction on cellular redox homeostasis are mainly mediated by a more reduced glutathione pool, rather than by decreased mitochondrial O2˙̄/H2O2 production, with a resultant lower extent of oxidative damage to macromolecules (62). Interestingly, at the cellular and tissue level, the exposure of cells to low glucose could lead to a more oxidized mitochondrial NAD pool and a concomitant decrease in mitochondrial protonmotive force (63, 64). The oxidation of the NAD pool and the drop in mitochondrial protonmotive force (and ΔpH) may decrease rates of mitochondrial O2˙̄/H2O2 production from site IF, site IQ, and upstream dehydrogenases under these conditions (21, 27, 32).

In contrast, overnutrition was suggested to be associated with a more reduced NAD pool, which may in turn lead to higher mitochondrial O2˙̄/H2O2 production at site IF of the electron transport chain and by upstream dehydrogenases (36, 64–67). Enhanced substrate supply may also increase protonmotive force (68), which may in turn favor the production of superoxide/H2O2 from site IQ. Availability of nutrients can also impact mitochondrial dynamics. How such changes affect formation of mitochondrial O2˙̄/H2O2 production remains to be investigated (69).

The contributions (under native conditions in mitochondria oxidizing different substrates and in conditions mimicking physical exercise) and capacities (after caloric restriction) of mitochondrial sites to O2˙̄/H2O2 production in response to the changes in bioenergetic state have been mainly investigated in isolated mitochondria. Despite the effort to characterize the involvement of mitochondrial enzyme complexes in O2˙̄/H2O2 production in cells and tissues (mostly by genetic or pharmacological modifications) under pathological conditions, the contributions of specific mitochondrial sites to physiological O2˙̄/H2O2 production and their roles in cell signaling in situ and in vivo have not yet been elucidated.

Novel tools to characterize contributions of mitochondrial sites to O2˙̄/H2O2 production in vitro and in situ

In experiments with isolated mitochondria, O2˙̄/H2O2 are released and can be detected relatively easily in the surrounding assay medium. The measurement of mitochondrial O2˙̄/H2O2 production, and more specifically O2˙̄/H2O2 production from particular mitochondrial sites, is far more problematic in cells and tissues because of the ambiguous specificity and cellular localization of current probes as well as the impaired bioenergetics upon genetic modifications (70–72). A novel way around the problem of direct measurements in cells is to use molecules that suppress electron leak at specific sites. High-throughput chemical screening has identified molecules that specifically suppress O2˙̄/H2O2 production from particular mitochondrial sites without influencing mitochondrial oxidative phosphorylation. S1QELs (Suppressors of site IQ Electron Leak) are small molecules that suppress mitochondrial O2˙̄ production from site IQ during reverse electron transport (73). S3QELs (Suppressors of site IIIQo Electron Leak) are small molecules that suppress mitochondrial O2˙̄ production from site IIIQo (74). These compounds are very potent (effective at nanomolar concentrations) and inhibit phenotypes dependent on O2˙̄ production from sites IQ and IIIQo in several cell and tissue models (73, 74). The high potency, high specificity, and lack of inhibition of mitochondrial electron flow and oxidative phosphorylation make them promising tools to study the engagement of these mitochondrial sites in physiological O2˙̄/H2O2 production in situ and in vivo. A good example is the use of S1QELs to examine the involvement of mitochondrial O2˙̄ production from site IQ in primary astrocytes under an unstressed condition (74). In this study, the activity of succinate dehydrogenase, which has iron–sulfur centers that are susceptible to oxidative damage by O2˙̄ in the mitochondrial matrix (75), was used as an indirect measure of mitochondrial O2˙̄ production. S1QELs preserved succinate dehydrogenase activity in primary astrocytes cultured at 3% oxygen, showing the generation of O2˙̄ from site IQ at physiologically relevant rates in resting, unstimulated cells in the absence of overt oxidative stress. The finding was further supported by a parallel protective effect of S1QELs on cellular aconitase activity (74).

Summary

In summary, the rates and the relative contributions of different mitochondrial sites of O2˙̄/H2O2 production change in response to changes in bioenergetic states, as a consequence of alterations of mitochondrial substrate availability and the reduction states of mitochondrial redox centers. Characterizing mitochondrial O2˙̄/H2O2 production in isolated mitochondria in vitro and in cells and tissues in situ and in vivo may help us understand its roles in cell signaling under both physiological and pathological conditions.

This is the first article in the Thematic Minireview Series “Redox metabolism and signaling.” The authors declare that they have no conflicts of interest with the contents of this article.

2
The abbreviations used are:
O2˙̄
superoxide
ROS
reactive oxygen species
ETF
electron-transferring flavoprotein.

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