Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

Background: Ten mitochondrial sites of superoxide/H₂O₂ generation are known, but their contributions in vivo are undefined.

Results: We assessed their rates ex vivo in conditions mimicking rest and exercise.

Conclusion: Sites I_Q and II_F generated half the signal at rest. During exercise, rates were lower, and site I_F dominated.

Significance: Contributing sites ex vivo probably reflect those in vivo.

Abstract

The sites and rates of mitochondrial production of superoxide and H₂O₂ in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site III_Qo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site I_F) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H₂O₂ using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site I_Q) and the flavin site in complex II (site II_F) each account for about a quarter of the total measured rate of H₂O₂ production. Site I_F, site III_Qo, and perhaps site E_F in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site I_F dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H₂O₂ in vivo.

Introduction

Mitochondrial generation of superoxide and hydrogen peroxide was discovered in the 1960s and 1970s (1, 2) and has been well studied (3–15). It is not a single process; the signal obtained is the sum of rates from different sites that prematurely reduce oxygen to superoxide or H₂O₂. Each site has its own unique properties. The sites can be distinguished and studied in situ by providing electrons from appropriate substrates and using specific electron transport inhibitors to isolate them pharmacologically. In this way, at least 10 distinct sites have been characterized in rat skeletal muscle mitochondria (16). These sites are represented as red circles in Fig. 1. In order of their maximum capacities, they are as follows: III_Qo⁴ in complex III (17); I_Q (18, 19) and II_F (20) in complexes I and II; O_F, P_F, and B_F in the 2-oxoglutarate, pyruvate, and branched-chain 2-oxoacid dehydrogenase complexes (16); G_Q in mitochondrial glycerol phosphate dehydrogenase (21); I_F in complex I (16); E_F in ETF/ETF:Q oxidoreductase (22); and D_Q in dihydroorotate dehydrogenase (23).

FIGURE 1.

Sites of superoxide and H₂O₂ production during electron flow from different blocks of metabolites through the mitochondrial electron transport chain. Metabolic substrates are grouped into five blocks colored to match Table 1: ketone bodies, amino acids, tricarboxylic acid (TCA) cycle, glycerol 3-phosphate (GP) shuttle, and β-oxidation. Unfilled boxes represent intermediate metabolites not added in the media. Electrons from the oxidation of these reduced substrates enter the mitochondrial electron transport chain through different isopotential groups of redox centers, denoted by the two planes, each operating at about the same redox potential (E_h): NADH/NAD⁺ at E_h ∼−280 mV and QH₂/Q at E_h ∼+20 mV (59). The flow of electrons through NADH to the Q pool at complex I and from the Q pool to cytochrome c (cyt c) at complex III is indicated by the large green arrows dropping down through the isopotential planes. Enzymes that feed electrons into each isopotential group are represented as ovals, electron transport inhibitors are drawn with red blunted arrows, and CN-POBS, a suppressor of electron leak at site I_Q that does not inhibit electron transport, is drawn with a green blunted arrow. Electrons from NAD-linked substrates enter the NADH/NAD⁺ pool through appropriate NAD-linked dehydrogenases (DH), including those for branched-chain 2-oxoacids (BCOADH), pyruvate (PDH), 2-oxoglutarate (OGDH), and others that are grouped together because there is no evidence that they produce superoxide/H₂O₂. Electrons from NADH flow into complex I (site I_F) and then drop down via site I_Q to QH₂/Q in the next isopotential pool, providing the energy to generate protonmotive force (pmf). Q oxidoreductases, including complex II, mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH), ETF:QOR, and dihydroorotate dehydrogenase (DHODH), can also pass electrons into the Q pool. Electrons flow from QH₂ through complex III to cytochrome c and finally to oxygen (not shown), again pumping protons and generating pmf. The redox state of NADH (outlined in blue) reports the redox state of the first isopotential group. The redox state of cytochrome b₅₆₆ (outlined in blue) reports the redox state of the second isopotential group (24). Red circles indicate sites of superoxide/H₂O₂ production: the flavin/lipoate of the dehydrogenases for branched-chain 2-oxoacids (B_F), pyruvate (P_F), and 2-oxoglutarate (O_F), the complex I flavin (I_F) and Q-binding site (I_Q), the flavin site of complex II (II_F), the quinone site of mitochondrial glycerol 3-phosphate dehydrogenase (G_Q), the flavin site of ETF:QOR (E_F), the quinone site of dihydroorotate dehydrogenase (D_Q), and the outer quinol-binding site of complex III (III_Qo).

The rates of superoxide/H₂O₂ production under native conditions (i.e. in the absence of added inhibitors) are much less than these maximum capacities (24). Therefore, the maximum capacities of the sites are not necessarily related to the actual engagement and rate of each site under native conditions. To establish the native rate from each site, we devised novel methods based on measurements of the redox states of endogenous reporters in isolated mitochondria oxidizing conventional substrates (24, 25). These studies led to two striking conclusions. First, the overall rates of H₂O₂ production differed 5-fold between different conventional substrates; they were much higher with succinate than with glutamate plus malate as substrate. Second, the relative contribution of each site was completely dependent on the substrate being oxidized. With succinate as substrate, site I_Q was dominant; with glutamate plus malate, sites I_F, III_Qo, and O_F all contributed; with palmitoylcarnitine, site II_F was also important; and with glycerol 3-phosphate, five sites contributed significantly, including G_Q (25). Thus, the relative and absolute contribution of each specific site to the production of superoxide/H₂O₂ in isolated mitochondria depends very strongly on which conventional substrate is being oxidized.

In complex systems, such as intact cells, different substrates are metabolized simultaneously. The main groups of substrates oxidized by mitochondria are represented in Fig. 1 and Table 1 by colored boxes. In skeletal muscle, oxidation of ketone bodies, amino acids, tricarboxylic acid cycle intermediates, glycerol 3-phosphate, and fatty acids feeds electrons into multiple sites in the matrix dehydrogenases and electron transport chain. Therefore, in vivo, it is likely that several sites produce superoxide/H₂O₂ simultaneously at different rates.

TABLE 1.

Concentrations of substrates and effectors during rest, mild aerobic exercise, and intense aerobic exercise in rat skeletal muscle cytosol and compositions of the three media mimicking these conditions ex vivo

Data are for all metabolites assumed to be relevant, from the literature for rat skeletal muscle as indicated (values for carnitine and acetylcarnitine include some human data; values for free Ca²⁺ include some mouse data). Where appropriate, calculations used Equation 3 of Ref. 60 and assumed that muscle wet weight is 77% water (60–64) and 81% intracellular (60–62, 64–66) or that 18% of muscle wet weight is protein (66, 67). Published values were corrected for plasma concentrations where appropriate and for the distribution of intracellular metabolites between cytosol and mitochondria, using the following matrix/cytosol ratios from heart and liver (or the assumed values for metabolites in parentheses): glycerol 3-phosphate, glutamine (dihydroxyacetone phosphate, taurine): 0 (68, 69); citrate, isocitrate: 10 (69–71); malate (succinate): 4 (69, 70, 72, 73); 2-oxoglutarate: 7 (69–73); pyruvate (acetoacetate, 3-hydroxybutyrate, arginine, lysine): 2 (70, 73); aspartate (neutral amino acids, acylcarnitines): 1 (69, 70, 72, 74); glutamate: 3.5 (69, 70, 72, 74); carnitine: 0.74 (75); ATP: 0.25 (69); phosphate: 5 (76). Mitochondrial volume was assumed to be 10% of intracellular (77–82). Values were rounded to convenient whole numbers. Total Mg²⁺ and Ca²⁺ concentrations to give the targeted free values were calculated using the software MaxChelator. Targeted free Ca²⁺ concentrations were 0.05 μm (rest), 1 μm (mild aerobic exercise), and 1 μm (intense aerobic exercise) (83–86). Targeted free Mg²⁺ concentrations were 600 μm (rest), 600 μm (mild aerobic exercise), and 1000 μm (intense aerobic exercise) (40, 87–89). Targeted Na⁺ concentrations were 16 mm for all media (65, 89). Media had K⁺ and Cl⁻ adjusted to give an osmolarity of 290 mosm (90–92). Total K⁺ concentrations were 80 mm (rest), 77 mm (mild aerobic exercise), and 74 mm (intense aerobic exercise). The “basic media” had the compositions shown; the “complex substrate mixes” contained all of the substrates in the colored boxes. *, no data found, so values are assumed; **, maximum value to avoid mitochondrial uncoupling in vitro.

The conditions experienced by muscle mitochondria within cells differ substantially between rest and exercise. In particular, ADP supply, free Ca²⁺, cytosolic pH, and the availability of different substrates are very different, and this will have profound effects on the mitochondrial production of superoxide and H₂O₂. During exercise, the levels of both reactive oxygen species and reactive nitrogen species are increased (26). The production of such species is crucial for the beneficial effects of exercise (27–29) and for force development (30). However, excess production is detrimental for skeletal muscle performance (30, 31). Mitochondria are leading candidates for the increased production of these reactive species during exercise (30, 32), although others argue for non-mitochondrial sources (26, 32).

Several different probes can be used in intact cells to report changes in reactive oxygen species (33, 34), but they cannot reliably distinguish the mitochondrial sites active under particular physiological or pathological conditions. This is because pharmacological or genetic manipulation of particular mitochondrial sites invariably alters the redox states of other sites, changing their contributions to overall production of superoxide/H₂O₂ and making interpretation unreliable. Also, these probes invariably compete with the endogenous antioxidant defenses and measure steady state levels rather than rates of radical production. On the other hand, the rate from each site can now be quantified using isolated mitochondria (24, 25). However, given the strong substrate dependence of their superoxide/H₂O₂ production, isolated mitochondria oxidizing conventional substrates are not sufficiently physiologically relevant.

To improve the physiological relevance of the more amenable mitochondrial system, in the present work, we develop an ex vivo system in which isolated muscle mitochondria are incubated acutely in novel complex media. These media contain the measured physiological concentrations of all metabolites and effectors assumed to be relevant in skeletal muscle cytosol at rest and during mild and intense aerobic exercise. Using this system, we quantify the contribution of each mitochondrial site to total H₂O₂ production to gain novel insights into the rates and sites of mitochondrial superoxide/H₂O₂ production in skeletal muscle during rest and exercise.

EXPERIMENTAL PROCEDURES

Animals, Mitochondria, and Reagents

Female Wistar rats were from Charles River Laboratories, age 5–10 weeks, and fed chow ad libitum with free access to water. Mitochondria were isolated from hind limb skeletal muscle at 4 °C in Chappell-Perry buffer (CP1; 100 mm KCl, 50 mm Tris, 2 mm EGTA, pH 7.4, at 4 °C) by standard procedures (35) and kept on ice during the assays (<5 h). Protein was measured by the biuret method. The animal protocol was approved by the Buck Institute Animal Care and Use Committee in accordance with IACUC standards. Reagents were from Sigma except for Amplex UltraRed, from Invitrogen.

Oxygen Consumption and Superoxide/H₂O₂ Production

Skeletal muscle mitochondria (0.3 mg of protein·ml⁻¹) were incubated at 37 °C for 4–5 min in the appropriate “basic medium” (Table 1) mimicking the cytosol of skeletal muscle during rest (basic “rest” medium plus oligomycin), mild aerobic exercise (basic “mild aerobic exercise” medium with no further additions), or intense aerobic exercise (basic “intense aerobic exercise” medium plus glucose and sufficient hexokinase after titration (about 0.08 units·ml⁻¹) to give 90% of the maximum state 3 oxygen consumption rate). ATP (6 mm) was injected into the chamber, and then after 1 min, the appropriate “complex substrate mix” was added (Table 1). Oxygen consumption rates were measured using a Clark-type electrode fitted in a water-jacketed chamber (Rank Brothers, Bottisham, UK). Rates of superoxide/H₂O₂ production were measured collectively as rates of H₂O₂ production as two superoxide molecules are dismutated by endogenous or exogenous superoxide dismutase to yield one H₂O₂. H₂O₂ was detected using 5 units·ml⁻¹ horseradish peroxidase and 50 μm Amplex UltraRed in the presence of 25 units·ml⁻¹ superoxide dismutase (36) in a Varian Cary Eclipse spectrofluorometer (λ_excitation = 560 nm, λ_emission = 590 nm) with constant stirring and calibrated with known amounts of H₂O₂ in the presence of all relevant additions because some of them quenched fluorescence (36).

NAD(P)H and Cytochrome b₅₆₆ Redox State

The reduction state of endogenous NAD(P)H was determined by autofluorescence in mitochondria incubated as described for H₂O₂ production (most of the signal is from bound NADH in the matrix, and NADPH hardly changes in the present experiments, but for transparency we call it “NAD(P)H”) using a Shimadzu RF5301-PC dual wavelength spectrophotometer at λ_excitation = 365 nm, λ_emission = 450 nm (18, 24). NAD(P)H was assumed to be 0% reduced after 5 min without added substrate. 100% reduction was established internally by adding saturating conventional substrate (e.g. 5 mm malate plus 5 mm glutamate) and 4 μm rotenone at the end of each run. Intermediate values of NAD(P)H reduction were measured at 3–4 min after the addition of the appropriate mix of substrates and were determined as percentage of reduced NAD(P)H relative to the 0 and 100% values. The reduction state of endogenous cytochrome b₅₆₆ was measured using 1.5 mg of mitochondrial protein·ml⁻¹ with constant stirring at 37 °C in an Olis DW 2 dual wavelength spectrophotometer at A_{566 nm}–A_{575 nm} (17). This signal reports ∼75% cytochrome b₅₆₆ and ∼25% cytochrome b₅₆₂ (17, 37). Cytochrome b₅₆₆ was assumed to be 0% reduced after 5 min without added substrate. 100% reduction was established in separate cuvettes with 5 mm succinate and 2 μm antimycin A. Intermediate values of cytochrome b₅₆₆ reduction were measured over 45 s, ∼20 s after the addition of the appropriate complex substrate mix, and were determined as percentage of reduced cytochrome b₅₆₆ relative to the 0 and 100% values.

Correction for Matrix Peroxidase Activity

H₂O₂ production rates in Figs. 3 and 12 (but not in other figures) were corrected for losses of H₂O₂ caused by peroxidase activity in the matrix to give a better estimate of actual superoxide/H₂O₂ production rates (38). Rates were mathematically corrected to those that would have been observed after pretreatment with 1-chloro-2,4-dinitrobenzene (CDNB) to deplete glutathione and decrease glutathione peroxidase and peroxiredoxin activity, using an empirical equation,

where ν is the rate of H₂O₂ production in pmol of H₂O₂·min⁻¹·mg protein⁻¹.

FIGURE 3.

Maximum capacities for superoxide/H₂O₂ production of the 10 characterized sites in the mitochondrial electron transport chain and matrix compared with the native ex vivo rates using isolated mitochondria incubated in the absence of inhibitors in media mimicking the cytosol of skeletal muscle during rest, mild aerobic exercise, or intense aerobic exercise. The sites in the NADH/NAD⁺ isopotential group (Fig. 1) are O_F (flavin site of the 2-oxoglutarate dehydrogenase complex), P_F (flavin site of the pyruvate dehydrogenase complex); B_F (flavin site of the branched-chain 2-oxoacid dehydrogenase complex); and I_F (flavin site of complex I). Site I_Q of complex I is between the two isopotential groups. The sites in the QH₂/Q isopotential groups are III_Qo (outer ubiquinone binding site of complex III); II_F (flavin site of complex II); G_Q (quinone site of site mitochondrial glycerol 3-phosphate dehydrogenase); E_F (flavin site of the electron-transferring flavoprotein/ETF:ubiquinone oxidoreductase system), and D_Q (quinone site of dihydroorotate dehydrogenase). The first nine bars are replotted from Ref. 16, and the D_Q bar is replotted from Ref. 23. Ex vivo native rates are from Fig. 2D. All rates were corrected for matrix peroxidases using Equation 1. Values are means ± S.E. (error bars) (n = 3–20).

FIGURE 12.

Contributions of different sites to superoxide and H₂O₂ production by isolated skeletal muscle mitochondria ex vivo in media mimicking rest, mild aerobic exercise, and intense aerobic exercise. Positive values taken from Figs. 8D, 9, and 11 were corrected for the losses of H₂O₂ caused by the activity of mitochondrial matrix peroxidases using Equation 1. Because of their topology, only 50% of the signal assessed for sites III_Qo and G_Q was corrected, slightly diminishing their contributions relative to other sites. Inverted error bars indicate the propagated errors for each site, and conventional error bars indicate the propagated sum of these errors. Values are means ± S.E. (error bars) (n = 3–20).

Due to the non-linearity of the curve, the correction is different at different overall rates. Therefore, the total H₂O₂ production rates were first corrected using Equation 1. The corrected rate from each site (and its S.E.) was then back-calculated based on its relative contribution in a given condition (Table 3). All sites produce superoxide/H₂O₂ exclusively into the mitochondrial matrix, except for III_Qo and G_Q, which generate ∼50% of the superoxide/H₂O₂ to the cytosol (24). For these sites, only 50% of the rate was corrected.

TABLE 3.

Corrections applied for changes in redox state of NAD(P)H and cytochrome b₅₆₆, and rate of superoxide/H₂O₂ production from each site during “rest,” “mild aerobic exercise,” and “intense aerobic exercise”

Δ control values in columns B and C were used to correct the observed changes in H₂O₂ production rate in column A for changes in the redox states of NAD(P)H and cytochrome b₅₆₆, as shown in the last column.

^a 2.5 μm CN-POBS decreased I_Q superoxide production by only 65%; therefore, values were scaled to account for CN-POBS potency.

^b 10 μm CN-POBS reduced I_Q superoxide production by only 75%; therefore, values were corrected for CN-POBS potency.

^c The relative contribution was calculated based on the CDNB corrected rates for each site. The S.E. was scaled according to the internal error for each site. DHAP, dihydroxyacetone phosphate; nd, not determined.

Curve Fitting

Data were fit by exponential functions in Figs. 5C, 6C, and 10 (C and F) to yield the parameter values in Equations 2–5, respectively,

where νH₂O₂ is the rate of H₂O₂ production.

FIGURE 5.

Contribution of site I_F at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, rate of superoxide production from site I_F, defined by the presence of 4 μm rotenone, 1.5 mm aspartate, and 2.5 mm ATP, at different malate concentrations. B, dependence of NAD(P)H reduction level on malate concentration under the same conditions (100% reduction was established by adding 5 mm malate plus 5 mm glutamate). C, calibration curve obtained by combining the y axes from A and B, showing the dependence of the rate of superoxide production from site I_F on the NAD(P)H reduction level. D, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contribution of site I_F assessed from the reduction state of NAD(P)H in Fig. 4 and the calibration curve in C (green; the inverted error bar indicates the propagated error for site I_F). Values are means ± S.E. (error bars) (n = 3–20). p < 0.0001 by Welch's t test.

FIGURE 6.

Contribution of site III_Qo at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, rate of superoxide production from site III_Qo, defined as the rate in the presence of 4 μm rotenone sensitive to 4 μm myxothiazol, at different ratios of succinate/malonate (total concentration 5 mm). Data were corrected for changes in the contribution of site I_F using changes in the NAD(P)H redox state and the calibration curve in Fig. 5C (Table 3) (24). B, dependence of cytochrome b₅₆₆ reduction state on the succinate/malonate ratio under the same conditions (100% reduction was established by adding 5 mm succinate plus 2 μm antimycin A). C, calibration curve obtained by combining the y axes from A and B showing the dependence of the rate of superoxide produced from site III_Qo on the cytochrome b₅₆₆ reduction level. D, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contributions of sites I_F (from Fig. 5D) and III_Qo (assessed from the reduction state of cytochrome b₅₆₆ in Fig. 4 and the calibration curve in C). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates the propagated sum of these errors. Values are means ± S.E. (error bars) (n = 3–20). p < 0.0001 by Welch's t test.

FIGURE 10.

Calibration of the reporters of sites I_F and III_Qo during “intense aerobic exercise.” Mitochondria were incubated in the basic medium mimicking intense aerobic exercise (Table 1). A–C, construction of the calibration curve showing the dependence of the rate of superoxide production from site I_F on NAD(P)H reduction level exactly as in Fig. 5, A–C, but using the basic medium mimicking intense aerobic exercise. D–F, construction of the calibration curve showing the dependence of the rate of superoxide production from site III_Qo on the cytochrome b₅₆₆ reduction level exactly as in Fig. 6, A–C, but using the basic medium mimicking intense aerobic exercise. Values are means ± S.E. (error bars) (n = 3).

Statistics

The calibration curves in Figs. 5C, 6C, and 10 (C and F) were used to calculate the rates of superoxide/H₂O₂ production at sites I_F and III_Qo, both directly when assessing the rates from these sites and indirectly when correcting for the effects on these sites of inhibition at other sites (Table 3). The errors in the calibration curves were taken into account when calculating the S.E. values of the assessed rates. This error was calculated by error propagation as described previously (24).

Because error propagation was used to include the uncertainty from the calibration curve in the assessed rates, the mean ± S.E. values plotted do not represent individual values. Therefore, the statistics were calculated using the averaged values ± S.E. and the number of observations. The significances of differences between the total measured rates and the assessed rates were calculated using Welch's t test. For the regular multiple comparison tests in Figs. 2 and 4, a one-way ANOVA was used, followed by Tukey's post hoc test. When comparing the values with omission of single substrates with the total values, Student's t test was used. p < 0.05 was considered significant.

FIGURE 2.

Oxygen consumption and H₂O₂ generation by isolated skeletal muscle mitochondria incubated in media mimicking the cytosol of skeletal muscle during rest, mild aerobic exercise, or intense aerobic exercise. A, representative traces of oxygen consumption. The dashed box indicates the interval over which the rates were analyzed. Numbers by the traces indicate mean rates in nmol of O·min⁻¹·mg of protein⁻¹. B, rates of oxygen consumption. The maximum (state 3) rate “Max” was measured in the presence of 20 mm glucose and excess (2.5 units·ml⁻¹) hexokinase. C, representative traces of H₂O₂ generation. The dashed box indicates the interval over which the rates were analyzed. Numbers by the traces indicate mean rates in pmol of H₂O₂·min⁻¹·mg of protein⁻¹. D, rates of H₂O₂ generation. In each panel, mitochondria were incubated in media mimicking rest, mild aerobic exercise, or intense aerobic exercise (Table 1), as indicated. Values are means ± S.E. (error bars) (n = 3–20 biological replicates). *, p < 0.05; ***, p < 0.0001, one-way ANOVA with Tukey's post hoc test.

FIGURE 4.

Reduction state of NAD(P)H and cytochrome b₅₆₆. Mitochondria were incubated in media mimicking rest, mild aerobic exercise, or intense aerobic exercise (Table 1), as indicated. Values are means ± S.E. (error bars) (n = 3–20). *, p < 0.05; **, p < 0.01; ***, p < 0.0001, one-way ANOVA with Tukey's post hoc test.

RESULTS

Media Mimicking Skeletal Muscle Cytosol during Rest, Mild Aerobic Exercise, and Intense Aerobic Exercise ex Vivo

In vivo, the substrates for mitochondrial metabolism come primarily from the catabolism of sugars, proteins, and fats. In the cytosol, they are available to the mitochondria as metabolites that can be categorized into five groups according to their origins: ketone bodies, amino acids, tricarboxylic acid cycle intermediates, glycerol 3-phosphate, and acylcarnitines (Table 1). The major metabolic fates of these substrates are indicated by the colored boxes leading to the mitochondrial dehydrogenases in Fig. 1. These metabolites connect to the 10 sites in skeletal muscle mitochondria known to have significant capacity to produce superoxide or H₂O₂. In Fig. 1, these sites are grouped in planes reflecting their operating redox potentials. The top plane represents the NADH/NAD⁺ isopotential group, containing the dehydrogenases that reduce NAD⁺ and the sites in complex I that oxidize NADH. From complex I, the electrons drop down in energy to a more positive redox potential in the ubiquinone pool. The bottom plane represents the QH₂/Q isopotential group, containing the ubiquinone oxidoreductases that reduce ubiquinone and the sites in complex III that oxidize ubiquinol. The electrons are then transferred to cytochrome c and on to the final acceptor, O₂, to generate H₂O (not shown).

Table 1 lists the consensus concentrations in rat skeletal muscle cytosol of all metabolites and effectors thought to be potentially relevant to mitochondrial electron transport and production of superoxide/H₂O₂. The values were taken from the extensive literature on rat skeletal muscle obtained mostly by freeze-clamp followed by enzymatic or chromatographic quantitation, both in vivo and in isolated muscle preparations. Where appropriate, they were corrected using consensus values for extracellular contamination and for the estimated compartmentation between the mitochondrial matrix and the cytosol. Values are listed for resting muscle and for two exercise conditions: submaximal stimulation, representing mild aerobic exercise, and extensive stimulation, representing intense aerobic exercise. We assumed that the exact K⁺ and Cl⁻ concentrations were unimportant and therefore used KCl to bring the osmolarity to the physiological value of 290 mosm.

These literature values enabled us to prepare three different media for mitochondrial incubations ex vivo, containing the relevant metabolites and effectors at the concentrations that would be encountered by mitochondria in vivo during rest and mild and intense aerobic exercise. ATP turnover and hence steady-state ADP concentrations were set based on respiration rates as described below. These media are the first that we know of to be carefully designed to mimic the substrate and effector mix in the cytosol of skeletal muscle cells during “rest,” “mild aerobic exercise,” and “intense aerobic exercise” (Table 1). They enabled us to assess the production of H₂O₂ from isolated mitochondria in an ex vivo system that closely mimicked the relevant aspects of the cytosol of rat muscle cells in vivo.

Setting ATP Demand during “Rest”, “Mild Aerobic Exercise,” and “Intense Aerobic Exercise”

To deplete endogenous substrates, rat skeletal muscle mitochondria were incubated for 4–5 min in the appropriate basic medium lacking all respiratory substrates (Table 1). ATP was added 1 min before respiration was initiated, to minimize the time available for ATP hydrolysis. To initiate respiration, the appropriate complex mix of substrates mimicking rest or mild or intense aerobic exercise (Table 1) was then added (Fig. 2A).

“Rest”

In resting muscle in vivo, the rates of respiration and ATP synthesis are low (39). To mimic rest ex vivo, a low rate of ATP synthesis by mitochondria incubated in the “rest” medium (Table 1) was achieved by including oligomycin in the medium to fully inhibit the mitochondrial ATP synthase. This was not ideal, because it implies no ATP turnover at rest, but it was necessary because the relatively high contaminating ATPase activity present in the mitochondrial preparation caused an intermediate respiration rate. The rate of oxygen consumption at “rest” was less than 10% of the maximum rate (Fig. 2, A and B, red).

“Mild Aerobic Exercise”

During exercise in vivo, respiratory rates increase due to increased ATP demand and altered substrate supply and concentrations of effectors (pH, Ca²⁺). Mild exercise induces ≤45% of whole body maximal O₂ consumption rate, VO_2max (39)_. To mimic mild aerobic exercise ex vivo, mitochondria were incubated in the basic “mild aerobic exercise” medium in the absence of oligomycin, followed by ATP and then the appropriate complex mix of relevant substrates (Table 1). The initial fast rate of respiration required to rephosphorylate ADP formed from the added ATP (Fig. 2A) was ignored. After this fast phase was complete and ATP/ADP settled to a steady state value, ATP demand and respiration rate ran at an intermediate rate limited by the supply of ADP from contaminating extramitochondrial ATPases. In this phase, the respiratory rate was 22% of the maximum rate (Fig. 2, A and B, green).

“Intense Aerobic Exercise”

Metabolic demand increases with exercise intensity. During intense exercise, respiration is ≥65% of VO_2max (39). ADP level increases significantly but is still an order of magnitude lower than the ATP level (40). Cytosolic pH drops to 6.7 due to increased CO₂ and lactate production. To mimic intense aerobic exercise ex vivo, mitochondria were incubated in the basic “intense aerobic exercise” medium, followed by ATP and then the appropriate complex mix of relevant substrates. To achieve high respiratory rates but keep ADP levels relatively low, in separate experiments, the O₂ consumption rate was titrated to 90% of the maximum rate using hexokinase in the presence of glucose to set the rate of extramitochondrial ATP hydrolysis high but still submaximal in each mitochondrial preparation, and this amount of hexokinase (about 0.08 units·ml⁻¹) was included in the “intense aerobic exercise” medium (Fig. 2, A and B, blue). Maximum phosphorylating respiration was set by adding excess hexokinase (Fig. 2B, white).

Mitochondrial H₂O₂ Production during “Rest,” “Mild Aerobic Exercise,” and “Intense Aerobic Exercise”

The rate of generation of superoxide and H₂O₂ was measured under the three conditions described above as the rate of extramitochondrial H₂O₂ production in the presence of exogenous superoxide dismutase to convert any superoxide to H₂O₂ for assay. Fig. 2C shows that rates were linear for 2–3 min after the addition of the complex substrate mixes and then decreased. Some metabolites were present at very low concentrations (Table 1) and were likely to be consumed quickly, so ideally we would measure initial rates, but because the system was not at steady-state over the first minute in the “exercise” medium (Fig. 2A), we calculated the rates as the pseudolinear rate between 1 and 3 min (dotted boxes in Fig. 2, A and C). Fig. 2D shows that the highest rate of H₂O₂ production was observed when mitochondria were incubated in the medium mimicking the cytosol of skeletal muscle at rest (336 pmol of H₂O₂·min⁻¹·mg of protein⁻¹). In the media mimicking mild aerobic exercise and intense aerobic exercise, the rates were significantly lower.

Unphysiological concentrations of conventional substrates and the presence of appropriate electron transport inhibitors favor high rates of mitochondrial superoxide/H₂O₂ production. These rates can be up to 2% of the total respiration rate of uninhibited resting mitochondria (2, 41). Under more realistic non-inhibited (native) conditions with conventional substrates, the percentage of electron leak to H₂O₂ is only ∼0.15% (42, 43). Table 2 shows the % electron leak when mitochondria were incubated under conditions mimicking rest, mild aerobic exercise, and intense aerobic exercise. At “rest,” the electron leak was 0.35%. During “exercise,” it was 10–35-fold lower: only 0.03% for “mild aerobic exercise” and 0.01% for “intense aerobic exercise.”

TABLE 2.

Electron leak during “rest,” “mild aerobic exercise,” and “intense aerobic exercise”

Rates were calculated per nmol e⁻ by multiplying the rates of H₂O₂ production in Fig. 2D in pmol of H₂O₂·min⁻¹·mg of protein⁻¹ by 0.002 and rates of oxygen consumption in Fig. 2B in nmol of O·min⁻¹·mg of protein⁻¹ by 2. Values are means ± S.E. (n = 3 for oxygen consumption rates; n = 3–20 for H₂O₂ production rates).

	“Rest”	“Mild aerobic exercise”	“Intense aerobic exercise”
Rate of H2O2 production (nmol e−·min−1·mg of protein−1)	0.68 ± 0.04	0.16 ± 0.01	0.10 ± 0.01
Rate of oxygen consumption (nmol e−·min−1·mg of protein−1)	196 ± 40	492 ± 88	2032 ± 73
Percentage electron leak (100·H2O2 production rate/respiration rate)	0.35%	0.03%	0.01%

The ex vivo data in Fig. 2D and Table 2 suggest that skeletal muscle mitochondria in vivo are unlikely to contribute to the overall increase in reactive oxygen species production observed during exercise, supporting the conclusions of others (26, 32). Instead, the mitochondrial production rate may decrease during exercise, because the redox centers that donate electrons to O₂ become more oxidized during exercise (see below).

Sites of Superoxide/H₂O₂ Production

Ten mitochondrial sites associated with the tricarboxylic acid cycle and electron transport chain are known to produce superoxide or H₂O₂ (Fig. 1). Their maximum capacities are shown in Fig. 3, on the same scale as the ex vivo rates from Fig. 2D after all of the values have been corrected for consumption of intramitochondrial H₂O₂ by matrix peroxidases. Clearly, the maximum capacities of several sites are much higher than the ex vivo rates, making it impossible to predict a priori which sites contribute most to the total signal ex vivo or in vivo. The rates of H₂O₂ production measured under the different conditions in Fig. 2D are the sums of the rates from different sites. Our goal here was to determine the contribution of each site during “rest,” “mild aerobic exercise,” and “intense aerobic exercise” in the tractable ex vivo system because no adequate methods exist to address this question in vivo or in intact cells. We assume that the sites contributing ex vivo in media containing the physiological cytosolic concentrations of all substrates and effectors thought to be relevant will approximate the sites contributing in vivo.

Conventionally, complexes I and III are thought to be the dominant sources of mitochondrial superoxide/H₂O₂ in isolated mitochondria and cells (10, 13). Complex I produces superoxide at two distinct sites: site I_Q, a high capacity site, most obviously active during reverse transfer of electrons from ubiquinol to NADH (18, 19, 44, 45); and site I_F (18), now known to have much lower capacity (16). Complex III can produce superoxide at high rates from site III_Qo (2, 17). The rate of superoxide production from site I_F depends on the redox state of the complex I flavin, which in turn has a unique relationship to the redox state of mitochondrial NAD(P)H. Similarly, the rate of superoxide production from site III_Qo is related to the redox state of cytochrome b₅₆₆ (17). The redox states of NAD(P)H and cytochrome b₅₆₆ were therefore used as endogenous reporters of the rates of superoxide production at sites I_F and III_Qo, respectively (24, 25).

Fig. 4 and Table 3 show the reduction levels of NAD(P)H and cytochrome b₅₆₆ when mitochondria were incubated in the three media. During “rest,” NAD(P)H was nearly 100% reduced, and cytochrome b₅₆₆ was 30% reduced. During “exercise,” both were significantly more oxidized. To assess the rate of superoxide production by sites I_F and III_Qo, calibration curves were built to establish the relationships between superoxide production from these sites and the reduction levels of the endogenous reporters.

Flavin of Complex I, Site I_F, during “Rest”

NAD(P)H redox state was calibrated as a reporter of the rate of superoxide production at site I_F, as described previously (24). Fig. 5A shows the observed rate of H₂O₂ production from site I_F as a function of the concentration of malate in the presence of rotenone (with added ATP and aspartate to minimize the contribution of site O_F (16)). Fig. 5B shows the NAD(P)H reduction state under the same conditions. Fig. 5C replots the data from Fig. 5, A and B, to show the dependence of the measured rate of H₂O₂ production arising from superoxide production at site I_F on the NAD(P)H reduction state. As shown in Fig. 4, NAD(P)H during “rest” was almost 100% reduced. From Fig. 5C, the contribution of site I_F during “rest” was assessed to be 65 ± 12 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 8). The contributions of site I_F and other relevant values developed below are summarized in Table 3.

Site I_F accounted for 20 ± 3% of the total rate of H₂O₂ production measured during “rest” (Fig. 5D). Therefore, other sites also produced superoxide/H₂O₂ at rest.

Outer Ubiquinone Binding Site of Complex III, Site III_Qo, during “Rest”

Cytochrome b₅₆₆ redox state was calibrated as a reporter of the rate of superoxide production at site III_Qo as described previously (24). Fig. 6A shows the observed myxothiazol-sensitive rate of H₂O₂ production from site III_Qo as a function of the ratio of added succinate and malonate in the presence of rotenone. At each point, the rate of H₂O₂ production from site I_F was calculated from the observed NAD(P)H reduction level using Fig. 5C and subtracted from the total observed rate to give the rate specifically from site III_Qo (Table 3). Fig. 6B shows the cytochrome b₅₆₆ redox state under the same conditions. Fig. 6C replots the data from Fig. 6, A and B, to show the dependence of the rate of H₂O₂ production arising from superoxide production at site III_Qo on the cytochrome b₅₆₆ reduction state. Fig. 4 and Table 3 show that cytochrome b₅₆₆ was 30% reduced during “rest.” Using the calibration curve in Fig. 6C, the contribution of site III_Qo was 55 ± 7 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 12) (Fig. 6D).

Site III_Qo accounted for 15 ± 2% of the total rate of H₂O₂ production measured during “rest” (Fig. 6D and Table 3). After accounting for the contributions of sites I_F and III_Qo to the total rate of H₂O₂ production, there was still a significant difference between the total rates measured and assessed (Fig. 6D). Therefore, other sites also produced superoxide/H₂O₂ at rest.

Ubiquinone Binding Site of Complex I, Site I_Q, during “Rest”

Site I_Q has a high capacity for superoxide production during reverse electron transport when succinate is oxidized (19, 43) (Fig. 3) and during forward electron flow from NAD-linked substrates under particular conditions (44). However, the importance of this site in vivo and under semiphysiological conditions (25, 46, 47) is unknown. The contribution of site I_Q during “rest” ex vivo was assessed by adding rotenone to inhibit electron transport at this site, followed by correction for consequent changes in the rate of superoxide production from sites I_F and III_Qo. Rotenone was added 1 min prior to the substrate mix and after correction significantly decreased the rate of superoxide/H₂O₂ production at “rest” (Fig. 7A). The rate attributed to site I_Q was 79 ± 23 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 6).

FIGURE 7.

Contributions of sites I_Q and II_F at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, total rate of H₂O₂ production and the rates in the presence of a I_Q electron transport inhibitor (4 μm rotenone) or a suppressor of I_Q superoxide production (2.5 μm CN-POBS) after correction for changes in the rates of sites I_F and III_Qo assessed by changes in NAD(P)H and cytochrome b₅₆₆ redox state (Table 3). The yellow hatched area represents the contribution of site I_Q. B, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contributions of sites I_F (from Fig. 5D), III_Qo (from Fig. 6D), and I_Q (from A). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates the propagated sum of these errors. C, total rate of H₂O₂ production and the rate in the presence of 4 μm malonate to inhibit the flavin site of complex II, after correction for changes in the rates of sites I_F, I_Q, and III_Qo (Table 3). The red hatched area represents the contribution of site II_F. D, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contributions of sites I_F (from Fig. 5D), III_Qo (from Fig. 6D), I_Q (from A), and II_F (from C). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates the propagated sum of these errors. Values are means ± S.E. (error bars) (n = 3–20). **, p < 0.005; ***, p < 0.0001 by one-way ANOVA followed by Tukey's post hoc test. *, p < 0.05 by Student's t test. Welch's t test was used to determine p = 0.003 and p = 0.39.

CN-POBS at 2.5 μm selectively blocks 65% of superoxide production from site I_Q with no effects on oxidative phosphorylation, although at higher concentrations, it is less selective (48). CN-POBS was added 1 min prior to the substrate mix and significantly decreased the rate of superoxide/H₂O₂ production at “rest.” The rate attributed to site I_Q after correction for the 65% effect of CN-POBS was 66 ± 29 pmol H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 6) (Fig. 7A), indistinguishable from the estimate made using rotenone.

From the data obtained with CN-POBS and corroborated using rotenone, site I_Q accounted for 23 ± 11% of the total rate of H₂O₂ production measured during “rest” (Fig. 7B and Table 3). This is the first evidence in any system that site I_Q may be an important contributor to mitochondrial superoxide/H₂O₂ production ex vivo and in vivo. The sum of the rates of superoxide/H₂O₂ production from sites I_F, III_Qo, and I_Q was significantly different from the total rate of H₂O₂ production measured during “rest” (Fig. 7B), suggesting that yet other sites also produced superoxide/H₂O₂ at rest.

Flavin of Complex II, Site II_F, during “Rest”

The flavin site of complex II, site II_F, has a high capacity for superoxide/H₂O₂ production (20) (Fig. 3). When downstream electron transport is prevented, there is a peak of superoxide/H₂O₂ production from this site at succinate concentrations close to the physiological range (20), indicating that site II_F might also contribute to H₂O₂ generation at rest. The contribution of site II_F during “rest” ex vivo was assessed by adding malonate to inhibit electron transport at this site, followed by correction for consequent changes in the rate of superoxide production from sites I_F, III_Qo, and I_Q. The addition of malonate significantly decreased the rates of H₂O₂ production at “rest” (Table 3). The change in rate attributed to site II_F after correction was 80 ± 35 pmol H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 7) (Fig. 7C and Table 3). Site II_F accounted for 24 ± 10% of the total rate of H₂O₂ production measured during “rest” (Fig. 7D and Table 3). This is the first evidence in any system that site II_F may be an important contributor to mitochondrial superoxide/H₂O₂ production ex vivo and in vivo.

The sum of the rates of superoxide/H₂O₂ production from sites I_F, III_Qo, I_Q, and II_F was not significantly different from the total rate of H₂O₂ production measured during “rest” (Fig. 7D). However, we still assessed the potential contributions of the remaining sites.

Electron-transferring Flavoprotein (ETF) and ETF:Q Oxidoreductase (ETF:QOR), Site E_F, during “Rest”

β-Oxidation of fatty acids, primarily palmitoylcarnitine, is an important source of ATP for resting skeletal muscle. During oxidation of palmitoylcarnitine, electrons are transferred partly to NAD⁺ and partly to ETF and then through ETF:QOR to the Q-pool. Superoxide/H₂O₂ production in the ETF/ETF:QOR system (site E_F) probably occurs at the flavin site of ETF (22). Site E_F produces superoxide/H₂O₂ at a maximum rate of 210 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (22). However, during oxidation of palmitoylcarnitine plus carnitine as the sole added substrate under native conditions without inhibitors, the observed H₂O₂ production was mainly from sites I_F, III_Qo, and II_F, leaving a small (but not statistically significant) shortfall that might have been from other sites, such as ETF (22). The contribution of site E_F during “rest” ex vivo was assessed by omitting palmitoylcarnitine from the substrate mix, followed by correction for consequent changes in the rate of superoxide production from sites I_F and III_Qo. Palmitoylcarnitine omission significantly decreased the rate of H₂O₂ production (Fig. 8A). There was no consequent change in the rates from sites I_F and III_Qo (Table 3). The possibility that palmitoylcarnitine withdrawal could alter the rate of superoxide/H₂O₂ production from site II_F was tested by adding malonate in a mix without palmitoylcarnitine, which decreased H₂O₂ production to the same extent as in the presence of palmitoylcarnitine, showing that palmitoylcarnitine omission did not affect the rate from site II_F (data not shown). The rate attributed to site E_F was 46 ± 21 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 8) (Fig. 8A and Table 3). Site E_F accounted for 13 ± 6% of the total rate of H₂O₂ production measured during “rest” (Fig. 8B and Table 3).

FIGURE 8.

Contributions of sites E_F, P_F, O_F, and G_Q at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, total rate of H₂O₂ production and the rate in the absence of palmitoylcarnitine after correction for changes in the rates of sites I_F and III_Qo (Table 3). The olive hatched area represents the contribution of site E_F. B, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contributions of sites I_F (from Fig. 5D), III_Qo (from Fig. 6D), I_Q (from Fig. 7A), II_F (from Fig. 7C), and E_F (from A). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates the propagated sum of these errors. C, total rate of H₂O₂ production and the rates in the absence of pyruvate, 2-oxoglutarate, or glycerol 3-phosphate plus dihydroxyacetone phosphate (DHAP) to assess the contributions of sites P_F, O_F, and G_Q. D, total rate of H₂O₂ production at “rest” (from Fig. 2D) and the contributions of sites I_F (from Fig. 5D); III_Qo (from Fig. 6D); I_Q (from Fig. 7A); II_F (from Fig. 7C); E_F (from A); and G_Q, P_F, and O_F (from C). Inverted error bars indicate the propagated errors for each site. Because some of the assessed contributions were negative, the sum of the contributions by each site was also plotted (error bar indicates the propagated sum of the errors for each assessed site) and used for the statistical test. Values are means ± S.E. (error bars) (n = 3–20). *, p < 0.05 by Student's t test. NS, not significantly different using one-way ANOVA followed by Tukey's post hoc test. Welch's t test was used to determine p = 0.42 and p = 0.15.

Other Sites, P_F, O_F, G_Q, B_F, and D_Q, during “Rest”

The pyruvate dehydrogenase complex, site P_F, and the 2-oxoglutarate dehydrogenase complex, site O_F, have the greatest capacities for superoxide/H₂O₂ production in the NADH/NAD⁺ isopotential group (16) (Figs. 1 and 3). Omission of pyruvate from the substrate mixture slightly slowed the rate of H₂O₂ production during “rest,” although this effect was not statistically significant (p = 0.5). Fig. 8C shows that after correcting for consequent changes at sites I_F, III_Qo, and I_Q (Table 3), site P_F was not significantly active at rest. Arithmetically, omission of pyruvate gave small negative rates for site P_F after correction (Fig. 8D and Table 3), presumably reflecting noise in the assays and small errors in the assumptions.

Similarly, omission of 2-oxoglutarate or the glycerol 3-phosphate/dihydroxyacetone phosphate couple from the substrate mixture resulted in non-significant small increases in the total rates of H₂O₂ production after correction for consequent changes in other sites (Fig. 8C and Table 3). Therefore, these sites were also unlikely to contribute to the measured rates of H₂O₂ production. Again, these assessments gave small negative rates for sites O_F and G_Q after correction(Fig. 8D and Table 3).

Site D_Q in dihydroorotate dehydrogenase (23) was considered to make zero contribution to total H₂O₂ production in our analysis because this site has low maximum capacity, the enzyme has low activity in skeletal muscle (49), and its substrate, dihydroorotate, is present at very low concentrations in rat skeletal muscle and was therefore not included in the substrate mix (Table 1). Similarly, site B_F of the branched-chain 2-oxoacid dehydrogenase (16) was considered to make zero contribution because (i) branched-chain 2-oxoacids are present at very low concentrations in rat skeletal muscle mitochondria at rest and were therefore not included in the substrate mix (Table 1) and (ii) the addition of the transaminase inhibitor aminooxyacetate at 1 mm did not alter the measured rate of H₂O₂ production (data not shown), indicating that transamination of the added branched-chain amino acids to generate branched-chain 2-oxoacids and produce superoxide/H₂O₂ at site B_F was not a major pathway.

Fig. 8D shows the final sum of the assessed rates of superoxide/H₂O₂ production from all of the sites, plotted both as a stack of positive and notionally negative contributions and as the sum of these values. This final value was indistinguishable from the total measured rate of H₂O₂ production (Fig. 8D and Table 3), so the observed rate of H₂O₂ production was entirely accounted for, within experimental error, by the sum of the assessed rates from each site.

In summary, when rat skeletal muscle mitochondria were incubated in a complex medium mimicking the cytosol of rat skeletal muscle at rest, containing physiological concentrations of all substrates and effectors thought to be relevant, the total rate of H₂O₂ production was 337 ± 21 pmol of H₂O₂·min⁻¹·mg of protein⁻¹ (mean ± S.E.; n = 11). Ranked by magnitude, the sites that contributed to this H₂O₂ production were sites II_F (24 ± 10%), I_Q (23 ± 11%), I_F (20 ± 3%), III_Qo (15 ± 2%), and E_F (13 ± 6%).

“Mild Aerobic Exercise”

During mild aerobic exercise, substrate concentrations are different, and the free Ca²⁺ concentration is 20-fold higher compared with rest (Table 1). When mitochondria were incubated in the medium mimicking mild aerobic exercise, ATP turnover was driven by extramitochondrial ATPases, respiration ran at 22% of the maximum rate (Fig. 2, A and B), and NAD(P)H and cytochrome b₅₆₆ reduction levels were significantly lower than at “rest” (Fig. 4 and Table 3). Fig. 2, C and D, shows that the measured rate of H₂O₂ production during “mild aerobic exercise” was only a quarter of the rate at “rest.”

The contribution of each site to superoxide/H₂O₂ production during “mild aerobic exercise” was assessed exactly as described above for “rest” (Table 3). The contributions of sites I_F and III_Qo were assessed using the calibration curves in Figs. 5C and 6C. Fig. 9 shows the final sum of the assessed rates of superoxide/H₂O₂ production from all sites, plotted both as a stack of positive and notionally negative contributions and as the sum of these values. This final value was indistinguishable from the total measured rate of H₂O₂ production (Fig. 9 and Table 3), so the observed rate of H₂O₂ production was entirely accounted for, within experimental error, by the sum of the assessed rates from each site. Compared with “rest,” the absolute contribution of each site decreased. The decrease was most marked for the sites in or connected to the QH₂/Q isopotential group (Fig. 1), reflecting their sensitivity to the reduction level of the Q pool, which dropped by 50% (Fig. 4). Site I_Q is also very sensitive to the protonmotive force (19), which will have decreased when ATP turnover increased. The absolute contribution of the only site in the NADH/NAD⁺ isopotential group that made a large contribution to H₂O₂ production at rest, site I_F, decreased much less, because the reduction level of NAD(P)H dropped by only 20% (Fig. 4). As a result, the relative contribution of site I_F to the rate of superoxide/H₂O₂ production increased significantly from 20% during “rest” to 44% during “mild aerobic exercise” (Table 3).

FIGURE 9.

Contributions of different sites during “mild aerobic exercise.” Mitochondria were incubated in medium mimicking mild aerobic exercise (Table 1). The total rate of H₂O₂ production (from Fig. 2D) and the contributions of sites I_F, III_Qo, I_Q, E_F, G_Q, P_F, II_F, and O_F were assessed in the same way as for “rest” in Fig. 8 using the calibration curves in Figs. 5C and 6C. Inverted error bars indicate the propagated errors for each site. Values are means ± S.E. (error bars) (n = 3–9). Because some of the assessed contributions were negative, the sum of the contributions by each site was also plotted (error bar indicates the propagated sum of the errors for each assessed site) and used for the statistical test. Welch's t test was used to determine p = 0.95.

In summary, when rat skeletal muscle mitochondria were incubated in a complex medium mimicking the cytosol of rat skeletal muscle during mild aerobic exercise, containing physiological concentrations of all substrates and effectors thought to be relevant, the total rate of H₂O₂ production decreased to about 25% of the rate in the medium mimicking rest. The sites that contributed to this H₂O₂ production were, ranked by magnitude, sites I_F (44 ± 4%), I_Q (18 ± 15%), III_Qo (15 ± 4%), and maybe E_F (12 ± 15%) and G_Q (5 ± 7%).

“Intense Aerobic Exercise”

During intense aerobic exercise, free Ca²⁺ concentration is high; ATP concentration is maintained almost constant (∼6 mm); ADP concentration, which is more than 1 order of magnitude lower than ATP concentration, can increase 5-fold; and creatine phosphate hydrolysis generates an increased concentration of P_i (40). CO₂ and lactate production increase, lowering intracellular pH (40, 50, 51), and substrate and effector concentrations also change (Table 1). When mitochondria were incubated in the medium mimicking intense aerobic exercise, ATP turnover was driven by sufficient extramitochondrial hexokinase to cause respiration to run at 90% of the maximum rate (Fig. 2, A and B), and NAD(P)H and cytochrome b₅₆₆ reduction levels were significantly lower than at “rest” (Fig. 4 and Table 3). NAD(P)H reduction level was also significantly lower than during “mild aerobic exercise” (Fig. 4 and Table 3). Fig. 2, C and D, shows that the measured rate of H₂O₂ production during “intense aerobic exercise” was only one-sixth of the rate at “rest,” but there was no significant difference in rates between “mild aerobic exercise” and “intense aerobic exercise.”

The contribution of each site to superoxide/H₂O₂ production during “intense aerobic exercise” was assessed as described above for “rest” and “mild aerobic exercise” (Table 3). The contributions of sites I_F and III_Qo were assessed using the new calibration curves in Fig. 10, C and F, because we found that the calibrations were different in this medium compared with the other two media (Figs. 5C and 6C), probably because of the lower pH.

Fig. 10, A and B, show the dependences of the observed rate of H₂O₂ production from site I_F and the NAD(P)H redox state on the concentration of malate in the medium mimicking intense aerobic exercise. Fig. 10C replots the data from Fig. 10, A and B, to show the dependence of the measured rate of H₂O₂ production arising from superoxide production at site I_F on NAD(P)H reduction state in this medium. Similarly, Fig. 10D shows the observed rate of H₂O₂ production from site III_Qo as a function of the ratio of added succinate and malonate in the presence of rotenone and myxothiazol. At each point, the rate of H₂O₂ production from site I_F was calculated from the observed NAD(P)H reduction level using Fig. 10C and subtracted from the total observed rate to give the rate specifically from site III_Qo. Fig. 10E shows the cytochrome b₅₆₆ redox state under the same conditions. Fig. 10F replots the data from Fig. 10, D and E, to show the dependence of the rate of H₂O₂ production arising from superoxide production at site III_Qo on the cytochrome b₅₆₆ reduction state in this medium.

Fig. 11 shows the final sum of the assessed rates of superoxide/H₂O₂ production from all sites, plotted both as a stack of positive and notionally negative contributions and as the sum of these values. This final value was indistinguishable from the total measured rate of H₂O₂ production (p = 0.46) (Fig. 11 and Table 3), so the observed rate of H₂O₂ production was entirely accounted for, within experimental error, by the sum of the assessed rates from each site. However, because of the low rate of H₂O₂ production during “intense aerobic exercise,” the experimental errors were relatively high, particularly for the sites making smaller contributions (Table 3). Under this condition, site I_F was the major contributor (99 ± 26%) and possibly the only one (Fig. 11). However, we could not discard the possibility that other sites were also active. Note that the total measured rates of H₂O₂ production were indistinguishable between “mild aerobic exercise” and “intense aerobic exercise” (Fig. 2D), but the sites contributing under each condition appeared to differ (Table 3).

FIGURE 11.

Contributions of different sites during “intense aerobic exercise.” Mitochondria were incubated in medium mimicking intense aerobic exercise (Table 1). The total rate of H₂O₂ production (from Fig. 2D) and the contributions of sites I_F, III_Qo, II_F, E_F, O_F, G_Q, I_Q, and P_F were assessed in the same way as for “rest” in Fig. 8 using the calibration curves in Fig. 10, C and F. Inverted error bars indicate the propagated errors for each site. Values are means ± S.E. (error bars) (n = 3). Because some of the assessed contributions were negative, the sum of the contributions by each site was also plotted (error bar indicates the propagated sum of the errors for each assessed site) and used for the statistical test. Welch's t test was used to determine p = 0.46.

In summary, when rat skeletal muscle mitochondria were incubated in a complex medium mimicking the cytosol of rat skeletal muscle during intense aerobic exercise, containing physiological concentrations of all substrates and effectors thought to be relevant, the total rate of H₂O₂ production decreased to about 15% of the rate in the medium mimicking rest. The main site that contributed to this H₂O₂ production was site I_F (99 ± 26% of the total measured rate and 42 ± 10% of the sum of assessed rates).

Rates of H₂O₂ Production after Correction for Losses of H₂O₂ in the Mitochondrial Matrix

Peroxidases in the mitochondrial matrix degrade some H₂O₂ before it can escape and be registered by the extramitochondrial horseradish peroxidase/Amplex UltraRed assay. The losses can be greatly decreased by depleting mitochondrial glutathione. Previous work has established the relationship between observed rates of H₂O₂ production and total rates after correcting for H₂O₂ losses (24, 38), so we corrected the data presented above using Equation 1 to give a more realistic estimate of the true total rates and the rates from each site. Most of the sites produce superoxide/H₂O₂ exclusively to the matrix, so their relative contributions will not change substantially after correction. Importantly, however, ∼50% of the superoxide/H₂O₂ from sites III_Qo and G_Q is produced toward the cytosol and avoids matrix peroxidase scavenging (17, 21). Therefore, the contribution of these sites becomes relatively smaller after this correction. Fig. 12 shows the corrected data and represents our best estimate of the total rate of mitochondrial superoxide/H₂O₂ production ex vivo and the contribution of each site during rest and mild and intense aerobic exercise. At “rest,” more than 50% of the total rate of superoxide/H₂O₂ production was shared between site I_Q and site II_F, previously not considered to be physiologically relevant. During “exercise,” superoxide/H₂O₂ production decreased substantially, and the low capacity site I_F accounted for half or more of the superoxide produced.

DISCUSSION

Mitochondria can produce superoxide or H₂O₂ from at least 10 different sites (16, 23). Which sites are the major producers in vivo is unknown. Currently, there are no methods available that can be used in intact cells or tissues or in vivo to identify unambiguously which sites are active or to measure the rates of production from each site. However, we recently developed methods to identify and quantify the rates from these sites in isolated mitochondria (24, 25). From these studies, we concluded that the overall rates and relative contributions of each site are strongly dependent on the substrate being oxidized. As a consequence, it is unreasonable to extrapolate from isolated mitochondria to tissues in vivo when the results in vitro are obtained using single conventional substrates at unphysiological concentrations.

The work described here represents a first step toward characterizing the mitochondrial sites of superoxide and H₂O₂ production in skeletal muscle in vivo. We analyzed the sites ex vivo in media designed to mimic the in vivo concentrations of all substrates and effectors we thought likely to be important in determining mitochondrial superoxide and H₂O₂ production in muscle at rest and during exercise. With this approach, extrapolations from ex vivo to in vivo are much more reasonable. Of course, the approach can still be criticized for assuming that other effects, such as fragmentation of mitochondria during isolation, do not substantially affect mitochondrial function and that other effectors that are not included in the media, such as components of unknown signaling pathways from the cytosol that do not work by altering the concentrations of the explicit metabolites, can be ignored. We used the approach specifically for rat skeletal muscle mitochondria, but it could be used with mitochondria from any cell type or any tissue from control or disease models as long as all metabolites and effectors are present at their physiological or pathological concentrations.

In the current paper, we identified and quantified for the first time all mitochondrial sites that are likely to contribute to mitochondrial superoxide and H₂O₂ production in skeletal muscle at rest and under mild aerobic and intense aerobic exercise. We found that the maximal capacities of the sites did not correlate with their native rates of superoxide and H₂O₂ production ex vivo. At “rest,” half of the total rate of H₂O₂ production was from sites I_Q and II_F, two sites whose relevance in vivo was not previously appreciated. Also, the low capacity site I_F produced H₂O₂ as fast as the highest capacity site, III_Qo.

In isolated mitochondria, site I_Q can produce superoxide/H₂O₂ at high rates during both the forward and reverse reactions (43, 44). The contribution of site I_Q in cells and in vivo is unknown. In some cells, the addition of rotenone decreases cellular production of reactive oxygen species (29, 52–54), which is consistent with a substantial contribution of site I_Q. However, if the cells were oxidizing predominantly NAD-linked substrates, the addition of rotenone would block reduction of ubiquinone and decrease superoxide/H₂O₂ production from sites in the QH₂/Q isopotential group, particularly sites III_Qo and II_F. If the cells were running reverse electron transport (29), the addition of rotenone would block reduction of NAD⁺ and decrease superoxide/H₂O₂ production from sites in the NADH/NAD⁺ isopotential group, particularly sites I_F, P_F, and O_F. These alternative explanations greatly weaken any conclusion from rotenone inhibition experiments that site I_Q is active in cells. Conversely, in many other cells, rotenone increases cellular production of reactive oxygen species (55–57), which appears to be inconsistent with a substantial role for site I_Q. However, if the cells were oxidizing primarily NAD-linked substrates, the addition of rotenone would cause reduction of the NADH/NAD⁺ isopotential group and increase superoxide/H₂O₂ production from sites I_F, P_F, O_F, etc., masking any decrease in the rate from site I_Q. In general, whether rotenone addition causes an increase or decrease in observed production of reactive oxygen species may depend upon the balance of rates and capacities of sites in the NADH/NAD⁺ and QH₂/Q isopotential groups and not only on the activity of site I_Q itself. These considerations highlight the problems of valid interpretation when using electron transport chain inhibitors or genetic manipulations to define mitochondrial sites of superoxide/H₂O₂ production in cells if changes in the redox states of all other relevant sites are not properly accounted for as in Table 3.

Here, we used two different approaches to assess the contribution of site I_Q. First, we used rotenone to inhibit the Q-site of complex I and corrected the observed overall changes in H₂O₂ production for the changes caused by alterations in the redox states of the NADH/NAD⁺ isopotential pool (site I_F) and QH₂/Q isopotential pool (site III_Qo), exposing the contribution of site I_Q. Second, we used CN-POBS, which specifically suppresses superoxide production from site I_Q without inhibiting electron transport (48) or altering the redox state of the NADH/NAD⁺ and QH₂/Q isopotential pools (Table 3). Both approaches gave the same result (Fig. 7A). This is the first evidence that site I_Q is active under a semiphysiological condition and therefore may also be significantly active in vivo.

The physiological contribution of superoxide/H₂O₂ production from site II_F has also been unappreciated, mostly because this site was not recognized as an important potential source until recently. However, the capacity of site II_F to produce superoxide/H₂O₂ in muscle mitochondria is great and is second only to site III_Qo (Fig. 3) (20). When succinate is used as a single substrate by isolated skeletal muscle mitochondria, the rate of production of superoxide/H₂O₂ by site II_F is highly dependent on the concentration of succinate, rising to a peak at about 400 μm (20), a concentration that approximates the physiological cytosolic level in skeletal muscle (Table 1), and then falling away at higher concentrations as succinate becomes inhibitory for this reaction. Our data indicate that at “rest,” ∼25% of the total H₂O₂ produced by muscle mitochondria originates from site II_F, indicating that this site is an important source of superoxide/H₂O₂ in vivo. Although the raw data are sparse, the concentration of succinate in skeletal muscle may rise from about 200 μm at rest to about 300 μm during exercise (Table 1). However, this increase ex vivo was not associated with a corresponding increase in the contribution of site II_F in “aerobic exercise” (Fig. 12), presumably because the activatory effect of higher succinate concentration was more than compensated for by the inhibitory effect of oxidation of the QH₂/Q pool during “exercise” (Fig. 4). Nonetheless, under conditions in which succinate concentration rises, such as hypoxia-reperfusion (58), but the QH₂/Q pool may not become oxidized, the contribution of site II_F in cardiac muscle or brain may be even greater.

The mechanisms underlying the decrease in total mitochondrial superoxide/H₂O₂ production during “aerobic exercise” are clear from our results. Despite increases in the concentrations of several substrates and effectors in the media mimicking exercise, including citrate, malate, pyruvate, succinate, glycerol 3-phosphate, acetylcarnitine, and free Ca²⁺ (Table 1), the redox centers in both the NADH/NAD⁺ and QH₂/Q isopotential groups became more oxidized (Fig. 4 and Table 3). This shows that the dominant effect of the media mimicking exercise was increased supply of ADP caused by turnover of ATP, leading to an increased respiration rate and a more oxidized electron transport chain. Because of the steep dependence of superoxide/H₂O₂ production on the redox state of electron transport chain components (Figs. 5, 6, and 10), this led to the steep decline in total mitochondrial superoxide/H₂O₂ production seen in “exercise” in Fig. 2.

Similarly, the mechanisms underlying the decreased relative contributions of some sites are also clear. Because the oxidation of the NADH/NAD⁺ pool was less severe than the oxidation of the QH₂/Q pool (Fig. 4 and Table 3), the decrease in the rate of superoxide production by site I_F was less than the decrease at the other major sites, which are all linked to the QH₂/Q pool, and the relative contribution of site I_F increased. Although complex I has a high capacity for superoxide production at site I_Q, the flavin site (site I_F) has one of the lowest capacities in skeletal muscle mitochondria (Fig. 3). During “aerobic exercise” when the protonmotive force decreases and the NADH and QH₂ pools become more oxidized, other sites contribute much less to overall H₂O₂ production, allowing the low capacity site I_F to dominate (Fig. 12).

CONCLUSIONS

There is a lack of information about the specific mitochondrial sites of superoxide and hydrogen peroxide production that are physiologically or pathologically active in vivo or in intact cells. Our data provide the first realistic estimate of the sites active in skeletal muscle in vivo under three physiological conditions, rest, mild aerobic exercise, and intense aerobic exercise, and provide the first evidence that in addition to the sites currently recognized (sites III_Qo and I_F), sites I_Q and II_F may also be very important. These results illuminate the specific sites that may need to be normalized to prevent excessive mitochondrial superoxide and H₂O₂ production both physiologically and in many disease states.