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Modulation of Mitochondrial Function by Hydrogen Peroxide*

  • Amy C. Nulton-Persson
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970
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  • Luke I. Szweda
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970
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  • Author Footnotes
    * This work was supported by Grant AG-16339 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:June 29, 2001DOI:https://doi.org/10.1074/jbc.M100320200
      During normal cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion (O⨪2) and subsequently hydrogen peroxide (H2O2). Because H2O2 increases in concentration under certain physiologic and pathophysiologic conditions and can oxidatively modify cellular components, it is critical to understand the response of mitochondria to H2O2. In the present study, treatment of isolated rat heart mitochondria with H2O2 resulted in a decline and subsequent recovery of state 3 NADH-linked respiration. Alterations in NADH levels induced by H2O2 closely paralleled changes in the rate of state 3 respiration. Assessment of electron transport chain complexes and Krebs cycle enzymes revealed that α-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and aconitase were susceptible to H2O2 inactivation. Of particular importance, KGDH and SDH activity returned to control levels, concurrent with the recovery of state 3 respiration. Inactivation is not because of direct interaction of H2O2 with KGDH and SDH. In addition, removal of H2O2alone is not sufficient for reactivation. Enzyme activity does not recover unless mitochondria remain intact. The sensitivity of KGDH and SDH to H2O2-mediated inactivation and the reversible nature of inactivation suggest a potential role for H2O2 in the regulation of KGDH and SDH.
      MOPS
      4-morpholinepropanesulfonic acid
      KGDH
      α-ketoglutarate dehydrogenase
      SDH
      succinate dehydrogenase
      The four-electron reduction of O2 to two molecules of H2O represents the final step in mitochondrial electron transport. Chance and Williams (
      • Chance B.
      • Williams G.R.
      ) first reported, however, that as much as 1–2% of oxygen consumed during mitochondrial respiration undergoes incomplete reduction to superoxide anion (O⨪2). Produced at the NADH-coenzyme Q reductase (Complex I) and cytochromec oxidase (Complex III) sites of the electron transport chain, superoxide is rapidly converted to hydrogen peroxide (H2O2) by superoxide dismutase (
      • Chance B.
      • Williams G.R.
      ,
      • Cadenas E.
      • Boveris A.
      • Ragan C.I.
      • Stoppani A.O.
      ,
      • Cadenas E.
      • Boveris A.
      ,
      • Turrens J.F.
      • Boveris A.
      ,
      • Nohl H.
      • Hegner D.
      ,
      • Nohl H.
      • Breuninger V.
      • Hegner D.
      ,
      • Cadenas E.
      • Davies K.J.
      ,
      • Boveris A.
      • Chance B.
      ,
      • Loschen G.
      • Azzi A.
      • Flohe L.
      ,
      • Boveris A.
      • Cadenas E.
      ). Interaction of H2O2 with metal ions, such as Fe2+, can result in the formation of metal-centered oxygen radicals and hydroxyl radical (OH). These species are highly reactive and can oxidatively damage protein, lipids, and DNA (
      • Beckman K.B.
      • Ames B.N.
      ,
      • Berlett B.S.
      • Stadtman E.R.
      ,
      • Esterbauer H.
      • Schaur R.J.
      • Zollner H.
      ,
      • Stadtman E.R.
      ). To maintain the delicate balance between production and removal of oxygen radicals, mitochondria maintain a vast array of antioxidant enzymes and low molecular weight scavengers. An important question emerges from these findings: does mitochondrial free radical production represent metabolic imperfection? If so, production of free radicals necessitates the expenditure of considerable energy and intracellular volume to prevent oxidative damage. Such imperfection is rarely tolerated by evolutionary constraints. Alternatively, oxygen radicals may serve a beneficial role in the regulation of metabolic function.
      To be considered signaling molecules, H2O2and/or oxygen radicals must exhibit rapid alterations in concentration and exert reversible effects commensurate with metabolic requirements. Evidence indicates that the rate of mitochondrial free radical production increases as a result of diminished electron transport (
      • Chance B.
      • Williams G.R.
      ,
      • Cadenas E.
      • Boveris A.
      • Ragan C.I.
      • Stoppani A.O.
      ,
      • Cadenas E.
      • Boveris A.
      ,
      • Turrens J.F.
      • Boveris A.
      ,
      • Nohl H.
      • Hegner D.
      ,
      • Nohl H.
      • Breuninger V.
      • Hegner D.
      ,
      • Cadenas E.
      • Davies K.J.
      ,
      • Boveris A.
      • Chance B.
      ,
      • Loschen G.
      • Azzi A.
      • Flohe L.
      ,
      • Boveris A.
      • Cadenas E.
      ). This would occur when demand for ATP declines or under certain conditions of stress that result in impairment of specific respiratory complexes (
      • Ambrosio G.
      • Zweier J.L.
      • Duilio C.
      • Kuppusamy P.
      • Santoro G.
      • Elia P.P.
      • Tritto I.
      • Cirillo P.
      • Condorelli M.
      • Chiariello M.
      • Flaherty J.T.
      ,
      • Bolli R.
      • Patel B.S.
      • Jeroudi M.O.
      • Lai E.K.
      • McCay P.B.
      ,
      • Lucas D.T.
      • Szweda L.I.
      ,
      • Lucas D.T.
      • Szweda L.I.
      ,
      • Otani H.
      • Tanaka H.
      • Inoue T.
      • Umemoto M.
      • Omoto K.
      • Tanaka K.
      • Sato T.
      • Osako T.
      • Masuda A.
      • Nonoyama A.
      • Kagawa T.
      ,
      • Ueta H.
      • Ogura R.
      • Sugiyama M.
      • Kagiyama A.
      • Shin G.
      ). The level of H2O2 and various free radicals is also dependent on the activity and concentration of a number of enzymatic and nonenzymatic antioxidant systems. Mitochondria are therefore capable of varying the concentration of oxygen radicals and H2O2 in response to alterations in metabolism. Furthermore, these species have been shown to react with and modify protein(s), thereby altering enzyme activity (
      • Berlett B.S.
      • Stadtman E.R.
      ). Although these phenomena have been investigated primarily in the context of free radical damage, it is clear that oxidative modification(s) to specific proteins can influence the activity of certain processes. Evidence for free radical-mediated alterations in function that are reversible and yield an appropriate regulatory response is required if oxygen radicals are to be considered signaling molecules within mitochondria.
      The purpose of this study was to gain further insight into the potential role of pro-oxidants in the regulation of mitochondrial function. Specifically, mitochondria were isolated from rat heart and treated with concentrations of H2O2 that result in reversible declines in the rate of state 3 respiration. The level of NAD(P)H, activities of electron transport chain, and Krebs cycle enzymes were monitored as a function of H2O2concentration and time of exposure. Results are presented that indicate that when NAD(P)H levels are diminished and maximal inhibition of respiration is observed, specific Krebs cycle enzymes exhibit declines in activity. Upon consumption of H2O2, NAD(P)H levels and the activities of certain of these enzymes return to control levels. Reversible H2O2-mediated inactivation of these enzymes requires that mitochondria remain intact. These results are discussed in light of the potential role of oxidants in the regulation of mitochondrial function and suggest future experiments necessary to further test these possibilities.

      MATERIALS AND METHODS

      Isolation of Subsarcolemmal Mitochondria from Rat Heart

      Harlan Sprague-Dawley rats (250–300 g) obtained from Zivic Miller Laboratories were anesthetized with sodium pentobarbitol and decapitated. Hearts were removed and immediately immersed and rinsed in ice cold buffer containing 180 mm KCl, 5.0 mm MOPS,1 and 2.0 mm EDTA, pH 7.4 (buffer A). Hearts (0.9–1.1 g) were then minced and homogenized in 20 ml of buffer A with a Polytron homogenizer (low setting, 2 s). The homogenate was centrifuged at 500 ×g for 5 min (5 °C), and the supernatant was filtered through cheesecloth. The mitochondrial pellet was obtained upon centrifugation of the supernatant at 5000 × g for 10 min (5 °C). After two rinses with ice-cold buffer, the mitochondria were resuspended into buffer A to a final concentration of ∼35.0 mg/ml. Protein determinations were made using the bicinchoninic acid method (Pierce), using bovine serum albumin as a standard.

      Incubation of Intact Mitochondria with H2O2

      Mitochondria were diluted to 0.25 mg/ml in buffer composed of 125 mm KCl and 5.0 mm KH2PO4, pH 7.25 (buffer B). Respiration was initiated by the addition of 15 mmα-ketoglutarate and allowed to proceed for 1.0 min. State 3 respiration was then induced by the addition of 2.0 mm ADP. One min after initiation of state 3 respiration, 12.5–100 μm H2O2 was added. All incubations were performed at room temperature.

      Evaluation of Mitochondrial Respiration

      The rate of mitochondrial oxygen consumption was monitored using a Clark-style oxygen electrode (Instech). Mitochondria (0.25 mg/ml in buffer B, 25 °C) were placed in the sealed oxygen chamber, and respiration was followed throughout the course of each experiment (see above).

      Assay for Mitochondrial NAD(P)H Levels

      NAD(P)H concentrations in intact mitochondria (0.25 mg/ml at 25 °C) were measured spectrofluorometrically (Shimadzu RF-5301 PC) with excitation and emission wavelengths of 340 and 460 nm, respectively. Incubations were carried out as described. Known quantities of NADH were added to 0.25 mg/ml mitochondria for calibration.

      Assay of NAD-linked Electron Transport Chain Complexes

      Activities of electron transport chain complexes I, III, and IV were evaluated after exposure of cardiac mitochondria to various experimental conditions. To measure Complex I activity, mitochondria were diluted to 0.05 mg/ml protein in buffer containing 25.0 mm KH2PO4 and 0.5 mm EDTA, pH 7.25 (buffer C), and sonicated (30 s, setting 3.0, 100% pulse rate, VWR Scientific). Complex I activity was assayed as the rate of NADH consumption (340 nm, ε = 6200m−1·cm−1) upon addition of 2 μg of antimycin A, 50 μm ubiquinone-1, and 75 μm NADH to a 1.0-ml volume of sonicated mitochondria (0.05 mg/ml mitochondrial protein). To measure Complex III activity, mitochondria were diluted to 0.05 mg/ml protein in buffer containing 35.0 mm KH2PO4, 2.0 mmNaCN, and 0.5 mm EDTA, pH 7.25, and disrupted with 0.05% Triton X-100. Samples were then placed in a sonicating water bath (Fisher) for 30 s. Complex III activity was determined as the rate of antimycin A-dependent reduction of cytochrome c (550 nm, ε = 18,500m−1·cm−1) upon addition of 60 μm decylubiquinol, 50 μm cytochromec, and 5.0 mm MgCl2 to sonicated mitochondria (0.01 mg/ml mitochondrial protein). For analysis of Complex IV activity, mitochondria were diluted to 0.05 mg/ml protein in buffer C and placed in a sonicating water bath (Fisher) for 10 s. Complex IV activity was measured as the rate of oxygen consumption upon addition of 5.0 mm ascorbate, 250 μm N,N,N′,N′-tetramethyl-1,4-phenylenediamine, and 10 μm cytochrome c to sonicated mitochondria (0.05 mg/ml mitochondrial protein). All assays were performed at room temperature.

      Assay of Krebs Cycle Enzymes

      Activities of Krebs cycle enzymes were evaluated after exposure of cardiac mitochondria to various experimental conditions. Mitochondria were then diluted to 0.05 mg/ml in 25.0 mm KH2PO4 and 0.5 mm EDTA, pH 7.25, containing 0.01% Triton X-100 and placed in a sonicating water bath (Fisher) for 30 s. Aconitase activity was assayed as the rate of NADP reduction (340 nm, ε = 6200m−1·cm−1) by isocitrate dehydrogenase upon addition of 5.0 mm sodium citrate, 0.6 mm MgCl2, 0.2 mm NADP+, 1.0 unit/ml isocitrate dehydrogenase to sonicated mitochondria (0.05 mg/ml mitochondrial protein). Isocitrate dehydrogenase activity was assayed as the rate of NAD reduction (340 nm, ε = 6200m−1·cm−1) upon addition of 5.0 mm MgCl2, 40 μm rotenone, 2.5 mm isocitrate, and 1.0 mm NAD to sonicated mitochondria (0.05 mg/ml mitochondrial protein). α-Ketoglutarate dehydrogenase (KGDH) activity was assayed as the rate of NAD+ reduction upon addition of 5.0 mmMgCl2, 40.0 μm rotenone, 2.5 mmα-ketoglutarate, 0.1 mm CoA, 0.2 mm thymine pyrophosphate, and 1.0 mm NAD+ to sonicated mitochondria (0.05 mg/ml mitochondrial protein). Succinate dehydrogenase activity was measured as the rate of 2,6-dichlorophenolindophenol reduction (600 nm, ε = 21,000 m−1·cm−1) upon the addition of 5.0 mm MgCl2, 2.0 mmKCN, 2.0 μg of antimycin A, 50.0 μm ubiquinone-1, 90.0 μm 2,6-dichlorophenolindophenol, and 25.0 mmsuccinate to sonicated mitochondria (0.05 mg/ml mitochondrial protein). Malate dehydrogenase activity was measured as the rate of NAD+ reduction upon addition of 40 μmrotenone, 5.0 mm MgCl2, 25 mmmalate, 1.0 unit/ml of citrate synthase, 0.3 mm acetyl CoA, and 10.0 mm NAD+ to sonicated mitochondria (0.0125 mg/ml mitochondrial protein). Purified citrate synthase was included in the malate dehydrogenase assay mixture to prevent accumulation of oxaloacetate, thereby shifting equilibrium in favor of the forward reaction. Citrate synthase activity was monitored by detection of 5,5′-dithiobis(nitrobenzoic acid)-reactive reduced coenzyme A (412 nm, ε = 13, 600m−1·cm−1) upon addition of 0.1 mm 5,5′-dithiobis(nitrobenzoic acid), 0.3 mmacetyl-CoA, and 0.5 mm oxaloacetate to sonicated mitochondria (0.025 mg/ml mitochondrial protein). All enzyme assays were performed at room temperature.

      RESULTS

      Effects of H2O2 on Mitochondrial Respiration

      The effects of H2O2 on NADH-linked state 3 (ADP-dependent) respiratory rates were evaluated using intact cardiac mitochondria with α-ketoglutarate as a respiratory substrate. At 1.0 min, ADP was added at a concentration of 2.0 mm to ensure maximum state 3 respiratory rates for the duration of the experiments. As shown in Fig.1, addition of H2O2 at 2.0 min resulted in a gradual decline in respiration. At ∼5.0 min, respiration leveled off and remained suppressed for a period of time dependent on the concentration of H2O2, at which point the rate of respiration returned to initial state 3 values. The times required for recovery of maximal rates of respiration were not linear with respect to H2O2 concentration (Fig. 1), indicative of enzymatic removal of H2O2. Similar effects of H2O2 were observed when intact mitochondria were allowed to respire in the presence of dinitrophenol, an uncoupler of mitochondrial respiration (not shown). Uncoupling agents collapse the proton gradient, thereby promoting maximum rates of mitochondrial respiration independent of ADP transport or ATP synthesis. If H2O2-mediated alterations in adenine nucleotide translocase or ATP synthase activities contribute to observed declines in state 3 respiration, the rate of uncoupled respiration would be greater than that of ADP-dependent respiration at a given concentration of H2O2. Therefore H2O2-mediated declines in mitochondrial respiration cannot be attributed to alterations in the activities of adenine nucleotide translocase or ATP synthase. To assess the effects of H2O2 on state 4 (ADP-independent) respiration, state 3 respiration was initiated utilizing concentrations of ADP (0.5 mm) that could be completely consumed. Under these conditions, state 4 respiratory rates were unaffected by H2O2 at all conditions tested (not shown). Thus exposure of mitochondria to H2O2 did not disrupt the integrity of the mitochondrial membrane.
      Figure thumbnail gr1
      Figure 1Effect of H2O2 on mitochondrial respiration. Mitochondria at a concentration of 0.25 mg/ml were assayed for oxygen consumption in the presence of 15 mm α-ketoglutarate. State 3 respiration was initiated with the addition of 2.0 mm ADP at 1.0 min. H2O2 (12.5, 25, 50, or 100 μm) was added at 2.0 min.

      Effects of H2O2 on Mitochondrial NAD(P)H Levels

      H2O2-mediated declines in mitochondrial respiration appeared due, in large part, to diminished levels of reducing equivalents available for electron transport. As determined by spectrofluorometric measurements, we observe an initial rapid decline in mitochondrial NADH concentration upon addition of ADP (1.0 min). This reflects the increased requirement for NADH to support maximal rates of respiration (state 3). Treatment of mitochondria with 12.5 μm H2O2 at 2.0 min resulted in a further decline in NAD(P)H levels (Fig.2). The time course over which the mitochondrial NAD(P)H content declined and recovered paralleled the H2O2-mediated loss and return of state 3 respiration (Fig. 2). These findings suggest that reversible inhibition of mitochondrial respiration is largely because of alterations in the steady state levels of reducing equivalents required for maximal rates of electron transport. Importantly, these experiments have defined conditions under which reversible H2O2-mediated inhibition of mitochondrial respiration and declines in reducing equivalents occur. This enables assessment of the response of various mitochondrial enzymes to H2O2 under conditions that do not lead to global oxidative damage.
      Figure thumbnail gr2
      Figure 2Effect of H2O2 on mitochondrial NAD(P)H levels. NAD(P)H production was initiated by the addition of 15 mm α-ketoglutarate to intact cardiac mitochondria (0.25 mg/ml). ADP (2.0 mm) was introduced at 1.0 min and 12.5 μm H2O2 at 2.0 min. NAD(P)H levels (○) were monitored spectrofluorometrically. Oxygen consumption (●) is the same as that shown in Fig. (12.5 μm H2O2).

      Effects of H2O2 on Specific Mitochondrial Enzymes

      To identify enzymes responsive to H2O2, intact cardiac mitochondria were treated with H2O2 in the presence of 15 mmα-ketoglutarate and 2.0 mm ADP. At indicated time points, the activities of specific enzymes were determined. As shown in TableI, the activities of Complexes I, III, and IV of the electron transport chain were not affected by treatment of mitochondria with 50 μm H2O2under the conditions of these experiments. Furthermore, the Krebs cycle enzymes citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase also were not affected by H2O2(Table II). Incubation of mitochondria with 50 μm H2O2 did, however, result in loss in KGDH, succinate dehydrogenase (SDH), and aconitase activities. KGDH, SDH, and aconitase activities declined 39, 37, and 96%, respectively. To test for the possibility of reversible inactivation, mitochondria were incubated with 12.5 or 100 μm H2O2 for varying durations of time. Mitochondria were then disrupted, and enzyme activities were assayed. The results of these experiments indicate that aconitase was significantly and irreversibly inactivated at both 12.5 and 100 μm H2O2 (Fig.3). In striking contrast, KGDH and SDH were reversibly inhibited upon exposure of mitochondria to H2O2 (Fig. 3). At each concentration of H2O2, the time-dependent inactivation and reactivation of KGDH and SDH reflected the time course over which the rate of state 3 mitochondrial respiration was reversibly inhibited. Thus we have provided evidence that treatment of intact mitochondria with micromolar concentrations of H2O2 selectively inactivates specific mitochondrial enzymes in a fully reversible fashion. It should be noted that mitochondria were incubated with α-ketoglutarate as a respiratory substrate. Under these conditions, acetyl-CoA is rapidly depleted, and the Krebs cycle does not progress through aconitase. Therefore whereas aconitase activity remains depressed, state 3 respiratory rates recover fully.
      Table IEffect of H2O2 on mitochondrial electron transport complex activities
      Enzyme0 μm[H2O2]50 μm[H2O2]
      Complex I189.9 ± 15.7191.1 ± 27.9
      Complex III3601.9 ± 88.63567.0 ± 391.5
      Complex IV2238.5 ± 135.22557.1 ± 157.7
      Intact mitochondria (0.5 mg/ml) were incubated in the absence and presence of 50 μm H2O2 for 7.5 min at 25 °C. Mitochondria were then disrupted, and enzyme activities were determined as described under “Materials and Methods.” Values are expressed as nmol/min/mg of mitochondrial protein and represent the means (n = 3) ± S.D.
      Table IIEffect of H2O2 on Krebs cycle enzyme activities
      Enzyme0 μm [H2O2]50 μm [H2O2]
      Citrate synthase1077.8 ± 144.91189.9 ± 44.7
      Aconitase167.2 ± 6.26.9 ± 1.4a
      Isocitrate dehydrogenase113.4 ± 7.1119.8 ± 3.1
      α-Ketoglutarate dehydrogenase230.2 ± 30.7140.0 ± 15.9b
      Succinate dehydrogenase129.4 ± 7.981.3 ± 9.1c
      Malate dehydrogenase1041.2 ± 82.31132.0 ± 240.7
      Intact mitochondria (0.5 mg/ml) were incubated in the absence and presence of 50 μm H2O2 for 7.5 min at 25 °C. Mitochondria were then disrupted, and enzyme activities determined as described under “Materials and Methods.” Values are expressed as nmol/min/mg of mitochondrial protein and represent the means (n = 3) ± S.D. p values (determined from paired t test) indicate statistically significant decreases in activity, as follows: ≤ 10−8(a), ≤ 0.0004 (b), ≤ 0.0002 (c).
      Figure thumbnail gr3
      Figure 3Fractional alterations in aconitase, KGDH , and SDH activities following addition of H2O2. Intact mitochondria (0.25 mg/ml) were incubated with α-ketoglutarate (15 mm). ADP (2.0 mm) was introduced at 1.0 min and 12.5 μm(●) or 100 μm (○) H2O2 at 2.0 min. At the times indicated on the abscissa, samples were diluted with hypotonic buffer containing 0.05% Triton X-100, sonicated, and assayed spectrophotometrically for enzyme activity.

      Conditions Necessary for Enzyme Inactivation

      To gain insight into potential mechanisms of KGDH and SDH inactivation and reactivation, conditions required for these processes were investigated. H2O2-mediated inactivation of KGDH and SDH did not require electron transport. KGDH and SDH exhibited the same degree of inactivation in the presence or absence of Complex I or Complex III inhibitors (rotenone and antimycin A, respectively). Inactivation of KGDH and SDH did, however, require α-ketoglutarate, a respiratory substrate. Furthermore, treatment of detergent-solubilized mitochondria with H2O2 did not result in KGDH or SDH inactivation. In contrast, aconitase was inactivated when intact, permeabilized, or detergent-solubilized mitochondria were incubated with H2O2. H2O2-mediated aconitase inactivation did not require α-ketoglutarate. These findings suggest that in intact mitochondria, H2O2 exerts effects on KGDH and SDH indirectly through redox-sensitive mechanism(s). However, inactivation of aconitase appears to occur via direct interaction of H2O2 with the enzyme.

      Conditions Necessary for Enzyme Reactivation

      Reactivation of KGDH and SDH is dependent on removal of H2O2and, as with enzyme inactivation, does not occur in disrupted mitochondria. This was demonstrated utilizing exogenously added catalase to remove H2O2. Mitochondria were incubated in the absence or presence of 50 μmH2O2 for 7.5 min. Catalase was then added to either intact or detergent-solubilized mitochondria for 4.0 min followed by measurement of KGDH and SDH activity. As shown in Fig. 4, KGDH and SDH activity recovered fully upon addition of catalase to intact, but not solubilized, mitochondria. These data clearly demonstrate that clearance of H2O2 is necessary but not sufficient for reactivation of KGDH and SDH. Furthermore, the requirement that mitochondria remain intact for enzyme reactivation indicates the involvement of a mitochondrial component in this process.
      Figure thumbnail gr4
      Figure 4Conditions required for reactivation of KGDH and SDH. Intact mitochondria (n = 4) (0.25 mg/ml) were incubated with α-ketoglutarate (15 mm). ADP (2.0 mm) was introduced at 1.0 min and 50 μmH2O2 at 2.0 min. Column 1, 11.5-min incubation in the absence of H2O2. Column 2, 11.5-min incubation in the presence of H2O2. Column 3, 7.5-min incubation in the presence of H2O2 followed by treatment with 0.25 mg/ml catalase for the remaining 4.0 min. Column 4, 7.5-min incubation in the presence of H2O2 followed by Triton X-100 solubilization of mitochondria and treatment with 0.25 mg/ml catalase for the remaining 4.0 min. Samples were diluted with hypotonic buffer containing 0.05% Triton X-100, sonicated, and assayed spectrophotometrically for enzyme activity.

      DISCUSSION

      Oxygen radicals and H2O2 have been viewed primarily from the perspective of the damage they may impart. It is becoming increasingly apparent, however, that by virtue of the ability to alter protein function (
      • Berlett B.S.
      • Stadtman E.R.
      ) and exhibit rapid alterations in concentration in response to physiological stimuli, these species satisfy certain prerequisites of signaling molecules (
      • Trimm J.L.
      • Salama G.
      • Abramson J.J.
      ,
      • Lee S.R.
      • Kwon K.S.
      • Kim S.R.
      • Rhee S.G.
      ,
      • Denu J.M.
      • Tanner K.G.
      ,
      • Barrett W.C.
      • DeGnore J.P.
      • Keng Y.F.
      • Zhang Z.Y.
      • Yim M.B.
      • Chock P.B.
      ,
      • Barrett W.C.
      • DeGnore J.P.
      • Konig S.
      • Fales H.M.
      • Keng Y.F.
      • Zhang Z.Y.
      • Yim M.B.
      • Chock P.B.
      ,
      • Saitoh M.
      • Nishitoh H.
      • Fujii M.
      • Takeda K.
      • Tobiume K.
      • Sawada Y.
      • Kawabata M.
      • Miyazono K.
      • Ichijo H.
      ,
      • Liu H.
      • Nishitoh H.
      • Ichijo H.
      • Kyriakis J.M.
      ) (for recent reviews, see Refs.
      • Gabbita S.P.
      • Robinson K.A.
      • Stewart C.A.
      • Floyd R.A.
      • Hensley K.
      ,
      • Finkel T.
      ,
      • Suzuki Y.J.
      • Forman H.J.
      • Sevanian A.
      ,
      • Kamata H.
      • Hirata H.
      ,
      • Rhee S.G.
      ). We have demonstrated that maximum rates of respiration and ATP synthesis are inhibited in a fully reversible fashion upon incubation of intact cardiac mitochondria with H2O2. State 3 respiratory rates reflect the availability of NADH for electron transport. These results are in general agreement with those reported for rat liver mitochondria treated with t-butyl hydroperoxide (
      • Rokutan K.
      • Kawai K.
      • Asada K.
      ). Further analysis of electron transport chain complexes and Krebs cycle enzymes revealed H2O2-dependent inactivation of SDH, KGDH, and aconitase. Thus the levels of NADH required for maximum rates of mitochondrial respiration would be limited not only by consumption of reducing equivalents for removal of H2O2 but also by inhibition of NADH production. In contrast to aconitase, the activities of SDH and KGDH recovered completely in parallel with maximum rates of respiration. Our results indicate that H2O2 mediates its effects on SDH and KGDH by modulating the redox status of the mitochondria. These results suggest that variations in the rate of H2O2 production may serve to regulate mitochondrial function.
      To be considered a regulatory molecule, H2O2must exert effects necessitated by metabolic requirements. Inactivation of KGDH and SDH would diminish reducing equivalents required for electron transport and limit generation of O⨪2 and H2O2. Thus reversible H2O2-induced inhibition of KGDH and SDH may serve an antioxidant function, preventing a pernicious rise in oxygen radical concentration. Alternatively, variations in H2O2 production may serve to regulate electron transport and ATP synthesis. As demand for ATP and electron transport declines, mitochondrial production of H2O2increases (
      • Chance B.
      • Williams G.R.
      ,
      • Cadenas E.
      • Boveris A.
      • Ragan C.I.
      • Stoppani A.O.
      ,
      • Cadenas E.
      • Boveris A.
      ,
      • Turrens J.F.
      • Boveris A.
      ,
      • Nohl H.
      • Hegner D.
      ,
      • Nohl H.
      • Breuninger V.
      • Hegner D.
      ,
      • Cadenas E.
      • Davies K.J.
      ,
      • Boveris A.
      • Chance B.
      ,
      • Loschen G.
      • Azzi A.
      • Flohe L.
      ,
      • Boveris A.
      • Cadenas E.
      ). This would in turn reduce the activities of KGDH and SDH under conditions when the requirement for NADH and FADH2 is diminished. Conversely, as the demand for ATP increases, production of H2O2 would be expected to decrease, resulting in reactivation of SDH and KGDH and the resumption of maximal rates of electron transport. Clearly, these potential roles for H2O2 within the mitochondria are not mutually exclusive. Reversible inactivation of KGDH and SDH would limit oxygen radical production under conditions of oxidative stress and may regulate ATP synthesis during normal metabolism. In this study, we utilized concentrations of H2O2 expected during conditions of oxidative stress. However, maximal inactivation of both KGDH and SDH (∼40%) was observed at the lowest concentration of H2O2 tested (12.5 μm). Future studies must address the response of mitochondria to H2O2 endogenously generated under diverse metabolic conditions.
      There is considerable recent precedence indicating that H2O2 and certain oxygen radicals regulate cellular function. It has been shown previously that H2O2 is an essential component of several signal transduction pathways. Mechanisms by which H2O2 and oxygen radicals exert their effects include reversible oxidation of key sulfhydryl residues and alterations in the interactions between redox-sensitive molecules and target proteins (
      • Trimm J.L.
      • Salama G.
      • Abramson J.J.
      ,
      • Lee S.R.
      • Kwon K.S.
      • Kim S.R.
      • Rhee S.G.
      ,
      • Denu J.M.
      • Tanner K.G.
      ,
      • Barrett W.C.
      • DeGnore J.P.
      • Keng Y.F.
      • Zhang Z.Y.
      • Yim M.B.
      • Chock P.B.
      ,
      • Barrett W.C.
      • DeGnore J.P.
      • Konig S.
      • Fales H.M.
      • Keng Y.F.
      • Zhang Z.Y.
      • Yim M.B.
      • Chock P.B.
      ,
      • Saitoh M.
      • Nishitoh H.
      • Fujii M.
      • Takeda K.
      • Tobiume K.
      • Sawada Y.
      • Kawabata M.
      • Miyazono K.
      • Ichijo H.
      ,
      • Liu H.
      • Nishitoh H.
      • Ichijo H.
      • Kyriakis J.M.
      ) (for recent reviews, see Refs.
      • Gabbita S.P.
      • Robinson K.A.
      • Stewart C.A.
      • Floyd R.A.
      • Hensley K.
      ,
      • Finkel T.
      ,
      • Suzuki Y.J.
      • Forman H.J.
      • Sevanian A.
      ,
      • Kamata H.
      • Hirata H.
      ,
      • Rhee S.G.
      ). To fully elucidate the role of H2O2 in mitochondria it is necessary to identify the mechanism through which SDH and KGDH are inhibited and reactivated. Inactivation of SDH and KGDH is not because of direct interaction with H2O2. In addition, removal of H2O2 alone is not sufficient for reactivation. These results indicate that SDH and KGDH are responsive to the redox status of the mitochondria. There are several possible mechanisms by which enzyme activity could be modulated. We are currently exploring the potential role of the redox-sensitive molecule glutathione and the susceptibility of SDH and KGDH to reversible glutathionylation. It is noteworthy that, under the conditions of our experiments, the magnitude to which both SDH and KGDH were inactivated was similar (40%), regardless of the concentration of H2O2 utilized. This observation coupled with the sensitivity of these enzymes to redox status suggests a common mechanism of reversible inactivation.
      The results of this study indicate that, in contrast to KGDH and SDH, H2O2 is able to interact directly with aconitase, resulting in enzyme inactivation. The susceptibility of aconitase to free radical inactivation is in keeping with previous studies (
      • Hausladen A.
      • Fridovich I.
      ,
      • Janero D.R.
      • Hreniuk D.
      ,
      • Verniquet F.
      • Gaillard J.
      • Neuburger M.
      • Douce R.
      ,
      • Flint D.H.
      • Tuminello J.F.
      • Emptage M.H.
      ,
      • Gardner P.R.
      • Fridovich I.
      ,
      • Gardner P.R.
      • Raineri I.
      • Epstein L.B.
      • White C.W.
      ,
      • Beinert H.
      • Kennedy M.C.
      • Stout C.D.
      ,
      • Yan L.J.
      • Levine R.L.
      • Sohal R.S.
      ,
      • Emptage M.H.
      • Dreyers J.L.
      • Kennedy M.C.
      • Beinert H.
      ,
      • Kennedy M.C.
      • Emptage M.H.
      • Dreyer J.L.
      • Beinert H.
      ,
      • Tong J.
      • Feinberg B.A.
      ). Aconitase contains an active site iron-sulfur [4Fe-4S]2+ complex. Using electronic spin resonance, it was determined that when purified mitochondrial aconitase is treated with superoxide (O⨪2) the [4Fe-4S]2+ cluster is oxidized to [3Fe-4S]1+, resulting in the release of Fe(II) and H2O2 (
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Kennedy M.C.
      ). Whereas this process renders the enzyme inactive, studies utilizing intact cells or purified enzyme revealed that reactivation occurs upon removal of oxidants and requires the presence of Fe(II) to reassemble [4Fe-4S]2+ (
      • Janero D.R.
      • Hreniuk D.
      ,
      • Gardner P.R.
      • Raineri I.
      • Epstein L.B.
      • White C.W.
      ,
      • Beinert H.
      • Kennedy M.C.
      • Stout C.D.
      ,
      • Emptage M.H.
      • Dreyers J.L.
      • Kennedy M.C.
      • Beinert H.
      ,
      • Kennedy M.C.
      • Emptage M.H.
      • Dreyer J.L.
      • Beinert H.
      ,
      • Tong J.
      • Feinberg B.A.
      ,
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Kennedy M.C.
      ). Aconitase was irreversibly inactivated under the conditions of our experiments. However, mitochondria were isolated in a buffer containing EDTA. It has been shown previously that EDTA prevents reactivation of aconitase through the depletion of labile iron (
      • Janero D.R.
      • Hreniuk D.
      ,
      • Gardner P.R.
      • Fridovich I.
      ). We have obtained preliminary results indicating that the addition of Fe(II) following H2O2 inactivation results in recovery of aconitase activity. Therefore, as suggested previously (
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Kennedy M.C.
      ), under in vivo conditions the activity of aconitase is likely reversibly affected by mitochondrial redox status. These results underscore the need to assess the potential for reversal of free radical-mediated enzyme inactivation. Because of incomplete removal of oxidants and/or the removal of key components required for reactivation, the reversible nature of inactivation may be easily overlooked. In the present study, aconitase activity remained depressed whereas the rate of state 3 respiration recovered completely. This is because, with α-ketoglutarate as a substrate, aconitase activity is not required for the production of NADH. Future studies that address the reversibility of aconitase inactivation and its relationship to state 3 respiratory rates must utilize substrates that result in the full use of the Krebs cycle.
      In summary, the oxidation-reduction reactions continuously carried out by respiring mitochondria would be expected to create a dynamic redox environment dependent on metabolic state. Maintenance of mitochondrial viability would therefore depend, in part, on the ability of mitochondria to sense changes in redox status and respond in a manner commensurate with metabolic requirements. In this study, we have provided clear evidence that overall mitochondrial respiration and the activities of specific mitochondrial enzymes are inhibited by the addition of H2O2 in a fully reversible fashion. Whereas these observations suggest a role for free radicals in regulation of mitochondrial function, direct evidence requires identification of mechanisms that lead to enzyme inactivation/reactivation. In addition, experiments designed to test the effects of exogenously added and endogenously produced H2O2 using a variety of respiratory substrates will further enhance our understanding of the process and its physiological significance. Enhanced free radical generation as well as loss of mitochondrial respiration and KGDH activity have been observed in Parkinson's disease (
      • Mizuno Y.
      • Ikebe S.
      • Hattori N.
      • Nakagawa-Hattori Y.
      • Mochizuki H.
      • Tanaka M.
      • Ozawa T.
      ), Alzheimer's disease (
      • Mastrogiacomo F.
      • Bergeron C.
      • Kish S.J.
      ,
      • Kish S.J.
      ), and cardiac ischemia/reperfusion injury (
      • Ambrosio G.
      • Zweier J.L.
      • Duilio C.
      • Kuppusamy P.
      • Santoro G.
      • Elia P.P.
      • Tritto I.
      • Cirillo P.
      • Condorelli M.
      • Chiariello M.
      • Flaherty J.T.
      ,
      • Bolli R.
      • Patel B.S.
      • Jeroudi M.O.
      • Lai E.K.
      • McCay P.B.
      ,
      • Lucas D.T.
      • Szweda L.I.
      ,
      • Lucas D.T.
      • Szweda L.I.
      ,
      • Otani H.
      • Tanaka H.
      • Inoue T.
      • Umemoto M.
      • Omoto K.
      • Tanaka K.
      • Sato T.
      • Osako T.
      • Masuda A.
      • Nonoyama A.
      • Kagawa T.
      ,
      • Ueta H.
      • Ogura R.
      • Sugiyama M.
      • Kagiyama A.
      • Shin G.
      ). Elucidation of the interplay between free radical processes and mitochondrial function would greatly benefit efforts to define specific molecular mechanisms by which free radicals contribute to the progression of these degenerative conditions.

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