Production of Reactive Oxygen Species by Mitochondria
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m304854200
ISSN1083-351X
AutoresQun Chen, Edwin J. Vazquez, Shadi Moghaddas, Charles L. Hoppel, Edward J. Lesnefsky,
Tópico(s)Electron Spin Resonance Studies
ResumoThe mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III. The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: ROS, reactive oxygen species; MOPS, 4-morpholinepropanesulfonic acid. contribute to a number of pathological processes including aging (1Herrero A. Barja G. Mech. 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Kerner J. Hoppel C.L. J. Mol. Cell. Cardiol. 2001; 33: 1065-1089Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 7Wallace D.C. Am. Heart J. (suppl.). 2000; 139: 70-85Crossref PubMed Scopus (179) Google Scholar). The rate of ROS production from mitochondria is increased in a variety of pathologic conditions including hypoxia (8Becker L.B. vanden Hoek T.L. Shao Z.H. Li C.Q. Schumacker P.T. Am. J. Physiol. 1999; 277: H2240-H2246PubMed Google Scholar), ischemia (9Kevin L.G. Camara A.K. Riess M.L. Novalija E. Stowe D.F. Am. J. Physiol. 2003; 284: H566-H574Crossref PubMed Scopus (232) Google Scholar), reperfusion (5Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiariello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar, 10Venditti P. Masullo P. Di Meo S. Cell Mol. Life Sci. 2001; 58: 1528-1537Crossref PubMed Scopus (63) Google Scholar), aging (1Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Crossref PubMed Scopus (173) Google Scholar, 2Moghaddas S. Hoppel C. Lesnefsky E.J. Arch. Biochem. Biophys. 2003; 414: 59-66Crossref PubMed Scopus (96) Google Scholar), and chemical inhibition of mitochondrial respiration (11Sugioka K. Nakano M. Totsune-Nakano H. Minakami H. Tero-Kubota S. Ikegami Y. Biochim. Biophys. Acta. 1988; 936: 377-385Crossref PubMed Scopus (119) Google Scholar, 12Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar). Complex I and complex III of the electron-transport chain are the major sites for ROS production (11Sugioka K. Nakano M. Totsune-Nakano H. Minakami H. Tero-Kubota S. Ikegami Y. Biochim. Biophys. Acta. 1988; 936: 377-385Crossref PubMed Scopus (119) Google Scholar, 12Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar). Complex I inhibition by rotenone can increase ROS generation in submitochondrial particles (12Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar, 13Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1055) Google Scholar). The oxidation of either complex I or complex II substrates in the presence of complex III inhibition with antimycin A increases ROS (1Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Crossref PubMed Scopus (173) Google Scholar, 13Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1055) Google Scholar, 14St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1211) Google Scholar). However, the major site of production of reactive oxygen species in mitochondria oxidizing complex I substrates remains unclear. In the pathologic condition of myocardial ischemia, administration of rotenone decreases the production of ROS (8Becker L.B. vanden Hoek T.L. Shao Z.H. Li C.Q. Schumacker P.T. Am. J. Physiol. 1999; 277: H2240-H2246PubMed Google Scholar) and ameliorates damage to mitochondria during ischemia in the isolated rat heart (15Lesnefsky E.J. Moghaddas S. Vazquez E. Hoppel C. FASEB J. 2003; 17 (abstr.): 518PubMed Google Scholar). These data suggest that rotenone inhibition of complex I decreases rather than increases ROS production by mitochondria during ischemia. Myocardial ischemia results in a decrease in oxidation through cytochrome oxidase in intact mitochondria (6Lesnefsky E.J. Moghaddas S. Tandler B. Kerner J. Hoppel C.L. J. Mol. Cell. Cardiol. 2001; 33: 1065-1089Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 16Lesnefsky E.J. Tandler B. Ye J. Slabe T.J. Turkaly J. Hoppel C.L. Am. J. Physiol. 1997; 273: H1544-H1554Crossref PubMed Google Scholar, 17Lesnefsky E.J. Slabe T.J. Stoll M.S. Minkler P.E. Hoppel C.L. Am. J. Physiol. 2001; 280: H2770-H2778Crossref PubMed Google Scholar, 18Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (200) Google Scholar). Although cytochrome oxidase is not a source of ROS (19Babcock G.T. Wikstrom M. Nature. 1992; 356: 301-309Crossref PubMed Scopus (1071) Google Scholar, 20Babcock G.T. Varotsis C. J. Bioenerg. Biomembr. 1993; 25: 71-80Crossref PubMed Scopus (48) Google Scholar, 21Varotsis C. Zhang Y. Appelman E.H. Babcock G.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 237-241Crossref PubMed Scopus (159) Google Scholar), inhibition of cytochrome oxidase may facilitate ROS production from complexes I or III (22Dawson T.L. Gores G.J. Nieminen A.L. Herman B. Lemasters J.J. Am. J. Physiol. 1993; 264: C961-C967Crossref PubMed Google Scholar). Cytochrome oxidase inhibition by KCN enhanced myocardial damage during reperfusion (5Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiariello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar), suggesting that blockade of electron flow at cytochrome oxidase increased both ROS generation and oxidative damage during reperfusion. Mitochondria and submitochondrial particles commonly are used to study ROS generation. Compared with mitochondria, submitochondrial particles lack matrix antioxidant enzymes (23Raha S. McEachern G.E. Myint A.T. Robinson B.H. Free Radic. Biol. Med. 2000; 29: 170-180Crossref PubMed Scopus (194) Google Scholar) and have a mixed orientation of the inner mitochondrial membrane (24Hoppel C. Cooper C. Arch. Biochem. Biophys. 1969; 135: 184-193Crossref PubMed Scopus (20) Google Scholar). These features may result in different findings when the production of ROS is compared between mitochondria and submitochondrial particles. In the present study, we used mitochondria and corresponding submitochondrial particles prepared from Sprague-Dawley rat heart to test the following: 1) if blockade of proximal electron flow decreases ROS generation from complex III in mitochondria and submitochondrial particles, 2) if complex IV inhibition enhances ROS generation during the oxidation of complex I or complex II substrates, 3) if matrix antioxidant defense dissipates ROS production from either complex I or complex III. Materials—All chemicals were reagent grade and purchased from Sigma. Supplies for the amplex red assay were obtained from Molecular Probes (Eugene, OR). Mitochondria Isolation—The Animal Care and Use Committee for the Louis Stokes Veterans Affairs Medical Center approved the protocol. Male Sprague-Dawley rats (400–500 g) were euthanized, and the hearts from those rats were isolated and put into buffer A (containing (in mm) 100 KCl, 50 MOPS, 1 EGTA, 5 MgSO4·7H2O, and 1 ATP, pH 7.4) at 4 °C. Cardiac subsarcolemmal mitochondria were isolated using the procedure of Palmer et al. (25Palmer J.W. Tandler B. Hoppel C.L. J. Biol. Chem. 1977; 252: 8731-8739Abstract Full Text PDF PubMed Google Scholar). Cardiac tissue was finely minced, placed in buffer A containing 0.2% bovine serum albumin, and homogenized with a Polytron tissue processor (Brinkmann Instruments) for 2.5 s at a rheostat setting of 6.0. The Polytron homogenate was centrifuged at 500 × g, the supernatant was saved for isolation of subsarcolemmal mitochondria, and the pellet was washed. The combined supernatants were centrifuged at 3000 × g to sediment subsarcolemmal mitochondria, which were washed twice and then suspended in KME (100 mm KCl, 50 mm MOPS, and 0.5 mm EGTA). Some of the mitochondria were used for studies of mitochondrial oxidation and H2O2 production, and the remainder was frozen at -70 °C for the preparation of submitochondrial particles. Mitochondrial protein concentration was determined by the Lowry method, using bovine serum albumin as a standard. Preparation of Submitochondrial Particles—Subsarcolemmal mitochondria from 2 to 3 rats were pooled together for the preparation of submitochondrial particles. Frozen mitochondria were thawed and diluted with 10 mm MOPS to a concentration of ∼10–20 mg of protein/ml. The mitochondria were sonicated for 20 s in a salt-ice-water bath (-4 °C) at 60% of maximal output in a Branson sonifier, followed by a 2-min interval. After 9 cycles, the suspension was centrifuged at 16,000 × g for 10 min. The supernatant was decanted and centrifuged at 150,000 × g for 45 min (24Hoppel C. Cooper C. Arch. Biochem. Biophys. 1969; 135: 184-193Crossref PubMed Scopus (20) Google Scholar, 26Lesnefsky E.J. Gudz T.I. Migita C.T. Ikeda-Saito M. Hassan M.O. Turkaly P.J. Hoppel C.L. Arch. Biochem. Biophys. 2001; 385: 117-128Crossref PubMed Scopus (133) Google Scholar). The pellet was resuspended in 10 mm MOPS, and the protein concentration was determined. Mitochondrial Oxidative Phosphorylation—Oxygen consumption in mitochondria was measured using a Clark-type oxygen electrode at 30 °C (16Lesnefsky E.J. Tandler B. Ye J. Slabe T.J. Turkaly J. Hoppel C.L. Am. J. Physiol. 1997; 273: H1544-H1554Crossref PubMed Google Scholar). Mitochondria were incubated in solution including 80 mm KCl, 50 mm MOPS, 1 mm EGTA, 5 mm KH2PO4, and 1 mg/ml bovine serum albumin at pH 7.4. Glutamate was used as substrate, and state 3 (ADP-stimulated), state 4 (ADP-limited) respiration, respiratory control ratios, and the ADP/O ratio were determined (16Lesnefsky E.J. Tandler B. Ye J. Slabe T.J. Turkaly J. Hoppel C.L. Am. J. Physiol. 1997; 273: H1544-H1554Crossref PubMed Google Scholar). Detection of H 2 O 2 Production—The rate of H2O2 production in mitochondria and submitochondrial particles was determined using the oxidation of the fluorogenic indicator amplex red in the presence of horseradish peroxidase (2Moghaddas S. Hoppel C. Lesnefsky E.J. Arch. Biochem. Biophys. 2003; 414: 59-66Crossref PubMed Scopus (96) Google Scholar). The concentrations of horseradish peroxidase and amplex red in the incubation were 0.1 unit/ml and 50 μm, respectively. Fluorescence was recorded in a microplate reader (1420 Victor2, PerkinElmer Life Sciences) with 530 nm excitation and 590 nm emission wavelengths. Standard curves obtained by adding known amounts of H2O2 to assay medium in the presence of the reactants (amplex red and horseradish peroxidase) were linear up to 2 μm. Background fluorescence was measured in the absence of submitochondrial particles or mitochondria and presented as fluorescence minus background (pmol/mg of protein/30 min). The addition of superoxide dismutase did not increase the rate of production of H2O2 indicating that under assay conditions, the non-enzymatic dismutation of superoxide to H2O2 was sufficiently rapid and complete. In a typical experiment, mitochondria or submitochondrial particles were incubated at 0.1 mg of protein/ml at 30 °C. H2O2 production was initiated in mitochondria using glutamate (10 mm), pyruvate (2.5 mm)/malate (2.5 mm), or succinate (5 mm) as substrates. Substrates used with submitochondrial particles were NADH (80 μm) or succinate (5 mm). Rotenone (2.4 μm), antimycin A (10 μm), and azide (10 mm) were added into incubation medium to inhibit the activities of complex I, complex III, and complex IV, respectively. Because thenoyltrifluoroacetone caused a high background fluorescence, malonate (2.5 mm), a succinate dehydrogenase inhibitor, was used to block complex II (27Echtay K.S. Murphy M.P. Smith R.A. Talbot D.A. Brand M.D. J. Biol. Chem. 2002; 277: 47129-47135Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Stigmatellin (6.6 μm) was used to test whether blockade of complex III at the Qo site, limiting electron flow into complex III, reduced H2O2 generation. The addition of catalase (643 units/ml) decreased fluorescence by 85–90% as expected and was used to dissipate H2O2 in the incubation system. The rate of H2O2 production was linear with respect to mg of mitochondrial protein. Statistical Analyses—Data are expressed as means ± S.E. of the mean. One-way analysis of variance with repeated measurements was used to assess the difference between different inhibitors in mitochondria. A difference of p < 0.05 was considered to be significant. Oxidative Phosphorylation—Oxidative phosphorylation was studied in mitochondria with glutamate as the substrate. The state 3 (0.2 mm ADP-stimulated), state 4, and maximal ADP (2 mm)-stimulated respiratory rates (n = 5) were 230 ± 4, 35 ± 3, and 260 ± 6 nAO min-1 mg protein-1, respectively, consistent with previous results (25Palmer J.W. Tandler B. Hoppel C.L. J. Biol. Chem. 1977; 252: 8731-8739Abstract Full Text PDF PubMed Google Scholar, 26Lesnefsky E.J. Gudz T.I. Migita C.T. Ikeda-Saito M. Hassan M.O. Turkaly P.J. Hoppel C.L. Arch. Biochem. Biophys. 2001; 385: 117-128Crossref PubMed Scopus (133) Google Scholar). Respiratory control ratio and ADP/O ratio were 7.1 ± 0.8 and 2.78 ± 0.12. These results indicated that mitochondrial respiration was tightly coupled. H 2 O 2 Production in Mitochondria—Oxidizing glutamate, mitochondria generated minimal H2O2 in the absence of inhibitors (Fig. 1A). Surprisingly, the complex I inhibitor rotenone did not lead to an additional increase in H2O2 production. Complex III inhibition by antimycin A markedly increased H2O2 production, as expected (11Sugioka K. Nakano M. Totsune-Nakano H. Minakami H. Tero-Kubota S. Ikegami Y. Biochim. Biophys. Acta. 1988; 936: 377-385Crossref PubMed Scopus (119) Google Scholar). Rotenone inhibition prevented antimycin A-induced H2O2 production in the mitochondria oxidizing glutamate (Fig. 1A). Stigmatellin attenuated the antimycin A-induced H2O2 production in mitochondria oxidizing glutamate (Table I). Azide inhibition did not increase H2O2 by mitochondria in the presence of glutamate (Table II). Catalase dissipated most of the ROS production by antimycin A indicating that the increase in fluorescence measured was because of H2O2 (Fig. 1A).Table IH2O2 generation in mitochondria oxidizing complex I and complex II substrates Values given are mean ± S.E., n = 5.No inhibitorAAAA + stigmatellinGlutamate (10 mm)20±10360±30ap < 0.05 compared to no inhibitor80±10bp < 0.05 compared to antimycin A (AA)Pyruvate (2.5 mm)/malate (2.5 mm)15±5480±60ap < 0.05 compared to no inhibitor70±10bp < 0.05 compared to antimycin A (AA)Succinate (5 mm)/rotenone (2.4 μm)20±10280±20ap < 0.05 compared to no inhibitor80±20bp < 0.05 compared to antimycin A (AA)a p < 0.05 compared to no inhibitorb p < 0.05 compared to antimycin A (AA) Open table in a new tab Table IIH2O2 generation in azide-treated mitochondria and submitochondrial particles oxidizing complex I and complex II substrates Submitochondrial particle data are the average value for triplicate measurements on one pool of submitochondrial particles. A second pool of submitochondrial particles replicated the results. Values given are mean ± S.E. Mitochondria, n = 5.No inhibitorAzide (10 mm)Subsarcolemmal mitochondriaGlutamate (10 μm)20±520±10Pyruvate (2.5 mm)/malate (2.5 mm)10±520±10Succinate (5 mM)/rotenone (2.4 μm)15±535±10Submitochondrial particlesNADH 80 μm50170Succinate (5 mm)/rotenone (2.4 μm)3040 Open table in a new tab Because rotenone did not increase H2O2 production in mitochondria oxidizing glutamate, pyruvate/malate was used as a second complex I substrate to confirm the results with glutamate. In agreement with the glutamate results, mitochondria oxidizing pyruvate/malate generated minimal H2O2 (Fig. 1B). Inhibition of complex I with rotenone did not increase H2O2 production in mitochondria oxidizing pyruvate/malate compared with the absence of inhibitors (Fig. 1B). Rotenone prevented antimycin A-mediated H2O2 generation in mitochondria oxidizing pyruvate/malate (Fig. 1B), as did stigmatellin (Table I). Azide did not increase H2O2 generation in mitochondria oxidizing pyruvate/malate (Table II). With succinate as substrate, mitochondria produced minimal H2O2 in the presence of rotenone (Fig. 1C). Antimycin A markedly increased H2O2 production in mitochondria oxidizing succinate in the presence of rotenone (Fig. 1C). Malonate, a complex II inhibitor, significantly decreased antimycin A-induced H2O2 generation in mitochondria oxidizing succinate (Fig. 1C). Stigmatellin attenuated antimycin A-induced H2O2 production in mitochondria oxidizing succinate (Table I). Azide did not increase H2O2 generation in mitochondria in the presence of succinate (Table II). H 2 O 2 Production in Submitochondrial Particles—Because submitochondrial particles are unable to oxidize glutamate or pyruvate/malate (24Hoppel C. Cooper C. Arch. Biochem. Biophys. 1969; 135: 184-193Crossref PubMed Scopus (20) Google Scholar), NADH was used as the complex I substrate. In contrast to the situation in mitochondria, rotenone inhibition markedly increased H2O2 production in submitochondrial particles oxidizing NADH (Fig. 2A). Similar to findings in mitochondria, antimycin A markedly increased H2O2 production in submitochondrial particles oxidizing NADH. In submitochondrial particles oxidizing NADH, rotenone/antimycin A produced the same amount of H2O2 as did rotenone alone. Interestingly, stigmatellin only partially blocked antimycin A-induced H2O2 generation in submitochondrial particles oxidizing NADH (Fig. 2A). In contrast to results obtained with mitochondria, azide markedly increased H2O2 production from submitochondrial particles oxidizing NADH (Table II). Antimycin A increased H2O2 production in submitochondrial particles oxidizing succinate, whereas stigmatellin attenuated antimycin A-induced H2O2 production (Fig. 2B). Complex IV inhibition with azide did not significantly increase H2O2 generation in submitochondrial particles oxidizing succinate in the presence of rotenone (Table II). In the present study, we found that the production of reactive oxygen species measured as H2O2 differed substantially in mitochondria and corresponding submitochondrial particles. In intact mitochondria, rotenone inhibition of complex I did not increase ROS production with complex I substrates. In contrast, rotenone markedly increased ROS production in submitochondrial particles oxidizing NADH. Inhibition of complex III with antimycin A increased the production of ROS during the oxidation of complex I substrates in both mitochondria and submitochondrial particles. Rotenone inhibition prevented antimycin A-induced ROS generation in mitochondria oxidizing complex I substrates. However, ROS production during NADH oxidation in the presence of rotenone and antimycin A remained increased in submitochondrial particles. Rotenone blocks complex I near the binding site for ubiquinol, the electron acceptor for complex I (28Okun J.G. Lummen P. Brandt U. J. Biol. Chem. 1999; 274: 2625-2630Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Blockade of complex I at this distal site in the complex increases the reduction of the NADH dehydrogenase site of complex I, increasing electron leak to ROS (12Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar). Thus, rotenone blockade should enhance, not decrease, oxyradical production by complex I. The observation of rotenone-enhanced production of H2O2 in submitochondrial particles oxidizing NADH is consistent with this mechanism and with previous observations (12Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar, 29Turrens J.F. Freeman B.A. Crapo J.D. Arch. Biochem. Biophys. 1982; 217: 411-421Crossref PubMed Scopus (284) Google Scholar). The NADH dehydrogenase site of electron leak from complex I is located in the matrix side of the inner mitochondrial membrane (30Grigorieff N. Curr. Opin. Struct. Biol. 1999; 9: 476-483Crossref PubMed Scopus (90) Google Scholar). Thus, oxidant production from complex I is directed into the mitochondrial matrix and would either result in mitochondrial damage or be inactivated by matrix-antioxidant enzyme systems (23Raha S. McEachern G.E. Myint A.T. Robinson B.H. Free Radic. Biol. Med. 2000; 29: 170-180Crossref PubMed Scopus (194) Google Scholar, 31Arai M. Imai H. Koumura T. Yoshida M. Emoto K. Umeda M. Chiba N. Nakagawa Y. J. Biol. Chem. 1999; 274: 4924-4933Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Thus, in mitochondria, ROS production from complex I is likely to be directed into the matrix and detoxified by matrix antioxidant defense systems (Fig. 3). MnSOD will form H2O2 from superoxide (23Raha S. McEachern G.E. Myint A.T. Robinson B.H. Free Radic. Biol. 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Biophys. 1969; 135: 184-193Crossref PubMed Scopus (20) Google Scholar). Thus, the NADH dehydrogenase can release superoxide into the bulk solution in the absence of antioxidant defense. The observation that rotenone inhibition significantly increased H2O2 production in submitochondrial particles oxidizing NADH supports the finding that complex I is capable of robust H2O2 production. The finding that rotenone inhibition led to only a minimal increase in H2O2 production in mitochondria suggests that complex I is not the major site of net ROS generation in mitochondria that are oxidizing complex I substrates. Complex III is a key site for ROS generation (11Sugioka K. Nakano M. Totsune-Nakano H. Minakami H. Tero-Kubota S. Ikegami Y. Biochim. Biophys. Acta. 1988; 936: 377-385Crossref PubMed Scopus (119) Google Scholar, 14St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. 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In the present study, antimycin A markedly increased H2O2 generation in both mitochondria and submitochondrial particles oxidizing complex I substrates, supporting the idea that the ROS from the complex III Qo center is basically unaffected by antioxidant defense. In the present study, rotenone inhibition prevented antimycin A-induced H2O2 production in mitochondria oxidizing complex I substrates. Complex II inhibition with malonate attenuated antimycin A-induced ROS generation in mitochondria oxidizing succinate. Stigmatellin blocks electron transport into complex III at the Qo center (34Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar) leading to a similar attenuation of ROS produced from the complex in the presence of antimycin A. These results suggest that complex III is the main site for net ROS generation in mitochondria, and blockade of electron flow upstream of complex III minimizes ROS production in mitochondria that are oxidizing complex I substrates. This observation suggests a likely mechanism for the observations that administration of rotenone (15Lesnefsky E.J. Moghaddas S. Vazquez E. Hoppel C. FASEB J. 2003; 17 (abstr.): 518PubMed Google Scholar) or a succinate-dehydrogenase inhibitor (39Ockaili R.A. Bhargava P. Kukreja R.C. Am. J. Physiol. 2001; 280: H2406-H2411PubMed Google Scholar) before myocardial ischemia occurs decreases damage to the myocardium. Myocardial ischemia decreases cytochrome oxidase activity (16Lesnefsky E.J. Tandler B. Ye J. Slabe T.J. Turkaly J. Hoppel C.L. Am. J. Physiol. 1997; 273: H1544-H1554Crossref PubMed Google Scholar, 17Lesnefsky E.J. Slabe T.J. Stoll M.S. Minkler P.E. Hoppel C.L. Am. J. Physiol. 2001; 280: H2770-H2778Crossref PubMed Google Scholar, 18Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (200) Google Scholar). Cytochrome oxidase inhibition augments myocardial oxidative damage (5Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiariello M. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar). Cytochrome oxidase inhibition does not increase ROS production from complex IV (19Babcock G.T. Wikstrom M. Nature. 1992; 356: 301-309Crossref PubMed Scopus (1071) Google Scholar, 20Babcock G.T. Varotsis C. J. Bioenerg. Biomembr. 1993; 25: 71-80Crossref PubMed Scopus (48) Google Scholar, 21Varotsis C. Zhang Y. Appelman E.H. Babcock G.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 237-241Crossref PubMed Scopus (159) Google Scholar) but leads to increased reduction of redox centers in complex I or complex III, enhancing electron leak and ROS generation from these complexes (22Dawson T.L. Gores G.J. Nieminen A.L. Herman B. Lemasters J.J. Am. J. Physiol. 1993; 264: C961-C967Crossref PubMed Google Scholar). In the current study, azide did not increase H2O2 production in mitochondria oxidizing either complex I or complex II substrates. This observation makes complex IV-driven H2O2 generation from the Qo center of complex III less likely. However, azide increased H2O2 generation in submitochondrial particles oxidizing complex I substrates, yet H2O2 production from submitochondrial particles oxidizing succinate was not increased. This observation suggests that azide probably enhances ROS generation from complex I. Because ROS generated at complex I are released into the matrix as discussed above, azide inhibition of complex IV did not enhance ROS generation in mitochondria. In contrast, inhibition of cytochrome oxidase enhanced ROS generation in submitochondrial particles oxidizing NADH, but not succinate, reinforcing the notion of complex I as the major site of ROS production when the electron-transport chain is inhibited at cytochrome oxidase. By introducing a relative block at the Qo site of complex III, inhibition of cytochrome oxidase may augment the production of ROS from complex I but not from complex III. When cytochrome oxidase is inhibited, cytochrome c is reduced (40Partridge R.S. Monroe S.M. Parks J.K. Johnson K. Parker Jr., W.D. Eaton G.R. Eaton S.S. Arch. Biochem. Biophys. 1994; 310: 210-217Crossref PubMed Scopus (55) Google Scholar), leading to an increased relative reduction of cytochrome c 1 in complex III (34Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar, 41Trumpower B.L. Biochim. Biophys. Acta. 2002; 1555: 166-173Crossref PubMed Scopus (77) Google Scholar). According to the structure-function model of electron flow through complex III (41Trumpower B.L. Biochim. Biophys. Acta. 2002; 1555: 166-173Crossref PubMed Scopus (77) Google Scholar, 42Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1046) Google Scholar), when cytochrome c 1 is reduced the iron-sulfur protein cannot dock with cytochrome c 1, leading to disruption of oxidation of ubiquinol at the Qo site, effectively blocking electron entry into complex III (34Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar, 41Trumpower B.L. Biochim. Biophys. Acta. 2002; 1555: 166-173Crossref PubMed Scopus (77) Google Scholar). A block at the Qo site could attenuate oxyradical production from complex III but increase the relative reduction of complex I, leading, in turn, to leak from the NADH dehydrogenase portion of the complex. This proposed mechanism is consistent with the data in the present study that inhibition of cytochrome oxidase enhances ROS production in submitochondrial particles but not in intact mitochondria with complex I substrates. During myocardial ischemia, complex III enhances the production of reactive oxygen species in myocytes (8Becker L.B. vanden Hoek T.L. Shao Z.H. Li C.Q. Schumacker P.T. Am. J. Physiol. 1999; 277: H2240-H2246PubMed Google Scholar) and the intact heart (9Kevin L.G. Camara A.K. Riess M.L. Novalija E. Stowe D.F. Am. J. Physiol. 2003; 284: H566-H574Crossref PubMed Scopus (232) Google Scholar). Complex III, probably via oxidative mechanisms, mediates damage to the electron-transport chain in hearts (15Lesnefsky E.J. Moghaddas S. Vazquez E. Hoppel C. FASEB J. 2003; 17 (abstr.): 518PubMed Google Scholar). The results of oxidant production following inhibition of cytochrome oxidase obtained in the present study in non-ischemic cardiac mitochondria, as discussed above, strongly suggest that inhibition of complex IV during ischemia cannot be the sole mechanism of enhanced mitochondrial-mediated production of oxidants during ischemia. Thus, ROS production from complex III during ischemia appears to require an additional defect that increases oxidant production from the Qo site. During ischemia, complex III sustains damage to the iron-sulfur peptide with loss of the electron paramagnetic resonance signal in this key component of ubiquinol oxidation at the Qo site (26Lesnefsky E.J. Gudz T.I. Migita C.T. Ikeda-Saito M. Hassan M.O. Turkaly P.J. Hoppel C.L. Arch. Biochem. Biophys. 2001; 385: 117-128Crossref PubMed Scopus (133) Google Scholar). Functional alterations of the Qo site can disrupt electron flux in this portion of complex III leading to “bypass reactions” of the Qo site that enhance ROS production (43Muller F. Crofts A.R. Kramer D.M. Biochemistry. 2002; 41: 7866-7874Crossref PubMed Scopus (140) Google Scholar). Thus, ischemic damage may enhance oxidant production from the Qo site of complex III that is blunted by intervention with rotenone (8Becker L.B. vanden Hoek T.L. Shao Z.H. Li C.Q. Schumacker P.T. Am. J. Physiol. 1999; 277: H2240-H2246PubMed Google Scholar, 15Lesnefsky E.J. Moghaddas S. Vazquez E. Hoppel C. FASEB J. 2003; 17 (abstr.): 518PubMed Google Scholar), which limits flow into complex III. Our findings suggest that when complex III is modified to increase oxidant production from the Qo site either by the chemical inhibitor antimycin A (11Sugioka K. Nakano M. Totsune-Nakano H. Minakami H. Tero-Kubota S. Ikegami Y. Biochim. Biophys. Acta. 1988; 936: 377-385Crossref PubMed Scopus (119) Google Scholar), or perhaps by ischemic damage (26Lesnefsky E.J. Gudz T.I. Migita C.T. Ikeda-Saito M. Hassan M.O. Turkaly P.J. Hoppel C.L. Arch. Biochem. Biophys. 2001; 385: 117-128Crossref PubMed Scopus (133) Google Scholar), that inhibition of electron flow upstream of complex III will decrease ROS generation. Interestingly, stigmatellin did not totally block antimycin A-induced ROS generation in submitochondrial particles oxidizing NADH. This suggests that stigmatellin alone causes ROS generation (23Raha S. McEachern G.E. Myint A.T. Robinson B.H. Free Radic. Biol. Med. 2000; 29: 170-180Crossref PubMed Scopus (194) Google Scholar). Stigmatellin blocks electron flow at the Qo site of complex III (34Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar) and will lead to the accumulation of electrons at complex I, enhancing ROS production from this upstream complex in a similar manner to rotenone. In conclusion, complex III is the primary site for net ROS generation in mitochondria and limiting electron flow into complex III prevents ROS production in these organelles. Complex III is the dominant site because ROS products are directed away from the antioxidant defenses of the matrix. In contrast, complex I releases oxidants in the proximity of defense enzyme systems. Inhibition of cytochrome oxidase enhances the production of ROS from certain upstream sites in the electron-transport chain, surprisingly mostly from complex I. We appreciate the editorial assistance of Dr. Bernard Tandler.
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