Complex III Releases Superoxide to Both Sides of the Inner Mitochondrial Membrane
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m407715200
ISSN1083-351X
AutoresFlorian L. Müller, Yuhong Liu, Holly Van Remmen,
Tópico(s)Traumatic Brain Injury and Neurovascular Disturbances
ResumoMechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or “topology.” In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ∼50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ∼50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result. Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or “topology.” In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ∼50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ∼50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result. The mitochondrial electron transport chain is the main source of ATP in the mammalian cell and thus is essential for life (1Scheffler I.E. Mitochondria. Wiley-Liss, New York1999Crossref Google Scholar). However, during energy transduction, a small number of electrons “leak” to oxygen prematurely (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar, 3Skulachev V.P. Q. Rev. Biophys. 1996; 29: 169-202Crossref PubMed Google Scholar, 4Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4830) Google Scholar), forming the oxygen free radical superoxide ((O2·¯)in its anionic form and(HO2·)in its protonated form), which has been implicated in the pathophysiology of a variety of diseases including Parkinson's, Huntington's, and Alzheimer's diseases as well as the aging process itself (5Beckman K.B. Ames B.N. Physiol. Rev. 1998; 78: 547-581Crossref PubMed Scopus (3142) Google Scholar, 6Beal M.F. Curr. Opin. Neurobiol. 1996; 6: 661-666Crossref PubMed Scopus (385) Google Scholar). The detrimental significance of mitochondrial electron transport chain-derived superoxide is well illustrated by the lethal phenotype of mice lacking the mitochondrial matrix superoxide dismutase (Sod2) gene (7Li Y. Huang T.T. Carlson E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Wallace D.C. Epstein C.J. Nat. Genet. 1995; 11: 376-381Crossref PubMed Scopus (1455) Google Scholar). While an increasing number of investigators have focused their attention on the potential pathological effects of mitochondrial superoxide (and its derivatives), there is a dearth of information on the mechanisms of deleterious superoxide formation by the electron transport chain. Indeed the state of knowledge has not changed much since the mid-1970s (4Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4830) Google Scholar). As such, diminishing the rate of mitochondrial free radical production remains an elusive therapeutic strategy in the treatment of disease in which superoxide is thought to be involved. In this work, we seek to expand this area of investigation. The basic facts of superoxide production can be summarized as follows. At the ultrastructural level, Complexes I and III are the main sites of mitochondrial superoxide production (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar, 8Barja G. J. Bioenerg. Biomembr. 1999; 31: 347-366Crossref PubMed Scopus (413) Google Scholar, 9Votyakova T.V. Reynolds I.J. J. Neurochem. 2001; 79: 266-277Crossref PubMed Scopus (510) Google Scholar). In Complex I, the most likely sites of electron leakage are the iron-sulfur clusters (Refs. 8Barja G. J. Bioenerg. Biomembr. 1999; 31: 347-366Crossref PubMed Scopus (413) Google Scholar and 10Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (400) Google Scholar, although some evidence also points to the flavin (10Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (400) Google Scholar)), while in Complex III, it is the Qo semiquinone (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar, 3Skulachev V.P. Q. Rev. Biophys. 1996; 29: 169-202Crossref PubMed Google Scholar, 11Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1070) Google Scholar, 12Muller F.L. Roberts A.G. Bowman M.K. Kramer D.M. Biochemistry. 2003; 42: 6493-6499Crossref PubMed Scopus (118) Google Scholar, 13Muller F. Crofts A.R. Kramer D.M. Biochemistry. 2002; 41: 7866-7874Crossref PubMed Scopus (141) Google Scholar). However, at the atomic level, the chemical details of either reaction remain completely unknown (for example, how does oxygen actually reach its reduction site?). An important area of controversy, directly relevant to understanding the mechanism of superoxide formation, is to which side of the inner mitochondrial membrane either Complex I or Complex III releases superoxide (either to the mitochondrial matrix side or the cytoplasmic side (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar)). Anionic superoxide (O2(aq)) is highly membrane-impermeable (14Gus'kova R.A. Ivanov I.I. Kol'tover V.K. Akhobadze V.V. Rubin A.B. Biochim. Biophys. Acta. 1984; 778: 579-585Crossref PubMed Scopus (92) Google Scholar, 15Korshunov S.S. Imlay J.A. Mol. Microbiol. 2002; 43: 95-106Crossref PubMed Scopus (125) Google Scholar, 16Takahashi M.A. Asada K. Arch. Biochem. Biophys. 1983; 226: 558-566Crossref PubMed Scopus (277) Google Scholar, 17Takahashi M. Asada K. Arch. Biochem. Biophys. 1988; 267: 714-722Crossref PubMed Scopus (122) Google Scholar) such that biologically it is highly compartmentalized, i.e. there is no flux between the pools of matrix and cytoplasmic superoxide (16Takahashi M.A. Asada K. Arch. Biochem. Biophys. 1983; 226: 558-566Crossref PubMed Scopus (277) Google Scholar, 17Takahashi M. Asada K. Arch. Biochem. Biophys. 1988; 267: 714-722Crossref PubMed Scopus (122) Google Scholar, 18Missirlis F. Hu J. Kirby K. Hilliker A.J. Rouault T.A. Phillips J.P. J. Biol. Chem. 2003; 278: 47365-47369Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Copin J. Gasche Y. Chan P.H. Free Radic. Biol. Med. 2000; 28: 1571-1576Crossref PubMed Scopus (76) Google Scholar). The first studies on electron transport chain-derived superoxide production concluded that most superoxide must be extruded to the matrix side since superoxide was readily released from antimycin A- or rotenone-treated submitochondrial particles (SMPs, 1The abbreviations used are: SMP, submitochondrial particle; Q, ubiquinone; SOD, superoxide dismutase; HRP, horseradish peroxidase; cyt c, cytochrome c; MCLA, modified Cypridina luciferin analog; DHE, dihydroethidine; IMS, intermembrane space; Qo site, ubiquinol oxidation site; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide. in which the matrix side faces the medium) but not from intact mitochondria (Refs. 20Loschen G. Azzi A. Richter C. Flohe L. FEBS Lett. 1974; 42: 68-72Crossref PubMed Scopus (505) Google Scholar, 21Boveris A. Cadenas E. FEBS Lett. 1975; 54: 311-314Crossref PubMed Scopus (334) Google Scholar, 22Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1365) Google Scholar, 23Takeshige K. Minakami S. Biochem. J. 1979; 180: 129-135Crossref PubMed Scopus (258) Google Scholar; for reviews, see Refs. 24Guillivi C. Boveris A. Cadenas E. Gilbert D. Colton C.A. Reactive Oxygen Species in Biological Systems. Kluwer Academic/Plenum Publishers, New York1999: 77-102Google Scholar and 25Turrens J.F. Biosci. Rep. 1997; 17: 3-8Crossref PubMed Scopus (754) Google Scholar). However, concomitant measurements of H2O2 production also revealed that only about half of the total electron leak in the presence of antimycin A (thus Complex III-derived (21Boveris A. Cadenas E. FEBS Lett. 1975; 54: 311-314Crossref PubMed Scopus (334) Google Scholar, 24Guillivi C. Boveris A. Cadenas E. Gilbert D. Colton C.A. Reactive Oxygen Species in Biological Systems. Kluwer Academic/Plenum Publishers, New York1999: 77-102Google Scholar)) could be explained by net (outward)(O2·¯)release from SMPs. The view that most superoxide production was directed toward the matrix was unchallenged until very recently when the x-ray structure of Complex III was solved (Ref. 26Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (939) Google Scholar; for a review, see Ref. 27Crofts A.R. Annu. Rev. Physiol. 2004; 66: 689-733Crossref PubMed Scopus (377) Google Scholar). F. L. M. pointed out that the x-ray structure of Complex III unambiguously shows the ubiquinol oxidation site (Qo site), the locus of superoxide production in Complex III (11Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1070) Google Scholar), to be located immediately adjacent to the intermembrane space (IMS) and quite distant from the matrix (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar). Based on this structural evidence, it was argued that some fraction of superoxide derived from the Qo site must be released to the IMS and that, if the data supporting superoxide release to the matrix were correct, it was the ability of Qo site-derived superoxide to reach the matrix that begged an explanation. Taking the latter data to be correct (21Boveris A. Cadenas E. FEBS Lett. 1975; 54: 311-314Crossref PubMed Scopus (334) Google Scholar, 24Guillivi C. Boveris A. Cadenas E. Gilbert D. Colton C.A. Reactive Oxygen Species in Biological Systems. Kluwer Academic/Plenum Publishers, New York1999: 77-102Google Scholar, 28Longo V.D. Liou L.L. Valentine J.S. Gralla E.B. Arch. Biochem. Biophys. 1999; 365: 131-142Crossref PubMed Scopus (185) Google Scholar, 29Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), we formulated a simple model that could explain the release of Qo site superoxide to both sides of the inner mitochondrial membrane (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar). Subsequently Han et al. (30Han D. Williams E. Cadenas E. Biochem. J. 2001; 353: 411-416Crossref PubMed Scopus (481) Google Scholar) demonstrated that a Qo site-dependent DMPO signal could indeed be detected in mitoplasts, which these investigators interpreted as superoxide release to the IMS. Han et al. (30Han D. Williams E. Cadenas E. Biochem. J. 2001; 353: 411-416Crossref PubMed Scopus (481) Google Scholar) were aware of the difficulties (2Muller F. J. Am. Aging Assoc. 2000; 23: 227-253PubMed Google Scholar) regarding the reconciliation of the locus of superoxide production (the Qo site) with the release of superoxide to the matrix. Later St. Pierre et al. (31St. 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 (1235) Google Scholar, 32Miwa S. St. Pierre J. Partridge L. Brand M.D. Free Radic. Biol. Med. 2003; 35: 938-948Crossref PubMed Scopus (265) Google Scholar) demonstrated that net extramitochondrial superoxide release could account for 25–75% of the total electron leak through Complex III. St. Pierre et al. (31St. 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 (1235) Google Scholar, 32Miwa S. St. Pierre J. Partridge L. Brand M.D. Free Radic. Biol. Med. 2003; 35: 938-948Crossref PubMed Scopus (265) Google Scholar) argued that all Complex III-derived superoxide was released to the IMS and that the 75–25% unaccounted for could be explained by the action of IMS CuZn-SOD. In later work, Han et al. (33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar) took a similar position, and the view that Complex III exclusively releases superoxide to the cytoplasmic side of the inner membrane (i.e. IMS) seems to be embraced by the mainstream of the scientific community as judged by a (leading) recent textbook (Ref. 34Nicholls D.G. Ferguson S.J. Bioenergetics 3. Academic Press, London2002: 128Google Scholar, p. 128). The contradictory data from SMPs indicating that Complex III can release superoxide into the matrix were not discussed in Refs. 31St. 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 (1235) Google Scholar, 33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, and 34Nicholls D.G. Ferguson S.J. Bioenergetics 3. Academic Press, London2002: 128Google Scholar; however, it might be argued that SMPs are too disrupted to properly resolve the topology of the sites of superoxide production (SMPs can be contaminated with unsealed vesicles, rightside out vesicles, and sheets (35Harmon H.J. J. Bioenerg. Biomembr. 1987; 19: 167-189Crossref PubMed Scopus (9) Google Scholar)). Alternatively, the technique used to reach the estimates of superoxide release in Refs. 31St. 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 (1235) Google Scholar and 33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, and thereby the conclusion that all superoxide from Complex III is released into the IMS (31St. 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 (1235) Google Scholar, 33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 34Nicholls D.G. Ferguson S.J. Bioenergetics 3. Academic Press, London2002: 128Google Scholar), is known to overestimate the flux of superoxide (36Kettle A.J. Carr A.C. Winterbourn C.C. Free Radic. Biol. Med. 1994; 17: 161-164Crossref PubMed Scopus (28) Google Scholar). In the present report (preliminary results were presented at the 2003 Society for Free Radical Biology and Medicine conference (37Muller F. Van Remmen H. Richardson A. Free Radic. Biol. Med. 2003; 35 (Abstr. 52): S26Google Scholar)), we address previous experimental shortcomings and we challenge the newly popular opinion that Complex III only releases superoxide to the cytoplasmic side of the inner membrane. Using a refined version of the assay of St. Pierre et al. (31St. 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 (1235) Google Scholar), we show that while superoxide derived from the Qo site of Complex III was indeed released from intact skeletal muscle mitochondria, this net extramitochondrial superoxide release accounts for no more than ∼50% of H2O2 production (by extension ∼50% of total superoxide production) from Complex III even in mitochondria lacking CuZn-SOD (from Sod1–/– mice (38Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Crossref PubMed Scopus (1048) Google Scholar)). To demonstrate that the remaining ∼50% of superoxide is released into the matrix, we avoided the use of SMPs (noting the potentially confounding factors), instead using the aconitase inhibition assay (39Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar) in intact mitochondria. Superoxide release by Complex III into the matrix was corroborated by the observation of a profound inhibition of aconitase activity, a sensitive target of superoxide exclusively located in that compartment. This inhibition of aconitase occurred even in the presence of exogenously added SOD and catalase, verifying that neither H2O2 nor re-entry of released superoxide could account for this effect. Our data are thus fully in agreement with initial conclusions from studies in SMPs (24Guillivi C. Boveris A. Cadenas E. Gilbert D. Colton C.A. Reactive Oxygen Species in Biological Systems. Kluwer Academic/Plenum Publishers, New York1999: 77-102Google Scholar), confirming that Complex III-derived superoxide can indeed reach the matrix. We discuss two models of superoxide production that explain this observation. Chemicals—Unless stated otherwise, all chemicals used in this study were obtained from Sigma. Animals—All mice used in this study were in the C57B6/J background and housed in the vivarium of the Audie L. Murphy Veterans Affairs Hospital. CuZn-SOD knock-out (Sod1–/–) mice were generated by the laboratory of C. Epstein (38Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Crossref PubMed Scopus (1048) Google Scholar). Mice used in this study were between 3 and 8 months of age. Animals were anesthetized and sacrificed by cervical dislocation. All procedures were approved by the subcommittee for animal studies at the Audie L. Murphy Veterans Affairs Hospital. Mitochondrial Purification—Mitochondria were purified from whole hind limb skeletal muscle (predominantly gastrocnemius and soleus) according to Chappell and Perry (40Chappell J.B. Perry S.V. Nature. 1954; 173: 1094-1095Crossref PubMed Scopus (152) Google Scholar, 41Ernster L. Nordenbrand K. Methods Enzymol. 1967; 10: 86-94Crossref Scopus (103) Google Scholar). Hind limb skeletal muscle was excised, weighed, bathed in 150 mm KCl, and placed in Chappell-Perry buffer with nagarse. The minced skeletal muscle was homogenized with an all glass homogenizer. The homogenate was centrifuged for 10 min at 600 × g, and the supernatant was passed through two cheesecloth layers and centrifuged at 14,000 × g for 10 min. The resultant pellet was washed once in modified Chappell-Perry buffer with 0.5% bovine serum albumin and once in modified Chappell-Perry buffer without bovine serum albumin. Mitochondria were used immediately. Respiration was measured with a Clark electrode as described by Estabrook (42Estabrook R.W. Methods Enzymol. 1974; 10: 41-47Crossref Scopus (1896) Google Scholar). The respiratory control ratio was ∼5.5 with glutamate/malate and ∼2.5 with succinate + rotenone, indicating intactness of the inner mitochondrial membrane. Protein concentration was measured with the Biuret method. H2O2 Production—Mitochondrial H2O2 release was measured with the Amplex™ red-horseradish peroxidase method (Molecular Probes, Eugene, OR (43Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1119) Google Scholar)). Horseradish peroxidase (HRP, 2 units/ml) catalyzes the H2O2-dependent oxidation of non-fluorescent Amplex red (80 μm) to fluorescent resorufin red (43Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1119) Google Scholar). Since HRP is a large protein that does not cross membranes, this assay only detects H2O2 that has been released from the mitochondria (it cannot measure H2O2 inside mitochondria). 200 units/ml CuZn-SOD was added to convert all(O2·¯)into H2O2, a necessity since(O2·¯)reacts very rapidly with HRP and HRP-Compound I, resulting in underestimation of the actual rate of H2O2 production (36Kettle A.J. Carr A.C. Winterbourn C.C. Free Radic. Biol. Med. 1994; 17: 161-164Crossref PubMed Scopus (28) Google Scholar, 44Bielski B.H. J. Phys. Chem. Ref. Data. 1985; 14: 1041-1091Crossref Scopus (1846) Google Scholar). Fluorescence was followed at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a Fluoroskan Ascent type 374 multiwell plate reader (Labsystems, Helsinki, Finland). The slope of the increase in fluorescence is converted to the rate of H2O2 production with a standard curve. Addition of 450 units/ml catalase decreased this slope by ∼99% (data not shown). We performed all assays at 30 °C in black 96-well plates. Substrates used were 9 mm succinate and 5 mm glutamate + malate. For each assay, one reaction well contained buffer only and another contained buffer with mitochondria to estimate the background oxidation rates of Amplex red and to estimate the rate of H2O2 release in mitochondria without substrate (state 1) (45Chance B. Williams G.R. Adv. Enzymol. Relat. Subj. Biochem. 1956; 17: 65-134PubMed Google Scholar). The reaction buffer consisted of 125 mm KCl, 10 mm HEPES, 5 mm MgCl2, 2mm K2HPO4, pH 7.44. In this .study we also used the Amplex red-H2O2 assay to estimate the rate of O2 production by measuring H2O2 production in the presence or absence of exogenously added SOD (31St. 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 (1235) Google Scholar, 32Miwa S. St. Pierre J. Partridge L. Brand M.D. Free Radic. Biol. Med. 2003; 35: 938-948Crossref PubMed Scopus (265) Google Scholar, 33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). A weakness of this approach is that superoxide production is overestimated because superoxide can both reduce and oxidize HRP (36Kettle A.J. Carr A.C. Winterbourn C.C. Free Radic. Biol. Med. 1994; 17: 161-164Crossref PubMed Scopus (28) Google Scholar, 44Bielski B.H. J. Phys. Chem. Ref. Data. 1985; 14: 1041-1091Crossref Scopus (1846) Google Scholar). To address this problem of the previous procedure (31St. 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 (1235) Google Scholar, 32Miwa S. St. Pierre J. Partridge L. Brand M.D. Free Radic. Biol. Med. 2003; 35: 938-948Crossref PubMed Scopus (265) Google Scholar, 33Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar), we added 20 μm acetylated cytochrome c (cyt c) to the Amplex red reaction buffer to act as a superoxide “sink” (instead of HRP). As such, in the absence of SOD, extramitochondrially released superoxide reduces acetylated cyt c (a reaction that does not yield H2O2 as a product). When SOD is added, extramitochondrial superoxide reacts with it, forming H2O2 instead. Thus, the increase in H2O2 formation upon SOD addition can be used to estimate net extramitochondrial superoxide release. Superoxide Production—Superoxide production was measured by three direct methods: 1) MCLA (modified Cypridina luciferin analog) chemiluminescence, 2) dihydroethidine (DHE) fluorescence, and 3) inhibition of aconitase activity. MCLA (2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]-pyrazin-3-one, Molecular Probes) is chemically very similar to coelenterazine (47Teranishi K. Shimomura O. Anal. Biochem. 1997; 249: 37-43Crossref PubMed Scopus (63) Google Scholar) but exhibits brighter luminescence in response to superoxide. MCLA reversibly reacts with superoxide, forming an adduct whose irreversible decay generates light (∼465 nm (48Nakano M. Cell. Mol. Neurobiol. 1998; 18: 565-579Crossref PubMed Scopus (75) Google Scholar)). The apparent rate constant of this reaction is ∼105m–1 s–1 (48Nakano M. Cell. Mol. Neurobiol. 1998; 18: 565-579Crossref PubMed Scopus (75) Google Scholar). Light emission was detected and quantified using a Fluoroskan-FL Ascent type 374 microplate luminometer (Labsystems) with opaque (white) 96-well plates. The photomultiplier was set to default with an integration time of 1000 ms. The MCLA signal was quantified as an integral of 20 s of continuous measurement and expressed as relative luminescence units/mg of mitochondrial protein. The reaction was conducted in 100 μl of reaction buffer containing ∼0.5 mg/ml mitochondrial protein. The reaction buffer contained 125 mm KCl, 10 mm HEPES, 5 mm MgCl2, 2 mm K2HPO4, and 5 μm MCLA. As a positive control, we used the xanthine/xanthine oxidase system (49McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). 1 mm xanthine + 0.1 unit/ml xanthine oxidase caused a ∼100-fold increase in chemiluminescent signal as compared with xanthine or xanthine oxidase alone, which did not differ significantly from reaction buffer alone. Addition of SOD (CuZn-SOD from erythrocytes) decreased the MCLA signal by over 98% (data not shown). As a confirmation of the MCLA assay, we used dihydroethidine (Molecular Probes). This compound is oxidized by superoxide to a fluorescent product with an excitation maximum at 498 nm and an emission maximum at 598 nm (50Benov L. Sztejnberg L. Fridovich I. Free Radic. Biol. Med. 1998; 25: 826-831Crossref PubMed Scopus (426) Google Scholar). DHE (50 μm in the same buffer as described above) was tested with xanthine + xanthine oxidase. Confirming previous work (50Benov L. Sztejnberg L. Fridovich I. Free Radic. Biol. Med. 1998; 25: 826-831Crossref PubMed Scopus (426) Google Scholar), a steady increase in fluorescence could be detected for which the slope (the rate of superoxide formation) was reduced to zero by addition of 100 units/ml SOD (data not shown). The third method of superoxide detection used in this work is the aconitase inhibition method developed by Gardner and Fridovich (29Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 39Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 51Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (247) Google Scholar). Aconitase catalyzes the reversible isomerization of citrate to isocitrate (cis-aconitate being the intermediate). A low redox potential 4Fe-4S iron-sulfur cluster is required for this activity, and superoxide reacts with the latter at around ∼106m–1 s–1 (51Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (247) Google Scholar, 52Hausladen A. Fridovich I. Methods Enzymol. 1996; 269: 37-41Crossref PubMed Google Scholar), inactivating the enzyme. While it is known that other reactive species, such as H2O2, can also inactivate aconitase, the reaction of aconitase with superoxide is several orders of magnitude faster than that with H2O2. Thus, aconitase activity is a sensitive index of superoxide levels both in vivo and in vitro (18Missirlis F. Hu J. Kirby K. Hilliker A.J. Rouault T.A. Phillips J.P. J. Biol. Chem. 2003; 278: 47365-47369Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Longo V.D. Liou L.L. Valentine J.S. Gralla E.B. Arch. Biochem. 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