Direct and Indirect Roles of Cytochrome b in the Mediation of Superoxide Generation and NO Catabolism by Mitochondrial Succinate-Cytochrome c Reductase

Mitochondrial superoxide (O2·¯) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2·¯ generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2·¯ generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2·¯ generation from SCR was detected as the spin adduct DEPMPO/·OOH. Inhibitors of the Qo site only marginally reduced (20–30%) this O2·¯ production, suggesting a secondary role of Qo·¯ in the mediation of O2·¯ generation. Addition of cyanide significantly decreased (∼70%) O2·¯ production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c1 reduction, and the reaction was mediated by the formation of an O2·¯ intermediate, indicating a direct role for cytochrome b in O2·¯ generation. In the presence of NO, DEPMPO/·OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2·¯. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2·¯. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2·¯ in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption. Mitochondrial superoxide (O2·¯) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2·¯ generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2·¯ generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2·¯ generation from SCR was detected as the spin adduct DEPMPO/·OOH. Inhibitors of the Qo site only marginally reduced (20–30%) this O2·¯ production, suggesting a secondary role of Qo·¯ in the mediation of O2·¯ generation. Addition of cyanide significantly decreased (∼70%) O2·¯ production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c1 reduction, and the reaction was mediated by the formation of an O2·¯ intermediate, indicating a direct role for cytochrome b in O2·¯ generation. In the presence of NO, DEPMPO/·OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2·¯. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2·¯. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2·¯ in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption. Mitochondria are the major cellular source of oxygen-free radical production (1Raha S. Robinson B.H. Trends Biochem. Sci. 2000; 25: 502-508Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 2Turrens J.F. J. Physiol. 2003; 552: 335-344Crossref PubMed Scopus (3420) Google Scholar). They are also an important target for the endothelium-derived relaxant factor, nitric oxide (NO) 2The abbreviations used are: NO, nitric oxide; SCR, succinate-cytochrome c reductase; SQR, succinate-ubiquinol reductase or Complex II; QCR, ubiquinol-cytochrome c reductase or Complex III; RISP, Rieske iron-sulfur protein; antimycin-SCR, native SCR treated by antimycin A; stigmatellin-SCR, native SCR treated by stigmatellin; SOD, superoxide dismutase; DEPMPO, 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide; PBS, phosphate-buffered saline; Q, ubiquinone; TTFA, thenoyl trifluoroacetone. 2The abbreviations used are: NO, nitric oxide; SCR, succinate-cytochrome c reductase; SQR, succinate-ubiquinol reductase or Complex II; QCR, ubiquinol-cytochrome c reductase or Complex III; RISP, Rieske iron-sulfur protein; antimycin-SCR, native SCR treated by antimycin A; stigmatellin-SCR, native SCR treated by stigmatellin; SOD, superoxide dismutase; DEPMPO, 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide; PBS, phosphate-buffered saline; Q, ubiquinone; TTFA, thenoyl trifluoroacetone. (3Moncada S. Erusalimsky J.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 214-220Crossref PubMed Scopus (654) Google Scholar, 4Ramachandran A. Levonen A.L. Brookes P.S. Ceaser E. Shiva S. Barone M.C. Darley-Usmar V. Free Radic. Biol. Med. 2002; 33: 1465-1474Crossref PubMed Scopus (130) Google Scholar). The generation of oxygen-free radical(s) and the effect of NO on mitochondria are particularly relevant under physiological conditions of low oxygen tension such as state IV respiration or certain pathological conditions such as inflammation and ischemia-reperfusion injury (5Zweier J.L. Flaherty J.T. Weisfeldt M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1404-1407Crossref PubMed Scopus (1012) Google Scholar, 6Zweier J.L. Kuppusamy P. Williams R. Rayburn B.K. Smith D. Weisfeldt M.L. Flaherty J.T. J. Biol. Chem. 1989; 264: 18890-18895Abstract Full Text PDF PubMed Google Scholar, 7Ambrosio G. Zweier J.L. Duilio C. Kuppusamy P. Santoro G. Elia P.P. Tritto I. Cirillo P. Condorelli M. Chiariello M. Williams R. Rayburn B.K. Smith D. Weisfeldt M.L. Flaherty J.T. J. Biol. Chem. 1993; 268: 18532-18541Abstract Full Text PDF PubMed Google Scholar). Under these conditions, a decrease in the rate of mitochondrial phosphorylation can increase the production of oxygen-free radicals, in the form of superoxide (O2·¯), from the early stages of the electron transport chain. Two segments of the electron transport chain have been implicated in O2·¯ generation. One, on the NADH dehydrogenase of Complex I, operates via electron leakage from the reduced flavin mononucleotide (8Cadenas E. Boveris A. Ragan C.I. Stoppani A.O. Arch. Biochem. Biophys. 1977; 180: 248-257Crossref PubMed Scopus (681) Google Scholar, 9Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1341) Google Scholar, 10Kudin A.P. Bimpong-Buta N.Y. Vielhaber S. Elger C.E. Kunz W.S. J. Biol. Chem. 2004; 279: 4127-4135Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 11Chen Y.R. Chen C.L. Zhang L. Green-Church K.B. Zweier J.L. J. Biol. Chem. 2005; 280: 37339-37348Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The other, on Complex III, mediates O2·¯ generation through the Q cycle mechanism (Fig. 1), in which electron leakage presumably results from the autoxidation of ubisemiquinone (12Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1055) Google Scholar) and reduced cytochrome bL (13Nohl H. Jordan W. Biochem. Biophys. Res. Commun. 1986; 138: 533-539Crossref PubMed Scopus (175) Google Scholar). In the Q cycle mechanism, there are two ubisemiquinones formed in different parts of the cycle. An unstable ubisemiquinone (Qo·¯) formed near the cytoplasmic site (Fig. 1) is considered to be the major source of O2·¯; therefore, inhibitors such as stigmatellin and myxothiazol that block the formation of Qo·¯ would be expected to decrease overall O2·¯ generation. The other ubisemiquinone (Qi·¯), formed near the matrix site, is stable and EPR-detectable (Fig. 1). Although generally Qi·¯ is not thought to be a source of O2·¯ production because of its stability, when an inhibitor such as antimycin A is used to block the Qi site a reverse electron flow will be induced, thus enhancing the formation of Qo·¯ and increasing O2·¯ generation (Fig. 1). Furthermore, electron leakage for O2·¯ production mediated by low potential cytochrome bL (or b566) was postulated as shown in the Q cycle pathway (Fig. 1) (13Nohl H. Jordan W. Biochem. Biophys. Res. Commun. 1986; 138: 533-539Crossref PubMed Scopus (175) Google Scholar, 14Gong X. Yu L. Xia D. Yu C.A. J. Biol. Chem. 2005; 280: 9251-9257Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), whereas the importance of this autoxidation mediated by the ferrocytochrome b moiety in overall O2·¯ generation remains unclear. NO produced from endothelial cells has the capacity to modulate mitochondrial functions in regulation of metabolism, respiration, and mitochondrial biogenesis (3Moncada S. Erusalimsky J.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 214-220Crossref PubMed Scopus (654) Google Scholar, 4Ramachandran A. Levonen A.L. Brookes P.S. Ceaser E. Shiva S. Barone M.C. Darley-Usmar V. Free Radic. Biol. Med. 2002; 33: 1465-1474Crossref PubMed Scopus (130) Google Scholar, 15Nisoli E. Clementi E. Paolucci C. Cozzi V. Tonello C. Sciorati C. Bracale R. Valerio A. Francolini M. Moncada S. Carruba M.O. Science. 2003; 299: 896-899Crossref PubMed Scopus (1000) Google Scholar, 16Zhao X. He G. Chen Y.R. Pandian R.P. Kuppusamy P. Zweier J.L. Circulation. 2005; 111: 2966-2972Crossref PubMed Scopus (107) Google Scholar). It is well known that within the electron transport chain NO serves as a physiological regulator of cellular respiration in vivo, primarily via reversible binding to the binuclear center of cytochrome c oxidase. Complex II and Complex III are also important targets of NO in respiratory regulation (17Geng Y. Hansson G.K. Holme E. Circ. Res. 1992; 71: 1268-1276Crossref PubMed Scopus (266) Google Scholar, 18Welter R. Yu L. Yu C.A. Arch. Biochem. Biophys. 1996; 331: 9-14Crossref PubMed Scopus (82) Google Scholar). However, an independent pathway for NO utilization in mitochondria has been implicated as well: the Q cycle mechanism (19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 20Cadenas E. Poderoso J.J. Antunes F. Boveris A. Free Radic. Res. 2001; 33: 747-756Crossref Scopus (60) Google Scholar) (Fig. 1). Specifically, (a) QH2 has been suggested as the primary target of NO, as NO can oxidize QH2 in vitro, resulting in ubsemiquinone and nitroxyl anion (NO–); and (b) ubisemiquinone at the Qo site provides a source of electron leakage, forming O2·¯, which is in turn trapped by NO to generate OONO– (Fig. 1). The mechanism of NO consumption, as mediated by the Q cycle of Complex III, is not well understood, especially with regard to interaction with O2·¯. An ideal model for investigation of this mechanism is the succinate-cytochrome c reductase (SCR) supercomplex purified from the bovine heart, comprising succinate-ubiquinone reductase (SQR or Complex II) and the cytochrome bc1 complex (QCR or Complex III) (21Yu L. Yu C.A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar, 22Gwak S.H. Yu L. Yu C.A. Biochemistry. 1986; 25: 7675-7682Crossref PubMed Scopus (28) Google Scholar). Physiologically, the SCR supercomplex mediates electron transfer from succinate to cytochrome c during mitochondrial respiration. The redox centers of SQR contain flavin adenine nucleotide (FAD), three iron-sulfur clusters (S-1, S-2, and S-3), cytochrome b560, and ubiquinone (Q). The redox centers of QCR consist of ubiquinol (QH2), cytochromes bL, bH (high potential b or b562), and c1, and the Rieske iron-sulfur cluster (RISP). Succinate serves as an electron donor for SQR to reduce FAD, after which an electron is transferred from the reduced FAD to the iron-sulfur clusters and cytochrome b560, and Q is reduced to QH2. The electron transfer from QH2 to cytochrome c is catalyzed by QCR and follows the Q cycle mechanism (Fig. 1). The use of succinate as an electron donor for SCR has the advantage of avoiding artifactual O2·¯ generation derived from ubiquinol autoxidation (23Zhang L. Yu L. Yu C.A. J. Biol. Chem. 1998; 273: 33972-33976Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). In this work we have used purified SCR as a model system to address the fundamental question of how the Q cycle mediates O2·¯ generation and NO consumption. We have: 1) used EPR to obtain direct evidence of SCR-mediated O2·¯ generation; 2) used electrochemical detection with an NO electrode to obtain direct evidence of SCR-mediated NO consumption; and 3) demonstrated that NO scavenges O2·¯ generated from the ferrocytochrome b moiety of SCR by forming OONO–. Reagents—Ammonium sulfate, antimycin A, diethylenetriaminepentaacetic acid, horse heart cytochrome c (highest grade available in commercial, and prepared without using trichloroacetic acid), Zn,Cu-superoxide dismutase (SOD), succinic acid, and sodium cholate were purchased from Sigma and used as received. Ascorbic acid, potassium cyanide, and sodium dithionite were purchased from Aldrich. Myxothiazol, stigmatellin, and thenoyl trifluoroacetone (TTFA) were from Fluka BioChemika (St. Louis, MO). The 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) spin trap was purchased from ALEXIS Biochemicals (San Diego, CA). Preparations of Mitochondrial Succinate-Cytochrome c Reductase (SCR)—Bovine heart mitochondrial SCR was prepared and assayed according to the published method (21Yu L. Yu C.A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar). The purified SCR contained 4–4.2 nmol of heme b per mg of protein and exhibited an activity of ∼1.5 μmol of cytochrome c reduced/min/mg of protein. Purified SCR was stored in 50 mm sodium/potassium phosphate buffer, pH 7.4, containing 0.25 m sucrose and 1 mm EDTA. Analytical Methods—Optical spectra were measured on a Shimadzu 2401 UV-visible recording spectrophotometer. The enzyme concentration (based on the heme b) of SCR was calculated from the differential spectrum between dithionite reduction and ferricyanide oxidation, using an extinction coefficient of 28.5 mm–1 cm–1 for the absorbance difference of A562 nm–A576 nm. The enzyme activity of SCR was assayed by measuring cytochrome c reduction. An appropriate amount of SCR was added to an assay mixture (1 ml) containing 50 mm phosphate buffer, pH 7.4, 0.3 mm EDTA, 19.8 mm succinate, and 50 μm ferricytochrome c. The SCR activity was determined by measuring the increase in absorbance at 550 nm. Electron Paramagnetic Resonance Experiments—EPR measurements were performed using the EPR Core Facilities at the Davis Heart and Lung Research Institute. Experiments were carried out on a Bruker EMX spectrometer operating at 9.86 GHz with 100 kHz modulation frequency at room temperature. The reaction mixture was transferred to a 50-μl capillary, which was then positioned in the HS cavity (Bruker Instrument, Billerica, MA). The sample was scanned using the following parameters: center field, 3510 G; sweep width, 140 G; power, 20 milliwatt; receiver gain, 2 × 105; modulation amplitude, 1 G; time of conversion, 81.92 ms; time constant, 327.68 ms. The spectral simulations were performed using the WinSim program developed by Duling (24Duling D.R. J. Magn. Reson. B. 1994; 104: 105-110Crossref PubMed Scopus (883) Google Scholar), National Institutes of Health, NIEHS. Nitric Oxide Consumption Rate by SCR—NO consumption rate was measured electrochemically at 37 °C in an electrochemical vial using a Free Radical Analyzer (World Precision Institute, Sarasota, FL) and Clark NO electrode (World Precision Institute, Sarasota, FL). For measuring the NO consumption rate under anaerobic conditions, a slow flow of argon gas was maintained in the space above the solution. The electrochemical detector continuously recorded the current collected by the electrode, which is proportional to the NO concentration in the solution. The sensor was calibrated with known concentrations of NO, using NO-equilibrated solutions as described in the literature (25Liu X. Miller M.J. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., J.R. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 26Lee C.I. Liu X. Zweier J.L. J. Biol. Chem. 2000; 275: 9369-9376Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The quantitation of the SCR-mediated NO consumption rate with succinate or without succinate was based on: (a) the initial rate of the kinetic curve from NO decay in PBS, or (b) the decrease of peak current from the kinetic curve of NO decay in PBS (27Chen Y.R. Chen C.L. Liu X. He G. Zweier J.L. Arch. Biochem. Biophys. 2005; 439: 200-210Crossref PubMed Scopus (7) Google Scholar). DEPMPO Spin Trapping of O2·¯ Generated from SCR in the Presence of Succinate—To obtain direct evidence for O2·¯ production mediated by SCR, we employed the EPR spin-trapping technique to measure O2·¯ generation unambiguously and directly. Of the available spin traps, DEPMPO is ideal for quantitating O2·¯ production by SCR based on the following advantages: (a) DEPMPO is 40-fold more sensitive than the cytochrome c assay for the detection of O2·¯ (28Roubaud V. Sankarapandi S. Kuppusamy P. Tordo P. Zweier J.L. Anal. Biochem. 1997; 247: 404-411Crossref PubMed Scopus (159) Google Scholar), (b) DEPMPO traps O2·¯ with an efficiency of 60–70% (28Roubaud V. Sankarapandi S. Kuppusamy P. Tordo P. Zweier J.L. Anal. Biochem. 1997; 247: 404-411Crossref PubMed Scopus (159) Google Scholar), and (c) the O2·¯ adduct of DEPMPO/·OOH is more stable than that of DMPO/·OOH (29Frejaville C. Karoui H. Tuccio B. Le Moigne F. Culcasi M. Pietri S. Lauricella R. Tordo P. J. Med. Chem. 1995; 38: 258-265Crossref PubMed Scopus (462) Google Scholar). When purified SCR (2 μm, based on the heme b concentration) was incubated with DEPMPO (20 mm) in PBS and the reaction was initiated by the addition of succinate (18 μm), a multi-line EPR spectrum was produced that was characteristic of DEPMPO/·OOH (Fig. 2A, solid line) based on the hyperfine coupling constants (isomer 1: aN = 13.14 G, aβH=11.04G, aγH=0.96G, aP = 49.96 G (80% relative concentration); isomer 2: aN = 13.18 G, aβH=12.59G, aγH=3.46G, aP = 48.2 G (20% relative concentration) (11Chen Y.R. Chen C.L. Zhang L. Green-Church K.B. Zweier J.L. J. Biol. Chem. 2005; 280: 37339-37348Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 29Frejaville C. Karoui H. Tuccio B. Le Moigne F. Culcasi M. Pietri S. Lauricella R. Tordo P. J. Med. Chem. 1995; 38: 258-265Crossref PubMed Scopus (462) Google Scholar)) obtained from the computer simulation (Fig. 2A, dashed line). Trapping of SCR-mediated O2·¯ by DEPMPO was thus highly specific and suitable for quantitative analysis. That the DEPMPO/·OOH adduct arose from the trapping of O2·¯ was confirmed by the addition of Zn,Cu-containing superoxide dismutase (SOD, 100 units/ml) to the reaction system (Fig. 2B); upon its addition the adduct formation was completely prevented. In the absence of SCR, no DEPMPO/·OOH was detected (Fig. 2C), indicating the enzymatic dependence of the DEPMPO adduct formation. When native SCR was replaced with heat-denatured (70 °C for 5 min) SCR, the formation of DEPMPO/·OOH was inhibited (Fig. 2D), indicating that the electron transfer activity in the enzyme is required for O2·¯ generation. The detection of the superoxide adduct of DEPMPO was strongly inhibited when succinate was omitted from the system (Fig. 2E), supporting a direct role for succinate as the electron donor for SCR-mediated O2·¯ production. In the presence of an electron acceptor, ferricytochrome c (cytochrome c, 6 μm), the production of O2·¯ by succinate-energized SCR was nearly completely inhibited (Fig. 2F), suggesting two possibilities: (a) electron leakage from SCR was efficiently prevented by a stoichiometric amount of cytochrome c under the conditions of enzyme turnover, i.e. in the presence of both succinate and cytochrome c; and (b) any O2·¯ produced by SCR was presumably quenched by cytochrome c reduction. The enzymatic activity of SQR in SCR is sensitive to TTFA, which inhibits electron transfer from SQR to QCR through binding to the S3 center and the cytochrome b560 binding protein in SQR (30Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Pretreatment of SCR with TTFA (10 mm) inhibited ∼87% of the electron transfer activity from succinate to cytochrome c but only 65% of the O2·¯ generation (Fig. 2G), whereas complete inhibition of electron transfer activity and O2·¯ generation can be achieved by higher dosage of TTFA (50 mm); implying that QCR controlled most of the SCR-mediated O2·¯ generation. Electron Leakage to Oxygen by SCR under the Conditions of Enzyme Turnover—To measure the electron leakage from SCR to oxygen under the conditions of enzyme turnover, Zn,Cu-SOD (0.5 units/μl) was added to the reaction mixture containing 36 pmol of SCR and 30 μm cytochrome c in PBS. The reduction of cytochrome c was subsequently induced by the addition of various amounts of succinate (30 μm to 1.2 mm). Electron leakage to oxygen under these turnover conditions was calculated as the amount of SOD-inhibitable cytochrome c reduction. The observed electron leakage was dependent upon the ratio of succinate/cytochrome c. There was no detectable O2·¯ generation under the ratio of 15 or below. Above a ratio of 15, the observed electron leakage increased as the succinate/cytochrome c increased, reaching a plateau at a maximum of 14% of the total cytochrome c reduction when the ratio was greater than 30 (data not shown). Involvement of the Q Cycle Mechanism in SCR-mediated O2·¯ Generation—Antimycin A is a well known inhibitor of QCR; it blocks the transfer of the second electron of QH2 from cytochrome bH to Q, and inhibits the formation of Qi·¯ (see Fig. 1). In the presence of antimycin A, electrons would accumulate in the high-potential heme b (bH) of QCR, and electron leakage to molecular oxygen should be accelerated due to enhanced formation of Qo·¯, a potential source of O2·¯. Two other inhibitors of QCR, myxothiazol and stigmatellin, block electron transfer at the Qo site. Myxothiazol is bound in the Qo pocket slightly toward low-potential heme bL, which causes the release the RISP into a mobile conformation (31Zhang 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 (918) Google Scholar, 32Esser L. Quinn B. Li Y.F. Zhang M. Elberry M. Yu L. Yu C.A. Xia D. J. Mol. Biol. 2004; 341: 281-302Crossref PubMed Scopus (227) Google Scholar). It inhibits the first electron transfer from QH2 to RISP (Fig. 1). Whereas stigmatellin is bound at a different subsite in the Qo pocket, which immobilizes the RISP in the b conformation (31Zhang 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 (918) Google Scholar, 32Esser L. Quinn B. Li Y.F. Zhang M. Elberry M. Yu L. Yu C.A. Xia D. J. Mol. Biol. 2004; 341: 281-302Crossref PubMed Scopus (227) Google Scholar). It interrupts the transfer of the first electron from the Qo site to cytochrome c1 (Fig. 1). Both inhibitors and QH2 are mutually competitive for binding, so either would prevent QH2 occupying the site, and therefore would prevent the formation of Qo·¯ (33Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar, 34Crofts A.R. Barquera B. Gennis R.B. Kuras R. Guergova-Kuras M. Berry E.A. Biochemistry. 1999; 38: 15807-15826Crossref PubMed Scopus (150) Google Scholar), presumably inhibiting O2·¯ generation. The above three inhibitors inhibited more than 99.9% of the electron transfer activity of SCR at concentrations the same as the enzyme. In spin trapping with DEPMPO in the absence of cytochrome c, their effects on O2·¯ generation by SCR varied. When SCR (2 μm) was pretreated with antimycin A (10 μm), O2·¯ generation by SCR was enhanced by up to 20%, whereas both stigmatellin and myxothiazol inhibited succinate-initiated O2·¯ production by up to 20–30% (Fig. 3A). These results are consistent with the idea that the ubisemiquinone at the Qo site (Qo·¯) contributes an appreciable fraction of the total O2·¯ generation. In the presence of cytochrome c at one-third the level of the succinate concentration (6 μm), DEPMPO/·OOH was not detected (Fig. 3B). However, O2·¯ generation was significantly stimulated when SCR was preincubated with any of the above three inhibitors (Fig. 3B). This stimulation could be due to blocking of the electron transfer pathway by the inhibitors, leading to electron leakage to molecular oxygen. Antimycin A was observed to induce marginally higher stimulation of O2·¯ production under the condition of enzyme turnover, presumably due to the formation of Qo·¯. As indicated in the Fig. 3A, the Qo site inhibitors, stigmatellin and myxothiazol, caused only partial inhibition of the SCR-mediated O2·¯ generation in the absence of cytochrome c. Furthermore, both inhibitors stimulated electron leakage and subsequent O2·¯ production under the conditions of enzyme turnover in the presence of cytochrome c (Fig. 3B). Therefore, other mechanisms apart from Qo·¯ must be involved in the mediation of O2·¯ production. To address the possible involvement of the heme components of SCR, we pretreated SCR with a high concentration of the heme protein inhibitor KCN (5 mm), which resulted in the inhibition of 67% of its electron transfer activity and 70% of its O2·¯ generating ability (Fig. 3A), suggesting that the heme components were critical for SCR-mediated O2·¯ generation. It is worth noting that cyanide (5 mm) inhibited the electron transfer activity of QCR, but did not affect the electron transfer activity of SQR. In further support of this hypothesis, the UV-visible absorption spectra of stigmatellin-inhibited SCR (stig-SCR in Fig. 4) following the addition of succinate (10 μm) were obtained. As indicated in the initial spectrum (1st scan) of Fig. 4A, stigmatellin blocked the reduction of cytochrome c1, but did not block the reduction of cytochrome b, which is the typical effect of the Qo site inhibitor on the spectrum of Complex III (33Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar). The obtained UV-visible absorption spectra further indicated that the decrease or oxidation of ferrocytochrome b was accompanied by an increase of ferrocytochrome c1 during repeat scanning (Fig. 4A, 2nd–8th scans). Oxidation of ferrocytochrome b (absorbance decrease in A562 nm) was even more obvious when a lower level of succinate (4 μm) was used as indicated in Fig. 4B. However, the observed spectral interchange between heme b and heme c1 was partially inhibited by DEPMPO (Fig. 4C, dashed line) and completely inhibited by SOD (Fig. 4C, dotted line). Under conditions of argon saturation, spectral interchange between heme b and heme c1 was not observed (Fig. 4D). These data imply that oxygen mediates the intra-molecular electron transfer of succinate-reduced SCR via the formation of an O2·¯ intermediate from the autoxidation of ferrocytochrome b (Equations 1 and 2). Ferrocytochromeb(Fe2+)+O2→ferricytochromeb(Fe3+)+O2·¯(Eq. 1) Ferricytochromec1(Fe3+)+O2·¯→ferrocytochromec1(Fe2+)+O2(Eq. 2) It is known that O2·¯ can be quenched by ferricytochrome c via one-electron reduction (rate constant, k ∼ 106 m–1 s–1) (35Butler J. Koppenol W.H. Margoliash E. J. Biol. Chem. 1982; 257: 10747-10750Abstract Full Text PDF PubMed Google Scholar). The difference in Em,7 between cytochrome c and cytochrome c1 is small (<10 mV), and both share an identical heme configuration (c-type heme). Therefore, cytochrome c1 should be as efficient as cytochrome c at oxidizing O2·¯. It is worth noting that similar intramolecular electron transfer mediated by O2·¯ as an intermediate was also observed in the case of native SCR, antimycin A-treated SCR, and myxothiazol-treated SCR (data not shown). Therefore, these data provide strong evidence that reduced cytochrome b is a source of O2·¯. This concept was further supported by measurement of the NO consumption rate by stigmatellin-inhibited SCR (see below). Interaction of NO with SCR Prevents O2·¯ Generation—Poderoso et al. (19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 36Poderoso J.J. Carreras M.C. Schopfer F. Lisdero C.L. Riobo N.A. Giulivi C. Boveris A.D. Boveris A. Cadenas E. Free Radic. Biol. Med. 1999; 26: 925-935Crossref PubMed Scopus (145) Google Scholar) have proposed that the Q cycle mechanism represents an independent pathway in mitochondria that competes with cytochrome c oxidase in NO utilization. Therefore, the Q cycle has been implicated as an important target for NO-mediated respiratory regulation. This cytochrome c oxidase-independent pathway is proposed to regulate O2·¯ generation by forming peroxynitrite (OONO–