Generation of Superoxide Anion by Succinate-Cytochromec Reductase from Bovine Heart Mitochondria

Production of superoxide anion (O⨪2), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo1,2-apyrazin-3-one hydrochloride (MCLA)-O⨪2 adduct, was observed during electron transfer from succinate to cytochrome c by reconstituted succinate-cytochrome c reductase-phospholipid vesicles replenished with succinate dehydrogenase. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or detergent to the reconstituted reductase-phospholipid vesicles abolished O⨪2production, suggesting that O⨪2 generation is caused by the membrane potential generated during electron transfer through the cytochrome bc 1 complex. Production of O⨪2 was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which ∼99.7% of the reductase activity was inhibited. The rate of O⨪2 production was closely related to the rate of antimycin-insensitive cytochrome c reduction. Factors affecting antimycin-insensitive reduction of cytochrome calso affected O⨪2 production and vice versa. When the oxygen concentration in the system was decreased, the rate of O⨪2production and cytochrome c reduction by antimycin-treated reductase decreased. When the concentrations of MCLA and cytochromec were increased, the rate of O⨪2 production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin-insensitive cytochrome creduction was sensitive to Qo site inhibitors such as 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. These results indicate that generation of O⨪2 during the oxidation of ubiquinol by the cytochrome bc 1 complex results from a leakage of the second electron of ubiquinol from its Q cycle electron transfer pathway to interact with oxygen. The electron-leaking site is located at the reduced cytochrome b 566 or ubisemiquinone of the Qo site because addition of MCLA to antimycin-treated cytochrome bc 1 complex, in the presence of catalytic amounts of succinate-cytochrome creductase, delayed cytochrome b reduction by succinate. In the presence of oxidized cytochrome c, purified succinate dehydrogenase also catalyzed oxidation of succinate to generate O⨪2. When succinate dehydrogenase was reconstituted with thebc 1 particles to form succinate-cytochrome c reductase, the production of O⨪2 diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O⨪2 in the absence of their immediate electron acceptor and in the presence of cytochrome c. Production of superoxide anion (O⨪2), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo1,2-apyrazin-3-one hydrochloride (MCLA)-O⨪2 adduct, was observed during electron transfer from succinate to cytochrome c by reconstituted succinate-cytochrome c reductase-phospholipid vesicles replenished with succinate dehydrogenase. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or detergent to the reconstituted reductase-phospholipid vesicles abolished O⨪2production, suggesting that O⨪2 generation is caused by the membrane potential generated during electron transfer through the cytochrome bc 1 complex. Production of O⨪2 was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which ∼99.7% of the reductase activity was inhibited. The rate of O⨪2 production was closely related to the rate of antimycin-insensitive cytochrome c reduction. Factors affecting antimycin-insensitive reduction of cytochrome calso affected O⨪2 production and vice versa. When the oxygen concentration in the system was decreased, the rate of O⨪2production and cytochrome c reduction by antimycin-treated reductase decreased. When the concentrations of MCLA and cytochromec were increased, the rate of O⨪2 production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin-insensitive cytochrome creduction was sensitive to Qo site inhibitors such as 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. These results indicate that generation of O⨪2 during the oxidation of ubiquinol by the cytochrome bc 1 complex results from a leakage of the second electron of ubiquinol from its Q cycle electron transfer pathway to interact with oxygen. The electron-leaking site is located at the reduced cytochrome b 566 or ubisemiquinone of the Qo site because addition of MCLA to antimycin-treated cytochrome bc 1 complex, in the presence of catalytic amounts of succinate-cytochrome creductase, delayed cytochrome b reduction by succinate. In the presence of oxidized cytochrome c, purified succinate dehydrogenase also catalyzed oxidation of succinate to generate O⨪2. When succinate dehydrogenase was reconstituted with thebc 1 particles to form succinate-cytochrome c reductase, the production of O⨪2 diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O⨪2 in the absence of their immediate electron acceptor and in the presence of cytochrome c. ubiquinone 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo1,2-apyrazin-3-one hydrochloride membrane-anchoring proteins of succinate-Q reductase xanthine oxidase phospholipid carbonyl cyanide p-trifluoromethoxyphenylhydrazone. During mitochondrial respiration, there is a continuous release of electrons from their normal pathway to molecular oxygen (O2) to form superoxide anion (O⨪2) (1Loschen G. Azzi A. Flohe L. FEBS Lett. 1973; 33: 84-88Crossref PubMed Scopus (158) Google Scholar, 2Loschen G. Azzi A. Richter C. Flohe L. FEBS Lett. 1974; 42: 68-72Crossref PubMed Scopus (504) Google Scholar, 3Nohl H. Hegner D. Biochem. J. 1978; 134: 707-714Google Scholar). O⨪2 subsequently dismutates to H2O2spontaneously or by the action of superoxide dismutases (4McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). Isolated mitochondria in state 4 generate 0.6–1.0 nmol of H2O2/min/mg of protein, accounting for ∼2% of O2 uptake under physiological conditions (5Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1225) Google Scholar). Production of O⨪2 during mitochondrial respiration is closely related to mitochondrial coupling efficiency. Reactions in two segments of the respiratory chain are responsible for univalent reduction of dioxygen to O⨪2. The one located in NADH-Q1 reductase is cyanide-insensitive. Production of O⨪2 is probably via autoxidation of the reduced flavin mononucleotide of NADH dehydrogenase (6Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1365) Google Scholar, 7Cadenas E. Boveris A. Ragan C.I. Stoppani A.O.M. Arch. Biochem. Biophys. 1977; 180: 248-257Crossref PubMed Scopus (696) Google Scholar). The other is located in the cytochromebc 1 complex (ubiquinol-cytochrome creductase). Two redox components of the cytochromebc 1 complex, ubisemiquinone (8Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1070) Google Scholar) and reduced cytochrome b 566 (9Nohl H. Jordan W. Biochem. Biophys. Res. Commun. 1986; 138: 533-539Crossref PubMed Scopus (176) Google Scholar), have been suggested as the autoxidizable factors causing O⨪2 production. To date, most information concerning mitochondrial O⨪2generation has been obtained from studies with intact heart mitochondria and electron transfer inhibitors. Production of O⨪2 in intact mitochondria is difficult to measure because O⨪2 has a very short half-life (2Loschen G. Azzi A. Richter C. Flohe L. FEBS Lett. 1974; 42: 68-72Crossref PubMed Scopus (504) Google Scholar) and cannot pass outward through the inner membrane. However, since heart mitochondria contain superoxide dismutase, but no catalase, and H2O2is a relatively stable species that readily penetrates the mitochondrial membrane, production of O⨪2 in intact heart mitochondria is generally determined by measuring H2O2 concentration in the suspending medium (6Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1365) Google Scholar). Studies of O⨪2 formation by purified electron transfer complexes should yield less ambiguous results, especially when production of O⨪2 is directly measured by chemiluminescence of the MCLA-O⨪2 adduct (10Nakano M. Methods Enzymol. 1990; 186: 585-591Crossref PubMed Scopus (132) Google Scholar). The availability of highly purified bovine heart mitochondrial succinate-cytochrome c reductase complex (11Yu L. Yu C.-A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar) and its subcomplexes, such as succinate-Q reductase (11Yu L. Yu C.-A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar), the cytochromebc 1 complex (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar), succinate dehydrogenase (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar), membrane-anchoring proteins of succinate-Q reductase (QPs) (17Yu L. Xu J.-X. Haley P.E. Yu C.-A. J. Biol. Chem. 1987; 262: 1137-1143Abstract Full Text PDF PubMed Google Scholar), and the bc 1 particles (QPs plus the cytochromebc 1 complex) (18Yu C.-A. Yu L. King T.E. J. Biol. Chem. 1974; 249: 4905-4910Abstract Full Text PDF PubMed Google Scholar), in our laboratory enabled us to systematically study O⨪2 generation in this region of the mitochondrial electron transfer chain. Purified bovine heart succinate-cytochrome c reductase complex (11Yu L. Yu C.-A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar), which catalyzes electron transfer from succinate to cytochrome c, is composed of succinate-Q reductase and the cytochromebc 1 complex. Succinate-Q reductase catalyzes electron transfer from succinate to ubiquinone, whereas the cytochromebc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c. The cytochromebc 1 complex, which contains four redox centers (cytochromes b 566, b 562, and c 1 and the Rieske iron-sulfur cluster), has recently been crystallized (12Yu C.-A. Xia J.Z. Kachurin A.M. Yu L. Kim H. Deisenhofer J. Biochim. Biophys. Acta. 1996; 1275: 47-53Crossref PubMed Scopus (86) Google Scholar), and its structure was determined at 2.9-Å resolution (13Xia D. Yu C.-A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (873) Google Scholar). It is generally accepted that proton and electron transfer in the cytochrome bc 1 complex follows the Q cycle mechanism (14Mitchell P. J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (924) Google Scholar, 15Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar). Succinate-Q reductase is composed of a two-subunit succinate dehydrogenase (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar) and a three-subunit membrane anchoring protein (QPs) (17Yu L. Xu J.-X. Haley P.E. Yu C.-A. J. Biol. Chem. 1987; 262: 1137-1143Abstract Full Text PDF PubMed Google Scholar). Four redox components of the succinate-cytochrome c reductase segment of the respiratory chain (reduced FAD, ubiquinol, ubisemiquinone, and reduced cytochrome b 566) have been suggested as the electron donors for O2. Reduced FAD is in succinate dehydrogenase; ubiquinol is produced by succinate-Q reductase; and ubisemiquinone and reduced cytochrome b 566 are generated during the catalytic cycle of the cytochromebc 1 complex. Herein we report the production of O⨪2 during electron transfer through succinate-cytochromec reductase, succinate-Q reductase, and succinate dehydrogenase under various conditions. Asolectin was obtained from Associate Concentrates (Woodside, New York) and purified according to Sone et al. (19Sone N. Yoshida M. Hirata H. Kagawa Y. J. Biochem. (Tokyo). 1977; 81: 519-528Crossref PubMed Scopus (81) Google Scholar). Horse heart cytochrome c (type III), xanthine oxidase, superoxide dismutase, and catalase were from Sigma. Antimycin A was from U. S. Biochemical Corp. MCLA was a gift from Dr. Anraku (University of Tokyo, Tokyo, Japan). Bovine heart mitochondrial succinate-cytochrome c reductase (11Yu L. Yu C.-A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar), succinate-Q reductase (11Yu L. Yu C.-A. J. Biol. Chem. 1982; 257: 2016-2021Abstract Full Text PDF PubMed Google Scholar), succinate dehydrogenase (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar), the cytochrome bc 1 complex (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar), thebc 1 particles (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar), and QPs (17Yu L. Xu J.-X. Haley P.E. Yu C.-A. J. Biol. Chem. 1987; 262: 1137-1143Abstract Full Text PDF PubMed Google Scholar) were prepared and assayed according to the reported methods. Protein-phospholipid vesicles were prepared by the cholate dialysis method (21Miki T. Miki M. Orii Y. J. Biol. Chem. 1994; 269: 1827-1833Abstract Full Text PDF PubMed Google Scholar, 22Beattie D.S. Villalobo A. J. Biol. Chem. 1982; 257: 14745-14752Abstract Full Text PDF PubMed Google Scholar). Spectral measurements were carried out with a Shimadzu UV-2101PC spectrophotometer at room temperature. O⨪2 generation was determined by measuring the chemiluminescence of MCLA-O⨪2 (10Nakano M. Methods Enzymol. 1990; 186: 585-591Crossref PubMed Scopus (132) Google Scholar). An assay mixture (1 ml) containing 50 mm Tris-Cl, pH 7.8, 0.1 mm EDTA, 4 μm MCLA, 7 mm sodium succinate, and 10 μm cytochrome c was treated with an appropriate amount of enzyme solution to start the reaction. Luminescence was measured with a Lumac/3M Biocounter (Model 2010A). O⨪2 generation is expressed in XO units. One XO unit is defined as the chemiluminescence generated by 1 unit of xanthine oxidase in 1 min, which equals 4.3 × 109 counts from a Lumac/3M Biocounter (Model M2010) in 1 ml of assay mixture containing 20 mm Tris-Cl, pH 7.4, 43 μm hypoxanthine, 4 μm MCLA, and 0.2 m sucrose. One unit of xanthine oxidase is defined as the amount of enzyme that converts 1 μmol of xanthine to uric acid/min at pH 7.5 at 25 °C. For measurement of the rate of oxygen-dependent antimycin-insensitive cytochrome c reduction, 1 ml of assay mixture containing 50 mm Tris-Cl, pH 7.8, 0.1 mm EDTA, 7 mm succinate, 0.6 μmantimycin, 50 μm cytochrome c, and 12 μm MCLA was placed in the main chamber, and a 10-μl aliquot of succinate-cytochrome c reductase (1.6 mg/ml) was in the side arm. The complete anaerobic conditions were achieved by repeated evacuating and flushing with argon. The reaction was started by tipping the succinate-cytochrome c reductase solution in the side arm into the main chamber. The assay mixtures containing different O2 concentrations were made by injecting various amounts of air-saturated assay mixture (assuming 250 through a into the anaerobic assay mixture in the main the reaction. For measurement of oxygen-dependent O⨪2 the assay mixtures containing concentrations of O2 were prepared as for cytochrome c reduction The concentrations of cytochrome c and MCLA were 10 and 4 of succinate-cytochromec reductase (1.6 mg/ml) were Purified succinate-cytochromec reductase complex, as was in the It catalyzed electron transfer from succinate to cytochrome c with a activity of 4 μmol of succinate oxidized of O⨪2 was produced during electron transfer through this complex of O⨪2 by purified succinate-cytochrome c reductase under various are defined under succinate-cytochrome c reductase; purified succinate dehydrogenase; that succinate dehydrogenase was to the succinate-cytochrome c at a XO are defined under succinate-cytochrome c reductase; purified succinate dehydrogenase; that succinate dehydrogenase was to the succinate-cytochrome c at a creductase). in a When purified succinate-cytochrome c reductase complex was into phospholipid vesicles by the cholate dialysis method (21Miki T. Miki M. Orii Y. J. Biol. Chem. 1994; 269: 1827-1833Abstract Full Text PDF PubMed Google Scholar, 22Beattie D.S. Villalobo A. J. Biol. Chem. 1982; 257: 14745-14752Abstract Full Text PDF PubMed Google Scholar), the vesicles succinate-cytochrome c, and c reductase addition of the to the vesicles succinate-cytochrome c reductase activity c reductase activity by that the succinate-Q reductase but the cytochrome bc 1 complex, is in the The was as succinate dehydrogenase because addition of succinate dehydrogenase to the vesicles succinate-cytochromec reductase activity to the as that of the of succinate dehydrogenase is to the of this enzyme in form under conditions (16Yu L. Yu C.-A. Biochim. Biophys. Acta. 1980; 591: 409-420Crossref PubMed Scopus (97) Google Scholar). dehydrogenase was from the bc 1 complex addition of a mixture of sodium during the of The detergent to complete of phospholipid during the of caused of succinate dehydrogenase from its anchoring proteins succinate dehydrogenase was with in the phospholipid during the dialysis of succinate by succinate-cytochrome replenished with a of succinate produced XO of of Addition of or detergent to the succinate vesicles the rate of O⨪2 production by suggesting that the membrane potential generated during electron transfer through the cytochromebc 1 complex region electron from the normal pathway to with O2 to form O⨪2. The O⨪2 production observed with vesicles was to the succinate dehydrogenase in the system and to of protein vesicles by or Production of O⨪2 by succinate dehydrogenase is It has been reported that the production of H2O2 in or heart mitochondria with was to by addition of E. Boveris A. Biochem. J. 1980; PubMed Scopus Google Scholar). The in the the succinate-cytochrome c reductase-phospholipid system and the mitochondrial system by the presence of and cytochrome c in the In the mitochondrial the presence of the of membrane the oxidation of cytochrome c by cytochromec oxidase to generate the oxidized cytochromec for the generation of O⨪2 from reduced In the of reconstituted succinate-cytochrome of membrane potential by addition of the activity of succinate-cytochrome c reductase to The most to membrane O⨪2 production by the cytochromebc 1 complex region is to O⨪2production by vesicles from purified cytochromebc 1 complex and When purified cytochrome bc 1 complex was in phospholipid the cytochromebc vesicles an oxidation of with an of However, of O⨪2 production by vesicles was difficult because the rate of production of O⨪2 from ubiquinol by cytochrome c was with that of the production. The that during oxidation of succinate by A succinate-cytochromec reductase O⨪2 is produced us a system to study generation of O⨪2 in the cytochromebc 1 complex region of the respiratory chain. When succinate-cytochrome c reductase was treated with of its activity was and XO of O⨪2 were produced of Antimycin is to transfer of the second electron of ubiquinol from cytochrome b to ubiquinone to form ubisemiquinone at the according to the Q cycle mechanism the reduction of cytochrome c observed with reductase from the electron of ubiquinol to cytochrome c via the iron-sulfur protein and cytochrome c The second electron of ubiquinol in antimycin-treated reductase probably out of its normal electron transfer pathway at reduced 566 or ubisemiquinone at the and with O2 to produce O⨪2. this is the one the rate of O⨪2 production to closely related to the rate of electron transfer to cytochromec in the antimycin-treated In other factors affecting the rate of O⨪2 production should also the rate of antimycin-insensitive cytochrome c reduction and vice versa. O2 is the for and MCLA with O⨪2 to produce the chemiluminescence their in O⨪2 production is c, the other is the electron acceptor for the electron of ubiquinol via the iron-sulfur protein and cytochromec The O⨪2production and cytochrome c reduction by antimycin-treated reductase by the of MCLA, and cytochrome c the rate of It should that in the antimycin-treated reductase, MCLA the of but also a of O⨪2 production. A O⨪2 production was observed in the xanthine oxidase Addition of MCLA has no cytochrome c reduction by O⨪2 generated by xanthine oxidase in the presence of catalase (4McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). the of MCLA concentration the rate of O⨪2 production and cytochrome creduction by antimycin-treated succinate-cytochrome When succinate was to reductase in the presence of concentrations of MCLA, the rate of O⨪2generation as the MCLA concentration increased. production of O⨪2 was observed when the MCLA concentration MCLA concentrations caused an in O⨪2 production. is to of the chemiluminescence of MCLA-O⨪2 by concentrations of MCLA and to a in the rate of O⨪2 production. cytochrome c as the electron acceptor and the of the reduction of cytochromec should as as the MCLA-O⨪2 adduct formation the measured chemiluminescence the was observed when a concentration of MCLA was The of the chemiluminescence of MCLA-O⨪2 by concentrations of MCLA was also observed with O⨪2 generated by the xanthine oxidase system The amount of chemiluminescence generated by a amount of xanthine oxidase as the MCLA concentration increased, a and decreased. that concentrations of MCLA of It should that MCLA at the concentrations an an xanthine oxidase when the activity was by formation or cytochrome c reduction in the presence of catalase (4McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). When cytochrome c reduction by succinate-cytochrome c reductase was determined in the presence of concentrations of MCLA, succinate as the rate of reduction when the MCLA concentration was increased, a at an MCLA concentration of by that MCLA as an electron for O⨪2 by It the second electron of ubiquinol from reduced 566 or ubisemiquinone at the the rate of O⨪2 production and transfer of the electron of ubiquinol to cytochromec via the iron-sulfur protein and cytochromec is by the that antimycin-insensitive cytochromec reduction was sensitive to Qo site inhibitors such as of MCLA concentration the rate of O⨪2 production by xanthine The amounts of xanthine oxidase were and μm was as For other the to of concentration O⨪2production by antimycin-treated succinate-cytochrome The conditions were the as for that the concentrations of were O⨪2 generation is XO of O⨪2 is to H2O2 by superoxide dismutase, was of to or the rate of O⨪2 generation and cytochrome c reduction by antimycin-treated reductase is affected by addition of superoxide To our addition of superoxide the antimycin-insensitive cytochrome c reduction the O⨪2 generation site is to superoxide since the rate of of affected by its that the O⨪2generation site is in a to MCLA, but to superoxide the other addition of superoxide to the and reductase reduced the chemiluminescence of the suggesting that superoxide is to of O⨪2 in the form of Addition of superoxide to the xanthine oxidase system the oxidation of hypoxanthine, but the chemiluminescence of as that MCLA-O⨪2 as a for superoxide the of the rate of O⨪2 production and antimycin-insensitive cytochrome c reduction by antimycin-treated succinate-cytochrome c When the O2 concentration was decreased, the rate of O⨪2production and cytochrome c reduction decreased. When O2 was O⨪2 production antimycin-insensitive cytochrome c reduction was that cytochrome creduction with O⨪2 production by antimycin-treated succinate-cytochrome c the of cytochromec concentration O⨪2 production and cytochromec reduction by antimycin-treated succinate-cytochromec When amounts of cytochrome to antimycin-treated succinate-cytochrome in the presence of 4 μm MCLA, O⨪2production as the concentration of cytochrome to When the cytochrome was O⨪2production decreased. results from the of the chemiluminescence of MCLA-O⨪2 by cytochrome c. the other the rate of cytochrome c reduction catalyzed by reductase as the concentration of cytochrome c increased. The cytochrome c for antimycin-treated succinate-cytochromec reductase in the presence of MCLA was to to that obtained for the reductase, that of cytochrome c to cytochrome c 1 is affected by or cytochrome c is an O⨪2 (10Nakano M. Methods Enzymol. 1990; 186: 585-591Crossref PubMed Scopus (132) Google Scholar), is less MCLA in O⨪2 produced in or in a the rate of electron to the cytochrome bc 1 complex, by of succinate-Q reductase activity in succinate-cytochromec reductase by or by the addition of amounts of succinate-cytochrome c reductase to purified cytochrome bc 1 complex, the of reduction of b and c 1 determined by methods. When succinate was to purified cytochrome bc in the presence of catalytic amounts of purified succinate-cytochrome c reductase, the reduction of cytochrome b at the as the reduction of cytochrome c When the cytochromebc 1 complex was treated with the reduction of cytochrome b the reduction of cytochrome c Addition of MCLA to the cytochrome bc 1 complex in cytochrome c 1 reduced cytochrome delayed reduction of the that the second electron of ubiquinol out to with oxygen to form as the MCLA-O⨪2 adduct, cytochrome b The that ubiquinol O⨪2 in the presence of cytochrome c us to the production of O⨪2 by succinate-Q Purified succinate-Q reductase, in the presence and absence of cytochrome c, produced and XO of of protein, Succinate-Q reductase with in the presence and absence of cytochromec produced and XO of of protein, Production of O⨪2 by succinate-Q reductase in the presence of cytochrome c and addition of its electron the cytochromebc 1 complex. is in with the that electron transfer through succinate-cytochromec reductase generate O⨪2. succinate-cytochrome c reductase is from succinate-Q reductase by addition of the cytochromebc 1 complex. These results indicate that electron transfer through succinate-Q reductase in the absence of the cytochrome bc 1 complex the oxidation of ubiquinol in the presence of cytochrome c. The production of O⨪2 through oxidation of ubiquinol by cytochrome c by a in which ubiquinol its potential electron to cytochrome c to generate a potential that O2 to O⨪2. is by the that no O⨪2 is from ubiquinol in the presence of reduced cytochrome of O⨪2 by succinate-Q reductase under various of succinate-Q reductase; c, cytochrome concentration of cytochrome c and was 10 concentration of cytochrome c and was 10 concentration of ubiquinone was Q c Q c succinate-Q reductase; c, cytochrome The concentration of cytochrome c and was 10 The concentration of ubiquinone was in a O⨪2 production was observed during succinate oxidation by the reconstituted succinate-cytochromec reductase system in the presence of succinate dehydrogenase O⨪2 production by succinate dehydrogenase under various conditions was The rate of O⨪2 production by succinate dehydrogenase from to XO of protein addition of oxidized cytochrome c Addition of the bc 1 particles (QPs the cytochrome bc 1 but the cytochromebc 1 complex, the rate of O⨪2 production by succinate dehydrogenase in the presence of cytochrome c. The the bc 1 particles and the cytochrome bc 1 complex is that the contains which electron transfer Q and succinate dehydrogenase. addition of the bc to succinate dehydrogenase succinate-cytochrome c the rate of O⨪2 production by succinate was the as that of the addition of the bc 1 particles to the succinate dehydrogenase O⨪2 production. These results indicate that reduced flavin in succinate in the absence of an immediate electron transfer a potential electron to cytochromec through iron-sulfur to generate a potential flavin which in O2 to form O⨪2. cytochrome in the generation of O⨪2 catalyzed by succinate dehydrogenase or succinate-Q reductase the concentration is cytochromec in O⨪2 formation in succinate-Q reductase in succinate of O⨪2 by succinate dehydrogenase under various purified succinate dehydrogenase; cytochrome reductase; succinate-cytochrome QPs purified succinate dehydrogenase; cytochrome reductase; succinate-cytochrome in a Dr. for of this