Generation of Superoxide Anion by Succinate-Cytochrome c Reductase from Bovine Heart Mitochondria*

Production of superoxide anion (O 2 .), measured as the chemiluminescence of the 2-methyl-6-( p -methoxyphe-nyl)-3,7-dihydroimidazo[1,2- a ]pyrazin-3-one hydrochlo-ride (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 2 . production, suggesting that O 2 . generation is caused by the membrane potential generated during electron transfer through the cytochrome bc 1 com- plex. 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 c also affected O 2 . production and vice versa. When the oxygen concentration in the system was decreased, the rate of O 2 . production and cytochrome c reduction by antimycin-treated reductase decreased. When the concentrations of MCLA and cytochrome c were increased, the rate of O 2 . production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin-insensitive cytochrome c

During mitochondrial respiration, there is a continuous release of electrons from their normal pathway to molecular oxygen (O 2 ) to form superoxide anion (O 2 . ) (1)(2)(3). O 2 . subsequently dismutates to H 2 O 2 spontaneously or by the action of superoxide dismutases (4). Isolated mitochondria in state 4 generate 0.6 -1.0 nmol of H 2 O 2 /min/mg of protein, accounting for ϳ2% of O 2 uptake under physiological conditions (5). 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-Q 1 reductase is cyanide-insensitive. Production of O 2 . is probably via autoxidation of the reduced flavin mononucleotide of NADH dehydrogenase (6,7). The other is located in the cytochrome bc 1 complex (ubiquinol-cytochrome c reductase). Two redox components of the cytochrome bc 1 complex, ubisemiquinone (8) and reduced cytochrome b 566 (9), have been suggested as the autoxidizable factors causing O 2 . production.
To date, most information concerning mitochondrial O 2 . generation 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 (2) and cannot pass outward through the inner membrane. However, since heart mitochondria contain superoxide dismutase, but no catalase, and H 2 O 2 is a relatively stable species that readily penetrates the mitochondrial membrane, production of O 2 . in intact heart mitochondria is generally determined by measuring H 2 O 2 concentration in the suspending medium (6). 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 (10).
The availability of highly purified bovine heart mitochondrial succinate-cytochrome c reductase complex (11) and its subcomplexes, such as succinate-Q reductase (11), the cytochrome bc 1 complex (16), succinate dehydrogenase (16), membrane-anchoring proteins of succinate-Q reductase (QPs) (17), and the bc 1 particles (QPs plus the cytochrome bc 1 complex) (18), 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 (11), which catalyzes electron transfer from succinate to cytochrome c, is composed of succinate-Q reductase and the cytochrome bc 1 complex. Succinate-Q reductase catalyzes electron transfer from succinate to ubiquinone, whereas the cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c. The cytochrome bc 1 complex, which contains four redox centers (cytochromes b 566 , b 562 , and c 1 and the Rieske iron-sulfur cluster), * This work was supported in part by Grant GM30721 from the National Institutes of Health; by grant-in-aid 9507873S from the American Heart Association (to L. Y.); and by Agricultural Experimental Station Project 1819, Oklahoma State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. has recently been crystallized (12), and its structure was determined at 2.9-Å resolution (13). It is generally accepted that proton and electron transfer in the cytochrome bc 1 complex follows the Q cycle mechanism (14,15). Succinate-Q reductase is composed of a two-subunit succinate dehydrogenase (16) and a threesubunit membrane anchoring protein (QPs) (17). 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 O 2 . 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 cytochrome bc 1 complex. Herein we report the production of O 2 . during electron transfer through succinatecytochrome c reductase, succinate-Q reductase, and succinate dehydrogenase under various conditions.

EXPERIMENTAL PROCEDURES
Asolectin was obtained from Associate Concentrates (Woodside, New York) and purified according to Sone et al. (19). 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 (11), succinate-Q reductase (11), succinate dehydrogenase (16), the cytochrome bc 1 complex (16), the bc 1 particles (16), and QPs (17) were prepared and assayed according to the reported methods. Protein-phospholipid vesicles were prepared by the cholate dialysis method (21,22). Spectral measurements were carried out with a Shimadzu UV-2101PC spectrophotometer at room temperature. One unit of xanthine oxidase is defined as the amount of enzyme that converts 1 mol 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 M antimycin, 50 M cytochrome c, and 12 M MCLA was placed in the main chamber, and a 10-l 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 O 2 concentrations were made by injecting various amounts of air-saturated assay mixture (assuming 250 M O 2 concentration) through a rubber plug into the anaerobic assay mixture in the main chamber before starting the reaction. For measurement of oxygen-dependent O 2 . production, the assay mixtures containing varying concentrations of O 2 were prepared as described for cytochrome c reduction assays. The concentrations of cytochrome c and MCLA used were 10 and 4 M, respectively. 5-l aliquots of succinatecytochrome c reductase (1.6 mg/ml) were used.  (Table I).

RESULTS AND DISCUSSION
When purified succinate-cytochrome c reductase complex was incorporated into phospholipid (PL) vesicles by the cholate dialysis method (21,22), the resulting protein-PL vesicles showed little succinate-cytochrome c, succinate-Q, and ubiquinol-cytochrome c reductase activities. Whereas addition of the protonophore FCCP to the reductase-PL vesicles enhanced succinate-cytochrome c reductase activity only slightly, it increased ubiquinol-cytochrome c reductase activity by 7-fold. This indicates that the succinate-Q reductase region, but not the cytochrome bc 1 complex, is inactivated in the reductase-PL vesicles. The inactivated component was identified as succinate dehydrogenase because addition of purified, reconstitutively active succinate dehydrogenase to the FCCP-treated reductase-PL vesicles increased antimycin-sensitive succinate-cytochrome c reductase activity to about the same level as that of the starting reductase. Inactivation of succinate dehydrogenase is due to the labile nature of this enzyme in soluble form under aerobic conditions (16). Succinate dehydrogenase was detached from the QPs-cytochrome bc 1 complex upon addition of a mixture of 2% sodium cholate/asolectin during the first step of reductase-PL vesicle preparation. The high detergent concentration, used to ensure complete dispersion of phospholipid during the preparation of reductase-PL vesicles, apparently caused solubilization of succinate dehydrogenase from its anchoring proteins (QPs). Soluble succinate dehydrogenase was inactivated before it could be re-associated with QPs, in the phospholipid bilayer, during the subsequent dialysis step.
Oxidation It has been reported that the production of H 2 O 2 in rat or pigeon heart mitochondria supplemented with antimycin was stimulated up to 13-fold by addition of protonophores (20). The difference in the protonophore sensitivity between the succinate-cytochrome c reductase-phospholipid vesicle system and the mitochondrial system can be explained by the presence of antimycin and endogenous cytochrome c in the latter. In the mitochondrial system, the presence of protonophores causes the disruption of membrane potential, thus stimulating the oxidation of endogenous cytochrome c by cytochrome c oxidase to generate the oxidized cytochrome c needed for the generation of O 2 . from reduced Q. In the case of reconstituted succinate-  cytochrome c reductase vesicles, disruption of membrane potential by addition of protonophores will simply stimulate the activity of succinate-cytochrome c reductase to reduce cytochrome c.
Production . were produced per mg of protein.
Antimycin is known to block transfer of the second electron of ubiquinol from cytochrome b 562 to ubiquinone to form ubisemiquinone at the Q i site, according to the Q cycle mechanism (Fig. 1). Therefore, the reduction of cytochrome c observed with antimycin-inhibited reductase must result from the first electron of ubiquinol being transferred to cytochrome c via the iron-sulfur protein and cytochrome c 1  production. Since cytochrome c acts both as the first electron acceptor and the scavenger of superoxide, the reduction of cytochrome c should be twice as much as the MCLA-O 2 . adduct formation if the measured chemiluminescence could stoichiometrically represent the later. This relation was only observed when a low concentration of MCLA was used (Fig. 2). The quenching of the chemiluminescence of MCLA-O 2 . by high concentrations of MCLA was also observed with O 2 . generated by the xanthine oxidase system (Fig. 3). The amount of chemiluminescence generated by a given amount of xanthine oxidase increased as the MCLA concentration increased, reached a  (Fig. 4)      Complex-By limiting the rate of electron input to the cytochrome bc 1 complex, either by partial inhibition of succinate-Q reductase activity in succinate-cytochrome c reductase preparations by malonate or by the addition of limiting amounts of succinate-cytochrome c reductase to purified cytochrome bc 1 complex, the sequence of reduction of cytochromes b and c 1 can be determined by conventional spectrophotometric methods. When succinate was added to purified cytochrome bc 1 complex in the presence of catalytic amounts of purified succinatecytochrome c reductase, the reduction of cytochrome b occurred at about the same time as the reduction of cytochrome c 1 . When the cytochrome bc 1 complex was treated with antimycin, the reduction of cytochrome b occurred before the reduction of cytochrome c 1. Addition of MCLA to the antimycin-inhibited cytochrome bc 1 complex resulted in cytochrome c 1 being reduced before cytochrome b.  (Table III) or succinate-Q reductase (Table II). At the same concentration used, ferricyanide is more effective than cytochrome c in stimulating O 2 . formation in succinate-Q reductase than in succinate dehydrogenase.