The mitochondrial cytochrome
bc1 complex (ubiquinol/cytochrome
c oxidoreductase) is generally thought to generate superoxide anion that participates in cell signaling and contributes to cellular damage in aging and degenerative disease. However, the isolated, detergent-solubilized
bc1 complex does not generate measurable amounts of superoxide except when inhibited by antimycin. In addition, indirect measurements of superoxide production by cells and isolated mitochondria have not clearly resolved the contribution of the
bc1 complex to the generation of superoxide by mitochondria
in vivo, nor did they establish the effect, if any, of membrane potential on superoxide formation by this enzyme complex. In this study we show that the yeast cytochrome
bc1 complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted
bc1 complex increased exponentially with increased magnitude of the membrane potential, a finding that is compatible with the suggestion that membrane potential inhibits electron transfer from the cytochrome
bL to
bH hemes, thereby promoting the formation of a ubisemiquinone radical that interacts with oxygen to generate superoxide. When the membrane potential was further increased, by the addition of nigericin or by the imposition of a diffusion potential, the rate of generation of superoxide was further accelerated and approached the rate obtained with antimycin. These findings suggest that the
bc1 complex may contribute significantly to superoxide generation by mitochondria
in vivo, and that the rate of superoxide generation can be controlled by modulation of the mitochondrial membrane potential.The mitochondrial oxidative phosphorylation system utilizes the energy derived from the oxidation of metabolic substrates to drive the synthesis of ATP. Electron transport through the NADH dehydrogenase complex, cytochrome
bc1 complex, and cytochrome
c oxidase complex is coupled to proton translocation across the mitochondrial inner membrane, thus generating a protonmotive force (Δp) consisting of a membrane potential (ΔΨ) and a pH gradient (ΔpH) that drives the synthesis of ATP by the ATP synthase (reviewed in Ref.
1).Several of the mitochondrial electron transport complexes produce free radical intermediates that interact with oxygen to generate superoxide (reviewed in Refs.
2,
3). Superoxide is a highly reactive compound that can lead to the formation of other free radicals and reactive compounds and thus damage directly or indirectly cellular proteins, DNA, and phospholipids. It is also believed that free radical damage is a major cause of aging and contributes to many degenerative diseases (reviewed in Ref.
4).Studies with isolated mitochondria have attempted to evaluate the contributions of the different mitochondrial energy-transducing complexes to this process (
5–
9). An early study with isolated rat heart mitochondria suggested that the
bc1 complex produces large amounts of superoxide, but only when the mitochondrial membrane potential is high (
10). This conclusion led to the suggestion that cells modulate the magnitude of the mitochondrial protonmotive force to protect the mitochondria from excess production of superoxide (
11).However, it was shown later that with high concentrations of succinate as a substrate and without rotenone (as in Ref.
10), most of the superoxide is generated by reverse electron transport through complex I (
8). Moreover, the rate of generation of superoxide by reverse electron transport through complex I was shown to be more strongly dependent on ΔpH than on Δψ (
12). It was also suggested that the contribution of the
bc1 complex to superoxide generation by mitochondria is negligible compared with that produced by reverse electron transport through complex I (
9), but it is not clear whether reverse electron transport is a significant process under most physiological conditions.Several groups have measured superoxide production by the detergent-solubilized
bc1 complex isolated from either yeast or beef heart. It was possible to observe superoxide production by the isolated, detergent-solubilized
bc1 complex that was mutated in key residues at the ubiquinol oxidation site (
13). However, the native enzyme did not produce measurable amounts of superoxide except when inhibited by antimycin or other
bc1 complex inhibitors (
14–
18). The mechanism of the antimycin-induced generation of superoxide by the
bc1 complex is fairly well understood within the framework of the Q cycle mechanism, shown in . Following the oxidation of ubiquinol at center P, as electrons recycle through the
b hemes, antimycin inhibits reduction of ubiquinone at center N and electrons back up in center P, resulting in the formation of a ubisemiquinone radical, which can interact with oxygen to form superoxide (
15,
18). It also can be predicted that the membrane potential would inhibit electron transfer from heme
bL to
bH and stimulate the production of superoxide by the
bc1 complex. However, it is not known whether this prediction actually manifests and, if so, how strong is the dependence of superoxide production by the
bc1 complex on the magnitude of membrane potential.
Open in a separate windowMechanistic basis for production of superoxide by the reconstituted cytochrome
bc1 complex. The figure shows the protonmotive Q cycle mechanism and the leak of electrons to oxygen that is presumably the source of superoxide formation by the reconstituted enzyme.
A shows the protonmotive Q cycle mechanism as it normally functions. Ubiquinol (
QH2) is oxidized at center P near the outer surface of the membrane or vesicle in a bifurcated reaction that transfers one electron to the Rieske iron-sulfur protein (
ISP) and one electron to the
bL heme of cytochrome
b. The electron on the iron-sulfur protein is then transferred to cytochrome
c1, and the electron on the
bL heme is transferred to the
bH heme, which then reduces ubiquinone (Q) to semiquinone at center N. When a second molecule of ubiquinol is oxidized, the electron that arrives on the
bH heme reduces semiquinone to ubiquinol.
B shows the formation of superoxide anion that results when electron transfer from the
bL to
bH heme is inhibited, either by an opposing membrane potential or by antimycin, which blocks reoxidation of the
bH heme, causing electrons to accumulate in the
bL heme. Superoxide anion is formed by reaction of oxygen with ubisemiquinone, which is formed either by transfer of one electron from ubiquinol to the iron-sulfur protein or by reduction of ubiquinone by the reduced
bL heme. In both panels
solid arrows indicate electron transfer reactions.
Dashed arrows indicate movement of ubiquinone and ubiquinol between reaction centers in the
bc1 complex, release and uptake of protons at center P and center N, or changes in redox status of ubiquinone, ubiquinol, and oxygen.
Solid bars in
B show the opposition of electron transfer from the
bL to
bH heme by the membrane potential and inhibition of
bH reoxidation by antimycin.We have attempted to resolve this issue by reconstitution of the yeast cytochrome
bc1 complex into phospholipid vesicles, followed by measuring the rate of superoxide generation in parallel with the magnitude of the membrane potential that is generated by the reconstituted enzyme. Our findings indicate that superoxide anion formation by the
bc1 complex
in situ depends strongly on membrane potential and can approach values similar to those promoted by antimycin.
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