Membrane Potential Greatly Enhances Superoxide Generation by the Cytochrome bc1 Complex Reconstituted into Phospholipid Vesicles

Abstract

The mitochondrial cytochrome bc 1 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 bc 1 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 bc 1 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 bc 1 complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted bc 1 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 b L to b H 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 bc 1 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 bc 1 complex, and cytochrome c oxidase complex is coupled to proton translocation across the mitochondrial inner membrane, thus generating a protonmotive force (⌬p) consisting of a membrane potential (⌬⌿) and a pH gradient (⌬pH) that drives the synthesis of ATP by the ATP synthase (reviewed in Ref.

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…Both reaction schemes require as an initiation a reduced state of heme b L , which is consistent with the observation that ROS are observed when the electron transfer between heme b L and heme b H is impeded by blocking the Q i site with antimycin or by increase in membrane potential (144,218). In the semiforward scheme, reduced heme b L is unable to take electron from USQ o and thus cannot convert it to UQ (103,145,189,209).…”

Hypoxic Pulmonary Vasoconstriction

“…On the basis of these and other data, it was proposed that under hypoxic conditions the interaction of UQCRB with cytochrome b could prolong QH · lifetime at the Qo site of complex III, thereby enhancing generation and release of ROS into the intermembrane space and cytosol (FIGURE 5B). Since terpestacin also caused mitochondrial depolarization (916) and ROS production by complex III was increased by hyperpolarization (1665), it was further speculated that hypoxic prolongation of QH · lifetime at Qo was due to decreased electron transfer from cytochrome b L to b H , perhaps as a result of mitochondrial hyperpolarization (916) (FIGURE 5B). Consistent with this possibility, two laboratories have reported that hypoxia caused mitochondrial hyperpolarization in PASMC (1278,1814); however, another found that hypoxia had no effect on ⌬⌿ M (2032).…”

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

“…Therefore, we propose that during hypoxia, terpestacin bound to UQCRB of Complex III may mimic a condition where partially oxidized ubiquinone pool slows down the forward electron transfer to induce a reverse electron transfer onto O 2 by reduced heme b L , resulting in the prolonged lifetime of SQ at the Q o pocket, thereby enhancing the probability of superoxide production as was the case with antimycin-induced ROS production (23,30). In addition, recent evidence has revealed that the membrane potential enhances the formation of superoxide from Complex III by opposing electron transfer from heme b L to b H (33). Indeed, the binding of terpestacin to UQCRB decreases the mitochondrial membrane potential as shown in Fig.…”