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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.

SUBMITTER: Rottenberg H

PROVIDER: S-EPMC2740544 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Membrane potential greatly enhances superoxide generation by the cytochrome bc1 complex reconstituted into phospholipid vesicles.

Rottenberg Hagai H Covian Raul R Trumpower Bernard L BL

The Journal of biological chemistry 20090528 29

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 res ...[more]

PMID: 19478336

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Project description:One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron-sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c(1) of cytochrome c(1). Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations - G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking "semireverse" electron transfer from heme bL to quinone. Here we examine another mutation from that group - G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that "pushing" ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the "pushing" effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

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Membrane potential greatly enhances superoxide generation by the cytochrome bc1 complex reconstituted into phospholipid vesicles.

Publications

Membrane potential greatly enhances superoxide generation by the cytochrome bc1 complex reconstituted into phospholipid vesicles.

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