Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

Abstract

The deleterious effects of H202 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by beta-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to Qo site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Qo) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.

Figure 1

Effect of H₂O₂ on the rate of O₂ uptake in state 4 (A), state 3 (B) or uncoupled state (C). Mitochondria were incubated during 30 min at 4°C in respiration medium (0.6 m sucrose, 2 mm EGTA, 4 mm KH₂PO₄, 10 mm Tris-maleate), with indicated amounts of H₂O₂. After pre-incubation, oxygen uptake was measured as described in Materials and methods. Data are presented as mean±SE of >3 independent experiments. Signifintly different when compared to control (0 mm H₂O₂) (*p<0.05, **p<0.01).

Figure 2

Influence of H₂O₂ decomposition on oxygen consumption determinations. Respiration medium (A) or respiration medium plus 0.9 mg mitochondria (B) were incubated during 30 min at 4°C with 1.0 mm H₂O₂. Then, oxygen production was evaluated with a Clark-type oxygen electrode as described in Materials and methods. (A). Experiment was repeated at least three times. Data are from a representative experiment.

Figure 3

Effect of H₂O₂ on partial reactions of ETC. Triton-solubilized mitochondria were incubated during 30 min at 4°C in 50 mm KH₂PO₄ buffer with indicated concentrations of H₂O₂ under conditions described in Materials and methods. Succinate-DCIP oxidoreductase (A), succinate-cytochrome c oxidoreductase (B), cytochrome oxidase (C) or cytochrome c oxidase (D) activities were measured as described in Materials and methods. Results are expressed as the mean±SE of >3 independent experiments. Significantly different when compared to control (0 mm H₂O₂) (*p<0.05, **p<0.01).

Figure 4

Effect of β-mercaptoethanol on the impairment induced by H₂O₂ on partial reactions of ETC. Mitochondria were incubated after H₂O₂ treatment with indicated concentrations of β-mercaptoethanol during 30 min at 4°C on 50 mm KH₂PO₄ buffer. Later, H₂O₂ and β-mercaptoethanol were removed twice by centrifugation and mitochondrial pellet re-suspended on fresh 50 mm KH₂PO₄ buffer. Concentrations of H₂O₂ used were: 1.0 mm H₂O₂ for succinate-DCIP oxidoreductase activity (A) and 0.5 mm H₂O₂ for succinate-cytochrome c oxidoreductase activity (B). Activities were measured as described in Materials and methods. Values from mitochondria incubated with 2.0 mmβ-mercaptoethanol (inset, panel A) or 1.0 mmβ-mercaptoethanol (inset, panel B) without H₂O₂ addition were taken as 100%. Results are expressed as the mean±SE of >3 independent experiments. Significantly different when compared to control (0 mm H₂O₂) (*p<0.05, **p<0.01).

Figure 5

Effect of mannitol and EDTA on the impairment induced by H₂O₂ on partial reactions of ETC. Triton-solubilized mitochondria were incubated during 15 min on 50 mm KH₂PO₄ buffer at 4°C with 10 mm mannitol or 50 μm EDTA previous to treatment during 30 min with 1.0 mm H₂O₂ for succinate-DCIP oxidoreductase activity (A) and 0.5 mm H₂O₂ for succinate-cytochrome c oxidoreductase activity (B). Activities were measured as described in Materials and methods. Values from incubated controls (mitochondria incubated during 45 min without EDTA, mannitol or H₂O₂ addition, data not shown) were taken as 100% (dotted line). Results are expressed as the mean±SE of >3 independent experiments. Significantly different when compared to 10 mm mannitol (*p<0.05, **p<0.01) and 50 μm EDTA (⁺p<0.05).

Figure 6

Effect of H₂O₂ on cytochromes levels of yeast mitochondria using dithionite as electron donor. Mitochondria were pre-incubated during 30 min on 50 mm KH₂PO₄ buffer at 4°C in the absence (solid line) or the presence of 0.5 mm H₂O₂ (dotted line) Later, H₂O₂ was removed twice by centrifugation and mitochondrial pellet re-suspended on fresh 50 mm KH₂PO₄ buffer. A small amount of dithionite was used as electron donor. Spectra recording was performed as described in Materials and methods. Experiments were repeated at least three times. Data are from a representative experiment.

Figure 7

Effect of H₂O₂ on difference absorption spectra of cytochromes from yeast mitochondria in the presence of bc₁ complex inhibitors. Mitochondria were pre-incubated during 30 min on 50 mm KH₂PO₄ buffer at 4°C in the absence or the presence of 0.5 mm H₂O₂. Later, H₂O₂ was removed twice by centrifugation and mitochondrial pellet re-suspended on fresh 50 mm KH₂PO₄ buffer. Inhibitors were added 5 min before spectra recording and 10 mm succinate was used as electron donor. Spectra were performed as described in Materials and methods. (A) Spectra in absence (solid line) or the presence (dotted lines) of antimycin A, (B) spectra in the presence of myxothiazol in control (solid line) and treated (dotted line) mitochondria, (C) spectra in the presence of stigmatellin in control (solid line) and treated (dotted line) mitochondria. Experiments were repeated at least three times. Data are from a representative experiment.

Figure 8

Effect of H₂O₂ on lipid peroxidation levels in yeast and rat mitochondria. Mitochondria were pre-incubated during 30 min on 50 mm KH₂PO₄ buffer at 4°C in the absence (black bars) or the presence (grey bars) of 0.5 mm H₂O₂. Measurements were made as described in Materials and methods. Data are presented as mean±SE of three independent experiments. Significantly different when compared to yeast mitochondria treated with H₂O₂ (*p<0.05, **p<0.01).