stigmatellin and malic-acid

Succinate-driven oxidation via complex II (CII) may have a significant contribution towards the high rates of production of reactive oxygen species (ROS) by mitochondria. Here, we show that the CII Q site inhibitor thenoyltrifluoroacetone (TTFA) blocks succinate + rotenone-driven ROS production, whereas the complex III (CIII) Qo inhibitor stigmatellin has no effect, indicating that CII, not CIII, is the ROS-producing site. The complex I (CI) inhibitor rotenone partially reduces the ROS production driven by high succinate levels (5 mm), which is commonly interpreted as being due to inhibition of a reverse electron flow from CII to CI. However, experimental evidence presented here contradicts the model of reverse electron flow. First, ROS levels produced using succinate + rotenone were significantly higher than those produced using glutamate + malate + rotenone. Second, in tumor mitochondria, succinate-driven ROS production was significantly increased (not decreased) by rotenone. Third, in liver mitochondria, rotenone had no effects on succinate-driven ROS production. Fourth, using isolated heart or hepatoma (AS-30D) mitochondria, the CII Qp anti-cancer drug mitochondrially targeted vitamin E succinate (MitoVES) induced elevated ROS production in the presence of low levels of succinate(0.5 mm), but rotenone had no effect. Using sub-mitochondrial particles, the Cu-based anti-cancer drug Casiopeina II-gly enhanced succinate-driven ROS production. Thus, the present results are inconsistent with and question the interpretation of reverse electron flow from CII to CI and the rotenone effect on ROS production supported by succinate oxidation. Instead, a thermodynamically more favorable explanation is that, in the absence of CIII or complex IV (CIV) inhibitors (which, when added, facilitate reverse electron flow by inducing accumulation of ubiquinol, the CI product), the CII redox centers are the major source of succinate-driven ROS production.

Topics: Animals; Cattle; Cell Line, Tumor; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Glutamic Acid; Hydrogen Peroxide; Malates; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Polyenes; Rats; Reactive Oxygen Species; Rotenone; Succinic Acid; Thenoyltrifluoroacetone

Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.

Topics: Antifungal Agents; Antimycin A; Electron Transport Complex III; Hydrogen Peroxide; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Polyenes; Rotenone; Saccharomyces cerevisiae; Succinic Acid; Superoxides; Thiazoles

The rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Biphenyl Compounds; Fumarates; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Onium Compounds; Polyenes; Proline; Rotenone; Substrate Specificity; Succinic Acid; Superoxides; Thiazoles; Trypanosoma brucei brucei; Ubiquinone; Uncoupling Agents