Inhibitors Around the Antimycin-Sensitive Site in the Respiratory Chain

1. Mitochondria isolated from Polytomella caeca contain cytochromes b, c+c1 and a+a3 and several flavoprotein species. 2. Electron transport is inhibited by antimycin A, rotenone, piericidin A and cyanide. 3. Spectral data indicate that antimycin A inhibits the reoxidation of reduced cytochrome b. 4. Various types of flavoprotein are characterized by simultaneous spectrophotometric and fluorimetric measurements on antimycin A-inhibited preparations and also by their absorption and fluorescence-emission spectra. 5. The rotenone-sensitive site lies between the two flavoproteins of the respiratory chain, designated FpD1 and FpD2. 6. Other flavoprotein species detected include those involved in the oxidation of succinate and externally added NADH; a large proportion of mitochondrial flavine is reduced by dithionite but not by known respiratory substrates. 7. The kinetics of flavoprotein and cytochrome reactions were studied.

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NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH–ubiquinone reductase preparation

Biochemical Journal ◽

10.1042/bj1800129 ◽

1979 ◽

Vol 180 (1) ◽

pp. 129-135 ◽

Cited By ~ 221

Author(s):

Koichiro Takeshige ◽

Shigeki Minakami

Keyword(s):

Respiratory Chain ◽

Complex I ◽

Inorganic Salts ◽

Superoxide Anions ◽

Submitochondrial Particles ◽

Bovine Heart ◽

Sensitive Site ◽

Effects Of Ph ◽

High Concentrations ◽

O2 Production

1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O2−) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O2− by NADH–ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O2− production by the mitochondrial inner membranes.

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