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

Abstract

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 (O(2) (-)) 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 O(2) (-) 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 O(2) (-) production by the mitochondrial inner membranes.

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate

“…Oxygen radical generation by the respiratory chain has been classically attributed to complex III semiquinone (Boveris and Cadenas 2000). However, in agreement with early information obtained using submitochondrial particles (Takeshige and Minakami 1979), complex I also contains an important ROS generator in the form of intact, functional and well-coupled heart and brain mitochondria (Herrero and Barja 1997;Barja and Herrero 1998;Barja 2004a). Furthermore, the respiratory complex responsible for the lower mitochondrial ROS generation by tissues in a long-lived species (the pigeon: MLSP = 35 years) in relation to that of a short-lived one (the rat: MLSP = 4 years) is complex I, not complex III.…”

A mitochondrial DNA clone is associated with increased risk for Alzheimer disease.

“…It was proposed that since most of the mitochondrial genome encodes complex I subunits, defects in mitochondrial protein synthesis are more likely to impair complex I (NADH dehydrogenase) than any other mitochondrial gene product (33). Drugs that inhibit complex I induce production of mitochondrial superoxide and induce cell death (34)(35)(36)(37)(38)(39). The brain toxicity of a complex I inhibitor is suppressed by MnSOD, the mitochondrial superoxide dismutase (33), supporting the notion that mitochondrial superoxide could be a significant neurotoxin, and we suggest that excess superoxide generation may be an important outcome of the 4336G mutation.…”

Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology

“…Under aerobic conditions, the respiratory chain is a potent source of free radical (33,37,38). A CI impairment leads to enhanced formation of ROS, as has been shown in submitochondrial particles (16,18) and in different cell systems (39,40). In the case of HXC, defects in CI may predispose the respiratory chain to produce excess superoxide because of structural or stoichiometric alterations in the subunits.…”