External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide

Abstract

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 bc₁ 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.

Introduction

Recent experimental evidence has indicated that the mitochondrial electron transport chain is an important source of reactive oxygen species (ROS), including the superoxide radical (O₂^•−), hydrogen peroxide (H₂O₂), and the hydroxyl radical (^•OH) 1, 2, 3. Mitochondria are present in high density in almost all cells where they consume more than 90% of the total oxygen used by the cell. One-electron carriers of the respiratory chain are able to donate one electron to molecular oxygen with the resultant formation of superoxide. The superoxide, thus formed, is dismutated to H₂O₂ either catalyzed by superoxide dismutase (SOD) or spontaneously by the following reaction:

$$\text{2O}{}_2{}^{\mathrm{•-}}\text{+ 2H}{}^{\mathrm{+}}\text{→ H}{}_2\text{O}{}_2\text{+ O}{}_2$$

The hydrogen peroxide thus produced can be converted to the highly toxic hydroxyl radical in the presence of Fe²⁺ ions by the Fenton reaction. In addition, the superoxide can react with nitric oxide to form peroxynitrite [4]. These ROS, in particular the hydroxyl radical and peroxynitrite, may cause extensive damage to DNA, protein, and lipid 5, 6, 7, 8. In addition, these radicals may be involved in signal activities of physiological relevance 9, 10.

The cytochrome bc₁ complex has been identified as a major site of superoxide generation in mitochondria 1, 2, 3. According to the widely accepted Q cycle mechanism to describe electron transfer through the bc₁ complex (Fig. 1), two ubiquinone-binding sites are present in the bc₁ complex [11]. The ubiquinol-oxidizing site, Q_O, is located on the P side of the membrane and the ubiquinone-reducing site, Q_i, is located on the N side of the membrane. Antimycin A binds to the high potential b heme (b_H) at the Q_i site, thus blocking electron transfer at center Q_i. Consequently, the ubisemiquinone anion will accumulate at the Q_O site (Fig. 1). This unstable semiquinone may then transfer one electron to molecular oxygen to form superoxide 1, 2, 3. By contrast, myxothiazol binds to the low potential b heme (b_L), thus blocking the oxidation of ubiquinol at the Q_O site, and consequently no ubisemiquinone will be formed at this site and no transfer of electrons to molecular oxygen will occur 1, 2, 3, 11, 12.

A second site of superoxide production in the mitochondrial electron transport chain is complex I, the rotenone-sensitive NADH:ubiquinone oxidoreductase 1, 2, 3, 13, 14, 15. Complex I contains FMN and several iron-sulfur centers that transfer electrons to ubiquinone. The addition of specific inhibitors of complex I such as rotenone have been reported to stimulate the production of superoxide in mammalian mitochondria 1, 2, 15.

In addition to complex I, the presence of rotenone-insensitive NADH dehydrogenases (also called alternative or type II NADH dehydrogenases) has been reported in the mitochondria of plants, yeast, and fungi, as well as in the cytoplasmic membranes of bacteria 16, 17, 18, 19, 20, 21, 22. These alternative NADH dehydrogenases provide an additional pathway for the transfer of reducing equivalents from NADH to the electron transport chain without the concomitant pumping of protons across the membrane. The mitochondria of the yeast Saccharomyces cerevisiae have been shown to contain three alternative NADH dehydrogenases 18, 19. One enzyme, the internal alternative NADH dehydrogenase, is located on the interior face of the inner membrane facing the matrix, while the two external alternative NADH dehydrogenases are located on the exterior face of the inner mitochondria membrane facing the cytoplasm. The internal NADH dehydrogenase of S. cerevisiae has been suggested to play a role in regulating the redox balance at the level of mitochondrial NADH produced by the citric acid cycle [23] in addition to oxidizing NADH produced by ethanol oxidation. The external NADH dehydrogenases of S. cerevisiae function in the re-oxidation of the cytosolic NADH produced by glycolysis 24, 25. Interestingly, the external NADH dehydrogenase present in the obligate aerobic yeast Yarrowia lipolytica, is not required for cell growth [20].

The function of these alternative NADH dehydrogenases in cell metabolism has not yet been fully elucidated. The yeast S. cerevisiae provides an excellent organism for the investigations of the role of alternative NADH dehydrogenases in the cell, as this yeast does not contain complex I. In the current study, we report that in addition to the cytochrome bc₁ complex, the external alternative NADH dehydrogenase(s) present in S. cerevisiae mitochondria is (are) also a potential site of superoxide radical production.