With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-␣. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-␣. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.

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Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.