Inhibition of complex III promotes loss of Ca²⁺ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite

In intact U937 cells, peroxynitrite promotes the mitochondrial formation of superoxide via a Ca²⁺-dependent mechanism involving inhibition of complex III. Superoxide then readily dismutates to H₂O₂ causing lesions on different biomolecules, including DNA. Here we show that formation of H₂O₂ and DNA damage are suppressed by inhibition of complex I (by rotenone) or ubisemiquinone formation (by myxothiazol), as well as by a variety of manipulations preventing either the mobilization of Ca²⁺ or its mitochondrial accumulation. In addition, complex III inhibitors promoted rotenone- or myxothiazol-sensitive formation of H₂O₂ and DNA strand scission in cells exposed to otherwise inactive concentrations of peroxynitrite. However, under these conditions, the intra-mitochondrial concentration of Ca²⁺ remained unchanged and the effects of peroxynitrite therefore take place via Ca²⁺-independent mechanisms. H₂O₂ formation was paralleled by, and causally linked to, the loss of mitochondrial membrane potential associated with the mitochondrial release of cytochrome c and AIF, and with the mitochondrial accumulation of Bax. These events, although Ca²⁺ independent, were rapidly followed by death mediated by mitochondrial permeability transition, generally considered a typical Ca²⁺-dependent event. Thus, enforced inhibition of complex III promotes the loss of Ca²⁺ dependence of those mitochondrial mechanisms regulating superoxide formation and mitochondrial permeability transition evoked by peroxynitrite.