Alterations to glutathione and nicotinamide nucleotides during the mitochondrial permeability transition induced by peroxynitrite

Peroxynitrite is a biologically important oxidant that damages mitochondria in a number of ways. We investigated the interaction of peroxynitrite with the mitochondrial glutathione pool by measuring the formation of oxidised glutathione and glutathione-protein mixed disulfides in mitochondria exposed to either peroxynitrite or tert-butylhydroperoxide. In contrast to tert-butylhydroperoxide, peroxynitrite converts 40-50% of mitochondrial glutathione to products other than disulfides, primarily higher oxidation states of sulfur. These data show that peroxynitrite interacts with mitochondria quite differently from oxidants commonly used in studying mitochondrial oxidative stress. Peroxynitrite also induces a permeability transition in the mitochondrial inner membrane, and here we show that this permeability transition is prevented by the NAD(P)H-linked substrates glutamate and malate and by the thiol reagent dithiothreitol. Glutamate and malate prevented complete oxidation of the NAD(P)H pool by peroxynitrite or tert-butylhydroperoxide but did not prevent oxidation of the mitochondrial glutathione pool or the formation of glutathione-protein mixed disulfides. This study is consistent with regulation of the permeability transition by critical protein thiol groups, whose redox state responds to that of the mitochondrial NAD(P)H pool, but which do not equilibrate directly with the mitochondrial glutathione pool.