Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2−) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO−) in the mitochondrial matrix. Intramitochondrial ONOO− effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M−1 s−1) of ONOO− with NADH (233 ± 27), ubiquinol-0 (485 ± 54) and GSH (183 ± 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO− was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2−) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25–2 μM ONOO− showed a O2− production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 μM ONOO− was observed by Western blot analysis. Protein nitration was prevented by the addition of 50–200 μM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO− was calculated, taking into account the rate constants and concentrations of ONOO− coreactants.