Reactions of peroxynitrite in the mitochondrial matrix 1 1 Paper dedicated to the memory of Prof. Lars Ernster, convener of ICRO-UNESCO in a series of ICRO courses in Buenos Aires (original) (raw)

Superoxide radical (O 2 Ϫ ) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO Ϫ ) in the mitochondrial matrix. Intramitochondrial ONOO Ϫ effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M Ϫ1 s Ϫ1 ) of ONOO Ϫ with NADH (233 Ϯ 27), ubiquinol-0 (485 Ϯ 54) and GSH (183 Ϯ 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO Ϫ was also followed in the presence of CO 2 , to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO 2 Ϫ ) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO 2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 M ONOO Ϫ showed a O 2 Ϫ production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 M ONOO Ϫ was observed by Western blot analysis. Protein nitration was prevented by the addition of 50 -200 M ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO Ϫ was calculated, taking into account the rate constants and concentrations of ONOO Ϫ coreactants.