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 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM 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.
Valdez
L B
LB
Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University Hospital, School of Medicine, University of Buenos Aires, Argentina. lbvaldez@ffyb.uba.ar
Alvarez
S
S
Arnaiz
S L
SL
Schöpfer
F
F
Carreras
M C
MC
Poderoso
J J
JJ
Boveris
A
A
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D015153:Blotting, Western; D002245:Carbon Dioxide / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D009566:Nitrates; D009243:NAD; D013481:Superoxides; D014451:Ubiquinone; D002245:Carbon Dioxide; C039642:peroxynitric acid; D014443:Tyrosine; D005978:Glutathione; C003741:ubiquinol; D001205:Ascorbic Acid