Reactions of peroxynitrite with mitochondrial reductants.

VALDEZ L, ARNAIZ SL, ALVAREZ S, SCHÖPFER F, PODEROSO JJ, BOVERIS A

Florianópolis, Brasil

Congreso; First Meeting of the South American Group for Free Radical Research; 1999

South American Group for Free Radical Research

Peroxynitrite (ONOO-) is produced through the diffusion-controlled reaction (1.9 x 1010 M-1 s-1) of nitric oxide (NO) and superoxide anion (O2-). At physiological pH, ONOO- protonates to yield peroxynitrous acid (ONOOH; pKa= 6.8), which rearranges itself to nitrate (NO3-) with a half life of about 1 sec in the absence of oxidable substrates. Peroxynitrite is a powerful oxidant that oxidizes the sulphydryl group of cysteine and glutathione, ascorbate, and the purine and pirimidine bases of DNA. Peroxynitrite cal aso cause nitration reactions, such as nitration of tyrosine residues in proteins. 3-Nitrotyrosine has been postulated to be a fingerprint for ONOO- reactions in tissues. The aim of this work was to study oxidation and nitration reactions of ONOO- with mitochondrial compounds. Rate constants for those reactions and the physiological steady-state concentration of ONOO- in the mitochondrial matrix, were also determined. A simple competition kinetic model involving the participation of ONOO-, the target molecule (NADH) and a competitive reductant of ONOO-, was used. The reaction medium consisted of 100 Mm phosphate buffer, 0.1 mM DTPA, pH 7.0, 100 mM NADH and 100-700 mM ONOO-. The decrease of NADH fluorescence was measured at 37°C (lexc-lem: 340-463 nm) after addition of ONOO-, in the absence and presence of reductants (AH2), such as ascorbic acid, ubiquinol-0 (UQ0H2), glutathione (GSH), carbon dioxide (CO2) and uric acid. In order to evaluate protein nitration, liver submitochondrial particles were exposed to 200 mM ONOO- in the absence or presence of above mentioned compounds. 3-Nitrotyrosine formation was evaluated by Western blot analysis. NADH was oxidized immediately by ONOO-; this oxidation was prevented by addition of reductants in different concentrations. The second oder rate constants (in M-1 s-1) for the reaction of ONOO- with AH2 were: 233±27 for NADH, 485±54 for UQ0H2, 183±12 for GSH, 207±18 for uric acid. Taking into account that ONOO- reacts with CO2 (5.8 x 104 M-1 s-1) leading to the formation of the nitrosoperoxocarboxylate adduct (ONOOCO2-), NADH oxidation by ONOO- was studied in the presence of 2.5 mM CO2, using the same methodology. A mitochondrial ONOO- steady-stateconcentration of 2 nM was estimated taking into account rate constants and intramitochondrial concentrations of the scavengers. Nitration of proteins was observed in liver submitochondrial particles in the presence of ONOO-. The addition of CO2 produced the same nitration effect than ONOO- alone. Other reductants minimized tyrosine nitration induced by ONOO- in the sequence: ascorbic acid> UQ0H2> uric acid> NADH @ GSH. Both UQ0H2 and GSH inhibited tyrosine nitration by ONOO- in a concentration-dependent manner. This study addresses the intramitochondrial pathways of ONOO- utilization and provides the rate constants for the reactions of ONOO- with NADH, ubiquinol (UQ0H2) and GSH. It also provides evidence of a significant nitration of mitochondrial proteins by ONOO- which is prevented by tested reductants. The time course of those actions have to be re-examined now in terms of the physiological concentration of ONOO- in the mitochondrial matrix.