NADH oxidation by peroxynitrite. I. Effects of reductant scavengers

VALDEZ L; ALVAREZ S; LORES ARNAIZ S; SCHÖPFER F; PODEROSO JJ; BOVERIS A

Santa Bárbara, EEUU

Congreso; World Congress “Oxidant and antioxidants in Biology”.; 1999

Oxygen Club of California

Nitric oxide (NO) and superoxide anion (O2.­) react to produce peroxynitrite (ONOO-) in a diffusion-controlled reaction with a rate constant of 0.67-2.0 x 1010 M-1s-1. The oxidation of NADH by ONOO- was followed fluorometrically to determine the second order rate constant by simple competition with ascorbate (569 M-1s-1). Other reductants, components of the mitochondrial matrix or related substances such as ubiquinol-0, glutathione, uric acid, melatonin, and carbon dioxide were also assayed as competitors for ONOO- scavenging.The reaction of ONOO- with NADH was followed at 37°C, using 340-463 nm as excitation and emission wavelengths, respectively. The reaction medium consisted of 50 mM phosphate buffer, 0.1 mM DTPA, pH 7.0, 100 mM NADH and 100-700 mM ONOO-. The second order rate constants (in M-1s-1) for the reaction of ONOO- with reductant scavengers were 233 ± 27 with NADH, 485 ± 54 with ubiquinol-0, 183 ± 12 with glutathione, 207 ± 18 with uric acid, 43 ± 3 with melatonin, and 52 ± 3 with carbon dioxide. A mitochondrial ONOO- steady-state concentration of 27 nM was calculated taking into account rate constants and intramitochondrial concentrations of the scavengers. Peroxynitrite is a powerful and versatile univalent oxidant (E (ONOO-­/·NO2) = +1.4 V) but a few biomolecules in the mitochondrial matrix are expected to be targets, due to kinetics conditions.