Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems.
Cytochrome c3+ has been extensively used for the detection of superoxide produced in biological systems due to its fast superoxide-mediated reduction to cytochrome c2+. However, another biomolecule which is sometimes cogenerated with superoxide, nitric oxide, reacts with superoxide at almost diffusion-controlled rates (6.7 x 10(9) M-1 s-1), leading to the production of a highly oxidizing species, peroxynitrite anion (ONOO-). In this work we report that peroxynitrite readily oxidizes cytochrome c2+ to cytochrome c3+ in an ascorbate-reversible manner. The reaction between peroxynitrite and cytochrome c2+ occurs with a second-order rate constant of 2.3 x 10(5) M-1 s-1. The pH dependence of the apparent second-order rate constants as well as the effect of different scavengers indicated that peroxynitrous acid (ONOOH) in the ground state was the actual species responsible of cytochrome c2+ oxidation. The activation enthalpy, free energy, and entropy were +10.8 kcal mol-1, +11.8 kcal mol-1, and -3.15 cal mol-1 K-1, respectively, in agreement with the proposed reaction mechanism. Additionally, our results imply that when quantitating superoxide by the cytochrome c3+ reduction method, the existence of a simultaneous generation of nitric oxide and peroxynitrite may lead to an underestimation of the rates of superoxide production.
Thomson
L
L
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Trujillo
M
M
Telleri
R
R
Radi
R
R
D003574:Cytochrome c Group / Q000378:metabolism*; D005609:Free Radicals; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism*
D003574:Cytochrome c Group; D005609:Free Radicals; D013481:Superoxides; D009569:Nitric Oxide