Nitroxides catalytically inhibit nitrite oxidation and heme inactivation induced by H2O2, nitrite and metmyoglobin or methemoglobin

Stable nitroxide radicals have multiple biological effects, although the mechanisms underlying them are not fully understood. Their protective effect against oxidative damage has been mainly attributed to scavenging deleterious radicals, oxidizing reduced metal ions and reducing oxyferryl centers of heme proteins. Yet, the potential of nitroxides to protect heme proteins against inactivation while suppressing or enhancing their catalytic activities has been largely overlooked. We have studied the effect of nitroxides, including TPO (2,2,6,6-tetramethylpiperidin-N-oxyl), 4-OH-TPO, 4-oxo-TPO and 3-carbamoyl proxyl, on the peroxidase-like activity of metmyoglobin (MbFe(III)) and methemoglobin (HbFe(III)) using nitrite as an electron donor by following heme absorption, H2O2 consumption, O-2 evolution and nitrite oxidation. The results demonstrate that the peroxidase-like activity is accompanied by a progressive heme inactivation where MbFeIII is far more resistant than HbFeIII. Nitroxides convert the peroxidase-like activity into catalase-like activity while inhibiting heme inactivation and nitrite oxidation in a dose-dependent manner. The nitroxide facilitates H2O2 dismutation, yet none of its reactions with any of the intermediates formed in these systems is rate-determining, and therefore its effect on the rate of the catalysis is hardly dependent on the kind of the nitroxide derivative and its concentration. The nitroxide at mu M concentrations range catalytically inhibits nitrite oxidation, and consequently prevents tyrosine nitration induced by heme protein/H2O2/ nitrite due to its fast oxidation by (NO2)-N-center dot forming the respective oxoammonium cation, which is reduced back to the nitroxide by H2O2 and by superoxide radical. The nitroxides are superior over common antioxidants, which their reaction with (NO2)-N-center dot always yields secondary radicals leading eventually to consumption of the antioxidant. A mechanism is proposed, and the kinetic simulations fit very well the experimental data in the case of MbFeIII where most of the rate constants of the reactions involved are independently known.