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, O₂ evolution and nitrite oxidation. The results demonstrate that the peroxidase-like activity is accompanied by a progressive heme inactivation where MbFe^III is far more resistant than HbFe^III. 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 µM concentrations range catalytically inhibits nitrite oxidation, and consequently prevents tyrosine nitration induced by heme protein/H₂O₂/nitrite due to its fast oxidation by ^•NO₂ 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 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 MbFe^III where most of the rate constants of the reactions involved are independently known.