Mechanistic insight into the catalytic inhibition by nitroxides of tyrosine oxidation and nitration.

Background: Nitroxide antioxidants (RNO) protect from injuries associated with oxidative stress. Tyrosine residues in proteins are major targets for oxidizing species giving rise to irreversible cross-linking and protein nitration, but the mechanisms underlying the protective activity of RNO on these processes are not sufficiently clear.

Methods: Tyrosine oxidation by the oxoammonium cation (RN=O) was studied by following the kinetics of RNO formation using EPR spectroscopy. Tyrosine oxidation and nitration were investigated using the peroxidase/HO system without and with nitrite. The inhibitory effect of RNO on these processes was studied by following the kinetics of the evolved O and accumulation of tyrosine oxidation and nitration products.

Results: Tyrosine ion is readily oxidized by RN=O, and the equilibrium constant of this reaction depends on RNO structure and reduction potential. RNO catalytically inhibits tyrosine oxidation and nitration since it scavenges both tyrosyl and NO radicals while recycling through RN=O reduction by HO, tyrosine and nitrite. The inhibitory effect of nitroxide on tyrosine oxidation and nitration increases as its reduction potential decreases where the 6-membered ring nitroxides are better catalysts than the 5-membered ones.

Conclusions: Nitroxides catalytically inhibit tyrosine oxidation and nitration. The proposed reaction mechanism adequately fits the results explaining the dependence of the nitroxide inhibitory effect on its reduction potential and on the concentrations of the reducing species present in the system.

General Significance: Nitroxides protect against both oxidative and nitrative damage. The proposed reaction mechanism further emphasizes the role of the reducing environment to the efficacy of these catalysts.