Mechanism of HRP-catalyzed nitrite oxidation by H₂O₂ revisited: Effect of nitroxides on enzyme inactivation and its catalytic activity

The peroxidative activity of horseradish peroxidase (HRP) undergoes progressive inactivation while catalyzing the oxidation of nitrite by H₂O₂. The extent of inactivation increases as the pH increases, [nitrite] decreases or [H₂O₂] increases, and is accompanied by a loss of the Soret peak of HRP along with yellow-greenish coloration of the solution. HRP-catalyzed nitrite oxidation by H₂O₂ involves not only the formation of compounds I and II as transient heme species, but also compound III, all of which in turn, oxidize nitrite yielding ^•NO₂. The rate constant of nitrite oxidation by compound III is at least 10-fold higher than that by compound II, which is also reducible by ^•NO₂ where its reduction by nitrite is the rate-determining step of the catalytic cycle. The extent of the loss of the Soret peak of HRP is lower than the loss of its peroxidative activity implying that deterioration of the heme moiety leading to iron release only partially contributes toward heme inactivation. Cyclic stable nitroxide radicals, such as 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO), 4-OH-TPO and 4-NH₂-TPO at µM concentrations detoxify ^•NO₂ thus protecting HRP against inactivation mediated by this radical. Hence, HRP inactivation proceeds via nitration of the porphyrin ring most probably through compound I reaction with ^•NO₂, which partially leads to deterioration of the heme moiety. The nitroxide acts catalytically since its oxidation by ^•NO₂ yields the respective oxoammonium cation, which is readily reduced back to the nitroxide by H₂O₂, superoxide ion radical, and nitrite. In addition, the nitroxide catalytically inhibits tyrosine nitration mediated by HRP/H₂O₂/nitrite reactions system as it efficiently competes with tyrosyl radical for ^•NO₂. The inhibition by nitroxides of tyrosine nitration is demonstrated also in the case of microperoxidase (MP-11) and cytochrome c revealing an additional role played by nitroxide antioxidants.