Kinetics and mechanism of ^•NO₂ reacting with various oxidation states of myoglobin

Nitrogen dioxide (^•NO₂) participates in a variety of biological reactions. Of great interest are the reactions of ^•NO₂ with oxymyoglobin and oxyhemoglobin, which are the predominant hemeproteins in biological systems. Although these reactions occur rapidly during the nitrite-catalyzed autoxidation of hemeproteins, their roles in systems producing ^•NO₂ in the presence of these hemeproteins have been greatly underestimated. In the present study, we employed pulse radiolysis to study directly the kinetics and mechanism of the reaction of oxymyoglobin (MbFe^IIO₂) with ^•NO₂. The rate constant of this reaction was determined to be (4.5 ± 0.3) × 10⁷ M^-1s ^-1, and is among the highest rate constants measured for ^•NO₂ with any biomolecule at pH 7.4. The interconversion among the various oxidation states of myoglobin that is prompted by nitrogen oxide species is remarkable. The reaction of MbFe ^IIO₂ with ^•NO₂ forms MbFe ^IIIOONO₂, which undergoes rapid heterolysis along the O-O bond to yield MbFe^V=O and NO₃^-. The perferryl-myoglobin (MbFe^V=O) transforms rapidly into the ferryl species that has a radical site on the globin (^•MbFe ^IV=O). The latter oxidizes another oxymyoglobin (10⁴ M^-1s^-1 < k₁₇ < 10⁷ M ^-1s^-1) and generates equal amounts of ferrylmyoglobin and metmyoglobin. At much longer times, the ferrylmyoglobin disappears through a relatively slow comproportionation with oxymyoglobin (k₁₈ = 21.3 ± 5.3 M^-1s^-1). Eventually, each ^•NO₂ radical converts three oxymyoglobin molecules into metmyoglobin. The same intermediate, namely MbFe^IIIOONO ₂, is also formed via the reaction peroxynitrate (O ₂NOO^-/O₂NOOH) with metmyoglobin (k₁₉ = (4.6 ± 0.3) × 10⁴M^-1s^-1). The reaction of ^•NO₂ with ferrylmyoglobin (k₂₀ = (1.2 ± 0.2) × 10⁷ M^-1s^-1) yields MbFe^IIIONO₂, which in turn dissociates (k ₁₂ = 190 ±20 s^-1) into metmyoglobin and NO ₃^-. This rate constant was found to be the same as that measured for the decay of the intermediate formed in the reaction of MbFe ^IIO₂ with ^•NO, which suggests that MbFe^IIIONO₂ is the intermediate observed in both processes. This conclusion is supported by thermokinetic arguments. The present results suggest that hemeproteins may detoxify ^•NO₂ and thus preempt deleterious processes, such as nitration of proteins. Such a possibility is substantiated by the observation that the reactions of ^•NO₂ with the various oxidation states of myoglobin lead to the formation of metmyoglobin, which, though not functional in the gas transport, is nevertheless nontoxic at physiological pH.