Mechanism of Reaction of Myeloperoxidase with Nitrite

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4–8. At 15 °C the rate of formation of the low spin MPO-nitrite complex is (2.5 ± 0.2) × 104m−1 s−1at pH 7 and (2.2 ± 0.7) × 106m−1 s−1at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 ± 0.1 mm at pH 7 and 31.3 ± 0.5 μm at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 °C is (2.0 ± 0.2) × 106m−1s−1 at pH 7 and (1.1 ± 0.2) × 107m−1s−1 at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 °C is (5.5 ± 0.1) × 102m−1s−1 at pH 7 and (8.9 ± 1.6) × 104m−1s−1 at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pKa of (4.3 ± 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.