Mechanism of reaction of myeloperoxidase with nitrite

Abstract

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) x 104 M-1 s-1 at pH 7 and (2.2 ± 0.7) x 106 M-1 s-1 at 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) x 106 M-1 s-1 at pH 7 and (1.1 ± 0.2) x 107 M-1 s-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) x 102 M-1 s-1 at pH 7 and (8.9 ± 1.6) x 104 M-1 S-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 pK(a) of (4.3 ± 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.