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 c larify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediate s, compound I and compound II, with nitrite under pre-steady state conditio ns by using sequential mixing stopped-flow analysis in the pH range 4-8, At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) M-1 s(-1) at pH: 7 and (2.2 +/- 0.7) x 10(6) 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 mu M at pH 5, Nitrite is oxidize d by two one-electron steps in the MPO peroxidase cycle. The second-order r ate constant of reduction of compound I to compound II at 15 degrees C is ( 2.0 +/- 0.2) x 10(6) M-1 s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) M-1 s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) M-1 s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) M-1 s(-1) at pH 5. pH dependence studies suggest that bot h complex formation between the ferric enzyme and nitrite and nitrite oxida tion 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 histi dine) is necessary for optimum nitrite binding and oxidation.