Interaction of myeloperoxidase with peroxynitrite

<jats:p>Polymorphonuclear neutrophils generate both nitric oxide and superoxide and these molecules can combine to form peroxynitrite. Neutrophils also contain myeloperoxidase which reacts with peroxynitrous acid (HOONO). On mixing myeloperoxidase with HOONO compound II was formed. Compound I could not be detected as an intermediate. The apparent second‐order rate constant of formation of compound II was strongly pH‐dependent (2.5×10<jats:sup>5</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> at pH 8.9 and 6.2×10<jats:sup>6</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> at pH 7.2). The p<jats:italic>K</jats:italic><jats:sub>a</jats:sub> of this effect is 6.9 and it was concluded that the enzyme reacts with the protonated form of the peroxide, that is peroxynitrous acid, with a pH‐independent second‐order rate constant of 2.0×10<jats:sup>7</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> at 12°C. The interaction of HOONO with lactoperoxidase was studied for comparison. As was observed for myeloperoxidase, compound I could not be detected as an intermediate. The apparent second‐order rate constant of compound II formation is pH‐dependent and is 3.3×10<jats:sup>5</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> at pH 7.4 and 8.4×10<jats:sup>4</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> at pH 9.0. In contrast, horseradish peroxidase reacts with HOONO to form compound I, which is subsequently followed by the formation of compound II. The second‐order rate constant for the formation of compound I is 3.2×10<jats:sup>6</jats:sup> M<jats:sup>−1</jats:sup>· s<jats:sup>−1</jats:sup> and is pH‐dependent, the p<jats:italic>K</jats:italic><jats:sub>a</jats:sub> for this effect is 6.8. Catalase (up to 3 μM) does not affect the rate of decomposition of peroxynitrite and no compound I formation is observed. Since nitrite may be present in the peroxynitrite preparation and to discriminate between the reaction of the enzyme with nitrite or peroxynitrite, the effect of nitrite on myeloperoxidase was studied. The dissociation constant for the myeloperoxidase‐nitrite complex is pH‐dependent and has values of 580 μM at pH 6.0 and 55 mM at pH 8.5.</jats:p>