Myeloperoxidase potentiates nitric oxide-mediated nitrosation

Nitrosation is an important reaction elicited by nitric oxide ( NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline ( IQ; 10 muM) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nM MPO, 0.25 muM H2O2/min, and 0.024 muM NO/min. Autoxidation was only detected at greater than or equal to1.2 muM NO/min. MPO potentiation was not affected by a 40-fold excess flux of H2O2 over NO or less than a 2.4-fold excess flux of NO over H2O2. Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO+. MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2. or an NO2.-like species. MPO nonnitrosative oxidation of IQ with 0.3 mM NO2- at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO2-. Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN3. This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.