Scavenging of reactive nitrogen species by oxygenated hemoglobin: globin radicals and nitrotyrosines distinguish nitrite from nitric oxide reaction

The reaction of NO and NO2− with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe2+Hb: one with deoxygenated Hb, in which the biological properties of NO are preserved, and another with oxygenated hemoglobin (oxyHb), in which both species are oxidizes to NO3−. In this study we compared the oxidative reactions of NO and NO2− and, in particular, the radical intermediates formed during transformation to NO3−. The reaction of NO2− with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO3−. Direct EPR and spin trapping studies showed that NO2−, but not NO, induced the formation of globin Tyr-, Trp-, and Cys-centered radicals. MS studies provided evidence of the formation of 2% nitrotyrosine in both the α and β subunits, suggesting that NO2 diffuses in part away from the heme and reacts with Tyr radicals. No nitrotyrosines were detected in the reaction of NO with oxyHb. Collectively, these results indicate that NO2− reaction with oxyHb causes an oxidative challenge not observed with NO. The differences in oxidation mechanisms of NO and NO2− are discussed.