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

Abstract The reaction of NO and NO 2 − with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe 2+ 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 NO 3 − . In this study we compared the oxidative reactions of NO and NO 2 − and, in particular, the radical intermediates formed during transformation to NO 3 − . The reaction of NO 2 − with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO 3 − . Direct EPR and spin trapping studies showed that NO 2 − , 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 NO 2 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 NO 2 − reaction with oxyHb causes an oxidative challenge not observed with NO. The differences in oxidation mechanisms of NO and NO 2 − are discussed.