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 approximately 2% nitrotyrosine in both the alpha and beta 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.