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

The reaction of (NO)-N-. and NO2- 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)-N-. 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)-N-. 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)-N-., induced the formation of globin Tyr-, Tip-, and Cys-centered radicals. MS studies provided evidence of the formation of similar to2% nitrotyrosine in both the alpha and beta subunits, suggesting that (NO2)-N-. 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)-N-.. The differences in oxidation mechanisms of (NO)-N-. and NO2- are discussed. (C) 2004 Elsevier Inc. All rights reserved.