Heme nitrosylation of deoxyhemoglobin by S-nitrosoglutathione requires copper

Abstract

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFeII) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFeII)/GSNO incubates shows prompt formation (<5 min) of ∼100% hemenitrosylated Hb (HbFeIINO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N′N″,N″-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFeIINO was detected when neocuproine was added to copper-depleted HbFeII/ GSNO incubates. Thus, HbFeIINO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFeII/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFeII was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysβ93 of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.