title: 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 (HbFe(II)) 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 (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) 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%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO 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 HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) 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.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec H3G 1M8, Canada.
authors.forename: Andrea A
authors.initials: AA
authors.lastname: Romeo
authors.forename: John A
authors.initials: JA
authors.lastname: Capobianco
authors.forename: Ann M
authors.initials: AM
authors.lastname: English
mesh_terms: D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020030:Nitric Oxide Donors / Q000097:blood*; D004369:Pentetic Acid / Q000494:pharmacology; D011485:Protein Binding; D026422:S-Nitrosoglutathione / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry
chemical_list: D002614:Chelating Agents; D006454:Hemoglobins; D020030:Nitric Oxide Donors; D006418:Heme; D026422:S-Nitrosoglutathione; D003300:Copper; D004369:Pentetic Acid; C100003:deoxyhemoglobin; D006441:Hemoglobin A