title: Oxygen binding to partially nitrosylated hemoglobin.
abstract: Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
authors.affiliation: Department of Bioscience, Aarhus University, DK-8000 Aarhus, Denmark. angela.fago@biology.au.dk
authors.forename: Angela
authors.initials: A
authors.lastname: Fago
authors.forename: Alvin L
authors.initials: AL
authors.lastname: Crumbliss
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hendrich
authors.forename: Linda L
authors.initials: LL
authors.lastname: Pearce
authors.forename: Jim
authors.initials: J
authors.lastname: Peterson
authors.forename: Robert
authors.initials: R
authors.lastname: Henkens
authors.forename: Celia
authors.initials: C
authors.lastname: Bonaventura
mesh_terms: D019794:2,3-Diphosphoglycerate / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006442:Glycated Hemoglobin / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism; D011485:Protein Binding
chemical_list: D019794:2,3-Diphosphoglycerate; D006442:Glycated Hemoglobin; D006418:Heme; D009569:Nitric Oxide; D010100:Oxygen; D010108:Oxyhemoglobins; D010833:Phytic Acid; C058377:hemoglobin A, glycosylated-nitric oxide complex
keywords: 2,3-diphosphoglycerate; Allostery; DPG; EDRF; EPR; Hb; IHP; NO; Nitric oxide; Oxygen binding curve; Pentacoordinate; S-nitrosated hemoglobin; SNO-Hb; electron paramagnetic resonance; endothelium-derived relaxing factor; hemoglobin; inositol hexaphosphate; nitric oxide