title: Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.
abstract: A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.
authors.affiliation: From the Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Michael Brad
authors.initials: MB
authors.lastname: Strader
authors.affiliation: From the Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Hicks
authors.affiliation: From the Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Tigist
authors.initials: T
authors.lastname: Kassa
authors.affiliation: the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
authors.forename: Eileen
authors.initials: E
authors.lastname: Singleton
authors.affiliation: the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
authors.forename: Jayashree
authors.initials: J
authors.lastname: Soman
authors.affiliation: the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
authors.forename: John S
authors.initials: JS
authors.lastname: Olson
authors.affiliation: the Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, and.
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Weiss
authors.affiliation: From the Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Todd L
authors.initials: TL
authors.lastname: Mollan
authors.affiliation: the Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.affiliation: From the Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, abdu.alayash@fda.hhs.gov.
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D018360:Crystallography, X-Ray; D005319:Fetal Hemoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006441:Hemoglobin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006455:Hemoglobins, Abnormal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D040901:Proteomics; D055672:Static Electricity
chemical_list: D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D021122:Protein Subunits; C062318:hemoglobin Evans; D001224:Aspartic Acid; D006418:Heme; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D008715:Methionine; D006861:Hydrogen Peroxide; D007501:Iron
keywords: Aspartate (Aspartic Acid); Heme; Hemoglobin; Hemoglobinopathies; Mutant; Oxidation of Methionine; Post-translational Modification (PTM)