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.
Highlights
Met(E11) mutation in Hb heme pocket undergoes modification to Asp in ␥ and  subunits
Subsequent post-translational modification of Met to Asp was reported in ␥ subunits of human fetal Hb Toms River (␥67(E11)Val 3 Met) and  subunits of adult Hb (HbA) Bristol-Alesha (67(E11)Val 3 Met) that were associated with hemolytic anemia
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)
Summary
Alayash‡2 From the ‡Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, the §Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, the ¶Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, and the ʈDepartment of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom
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