Scientific Publications by FDA Staff

J Biol Chem 2014 Aug 8;289(32):22342-57

Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: A novel subunit specific mechanism in hemoglobin.

Strader MB, Hicks WA, Kassa T, Singleton E, Soman J, Olson JS, Weiss MJ, Mollan TL, Wilson MT, Alayash AI

A pathogenic Val 67 ¿ Met 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 ¿ subunits of HbF Toms River (¿ 67(E11)Val¿Met) and ß subunits of HbA Bristol-Alesha (ß67(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 Asp was enhanced during the spontaneous autooxidation of the mutants relative to wildtype HbA and HbF, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2 18O2, we verified incorporation of 18O 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 a Met(E11) position in the corresponding HbA Evans (a62(E11)Val¿Met). The crystal structures of the 3 recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autooxidation of the Hb mutant crystals lead to electron density maps indicative of AspE11 formation in ß subunits, but not in a subunits. This novel post-translational modification highlights the non-equivalence of human Hb a, ß, and ¿ subunits with respect to redox reactivity and may have direct implications to a/ß hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.