α-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of a-subunits of human HbA with hydrogen peroxide

Abstract

α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobin A (HbA) and modulates heme iron oxidation and subunit folding states. Although AHSP-αHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. Both metHbA and isolated met-β-subunits undergo further oxidation in the presence of hydrogen peroxide (H2O2) to form ferryl heme species. Surprisingly, much lower levels of H2O2-induced ferryl heme are produced by free met-α-subunits as compared with met-β-subunits, and no ferryl heme is detected in H2O 2-treated AHSP-met-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSP-met-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPR data suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and β-subunits. In contrast, treatment of free α-subunits with H2O2 yields much smaller radical signals, and no radicals are detected when H2O 2 is added to AHSP-α-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from +40 to -78 mV in 1 M glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSP-met-α complex markedly decreases the rate of the initial H2O2 reaction with iron and thus provides α-subunits protection against damaging oxidative reactions. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.