Substitutions in the subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation

Abstract

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, E6V) remains longer in a highly oxidizing ferryl form (HbFe4O) and induces irreversible oxidation of “hot-spot” amino acids, including Cys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the cleft of Hb Providence; and the third had both of these substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS F41Y and F41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of Cys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS K82D and F41Y/K82D, respectively. Under hypoxia, K82D and F41Y/K82D HbS substitutions increased the delay time by 250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS F41Y, respectively. Moreover, at 60 °C, rHbS K82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS F41Y/K82D variant. We conclude that the K82D and F41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.