Meng, FantaoKassa, TigistStrader, Michael BradSoman, JayashreeOlson, John S.Alayash, Abdu I.2019-12-112019-12-112019Meng, Fantao, Kassa, Tigist, Strader, Michael Brad, et al.. "Substitutions in the β subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation." <i>Journal of Biological Chemistry,</i> 294, no. 11 (2019) American Society for Biochemistry and Molecular Biology: 4145-4159. https://doi.org/10.1074/jbc.RA118.006452.https://hdl.handle.net/1911/107838After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of “hot-spot” amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β 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 βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-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 βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.engFinal version free via Creative Commons CC-BY license.Journal article2019-Meng-4145-59https://doi.org/10.1074/jbc.RA118.006452