Substitutions in the β 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, 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.

Effects of α subunit substitutions on the oxidation of βCys93 and the stability of sickle cell hemoglobin

“…Methods controlling Hb-mediated oxidative pathways in SCD have focused in recent years on the use of small molecule drugs or site-directed mutagenesis which specifically targets the βCys93 ‘hot spot’. This residue is readily and irreversibly oxidized in the presence of H 2 O 2 to cysteic acid resulting in the unfolding and destabilization of the protein [ 4 , 9 ]. The reactivity for the sulfur-containing amino acids, βCys is consistently more exposed to the surface according to Accessible Surface Area Calculations (ASA) [ 4 ] and therefore is more amenable to reaction with H 2 O 2 .…”

“…The substitution of the native βLys-82 for Asp-82, is located ∼18.3 Å away from the β -heme and is not known to be engaged in electron transfer processes. However, the higher oxidative stability caused by the K82D mutation has been attributed to cause changes in reactivity of the βCys93 side chain [ 9 ], which may be due to either indirect electrostatic effects or alterations in the local dynamics of the structure.…”

“…The rates of autoxidation for these mutants were also reported to be similar to that of HbS, but their oxidative side reactions are more profound than that of HbS [ 10 ]. If the effects were additive, it would be interesting to consider the favorable properties of an HbS variant with αH20Q/H50Q or αH50Q/H20R and βF41Y/K82D substitutions that we reported earlier [ 9 ]. Such an HbS is likely to be as soluble and resistant to oxidation and should therefore provide targeted gene therapeutic modalities.…”

“…Whereas the second contained Asp replacement of Lys (K82D) found in the β cleft of Hb Providence. The third construct contained both β subunit variants [ 9 ]. In the presence of H 2 O 2 , βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr41 side chain.…”

“…Mass spectrometric analysis of βCys93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and a dramatic 3- to 8-fold inhibition of cysteic acid formation in the rHbS βK82D/βE6 V and βF41Y/K82D/βE6 V protein variants, respectively. Substitutions of βK82D/βE6 V and βF41Y/βE6 V not only added significant resistance to oxidative stress, but also provided an additional anti-sickling property by delaying polymerization of HbS [ 9 ].…”