Isolated Hb Providence β82Asn and β82Asp Fractions Are More Stable than Native HbA₀ under Oxidative Stress Conditions

We have previously shown that hydrogen peroxide (H₂O₂) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H₂O₂ when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H₂O₂. However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α–α dimer formation in the presence of excess H₂O₂. Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H₂O₂ and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle