Analysis of the Global Architecture of Hemoglobin A₂ by Heme Binding Studies and Molecular Modeling

Abstract

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A₂ was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5−13 × 10⁷ M⁻¹ s⁻¹), local structural rearrangement (0.21−0.23 s⁻¹), global αδ structural event (0.071−0.098 s⁻¹), and formation of the Fe–His bond (0.009−0.012 s⁻¹), respectively. Kinetic differences observed between apohemoglobin A₂ and apohemoglobin A (previously studied) prompted an analysis of the structures of β and δ chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.