Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate*

Background: α-Hemoglobin stabilizing protein (AHSP) facilitates hemoglobin production. Results: AHSP preferentially binds to ferric versus ferrous α subunits and induces reversible structural alterations within seconds of binding. Conclusion: AHSP exerts its effects by stabilizing a ferric α folding intermediate and inhibiting its participation in hemoglobin assembly. Significance: AHSP is a molecular chaperone for ferric α-globin. Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k′AHSP) and dissociation (kAHSP) rate constants of ≈10 μm−1 s−1 and 0.2 s−1, respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp29-Pro30 peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe3+)-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro30 conformer. Both wild-type and Pro30-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe2+)-α. The dissociation rate of the met-α·AHSP complex (kAHSP ≈ 0.002 s−1) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.