Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

Background: AHSP is an eiythroid molecular chaperone of the alpha-hemoglobin chains (alpha-Hb). Upon AHSP binding, native ferric alpha-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7). Methods: Recombinant AHSP, WT alpha-Hb:AHSP and alpha-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated alpha-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of alpha-Hb bound to AHSP as well as alpha-Hb release from the alpha-Hb:AHSP complex were measured by time-resolved absorption spectroscopy. Results: AHSP binding to alpha-Hb is kinetically controlled to prevail over direct binding with beta-chains and is also thermodynamically controlled by the alpha-Hb redox state and not the liganded state of the ferrous alpha-Hb. The dramatic instability of isolated ferric alpha-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in alpha-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric alpha-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to alpha-Hb within the Hb-tetramer. Conclusion: alpha-Hb reduction could trigger its release from AHSP toward its final Hb beta-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis. General significance: The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia. (C) 2013 Elsevier B.V. All rights reserved.