Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state.
BACKGROUND
AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).


METHODS
Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.


RESULTS
AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.


CONCLUSIONS
α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.


CONCLUSIONS
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.
INSERM, U779, University Paris 11, 78 rue du General Leclerc, 94275 Le Kremlin Bicêtre, France. Electronic address: laurent.kiger@inserm.fr.
Laurent
L
Kiger
Corinne
C
Vasseur
Elisa
E
Domingues-Hamdi
Gilles
G
Truan
Michael C
MC
Marden
Véronique
V
Baudin-Creuza
D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006441:Hemoglobin A / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C468768:AHSP protein, human; D001798:Blood Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D006441:Hemoglobin A; D010100:Oxygen
AHSP; AHSP(WT); AHSP:α-Hb; Alpha-hemoglobin stabilizing protein (AHSP); Chaperone; Cyt b5; Erythropoiesis; GST; Hb; Heme hexacoordination; Hemoglobin; MetHb; Ngb; PBS; RBCs; ROS; alpha hemoglobin-stabilizing protein; complex formed between WT α-Hb chain and recombinant human wild type AHSP; ferric heme; glutathione S-transferase; hemin; human adult hemoglobin; neuroglobin; oxidized Hb; phosphate-buffered saline; reactive oxygen species; recombinant human wild type AHSP with an N-terminal Gly-Pro-Leu-Gly-Ser peptide; recombinant soluble domain of human membrane-bound cytochrome b5; red blood cells