Kinetics of α-globin binding to α-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate.
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 (k(AHSP)) 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 Asp(29)-Pro(30) 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(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 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.
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA.
Todd L
TL
Mollan
Eugene
E
Khandros
Mitchell J
MJ
Weiss
John S
JS
Olson
D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013379:Substrate Specificity; D055542:alpha-Globins / Q000737:chemistry* / Q000378:metabolism*
C468768:AHSP protein, human; D001798:Blood Proteins; D006420:Hemeproteins; D018832:Molecular Chaperones; D055542:alpha-Globins; C001332:hemichrome; D006441:Hemoglobin A