Molecular Mechanism of AHSP-Mediated Stabilization of α-Hemoglobin

Abstract

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.

α-Hemoglobin Stabilizing Protein (AHSP) Markedly Decreases the Redox Potential and Reactivity of α-Subunits of Human HbA with Hydrogen Peroxide

“…Inhibition of H 2 O 2 Reactions by AHSP-Early structural work on AHSP⅐␣-subunit complexes led to the proposal that the bishistidyl conformation strongly inhibits ferric-ferryl redox cycling following exposure to H 2 O 2 (15,52). It was estimated that at least 33% of the heme in ferrous ␣⌷ 2 ⅐AHSP complexes converts to the ferryl heme form following H 2 O 2 exposure, whereas less than 10% of the heme in ferric met-␣⅐AHSP complexes converts to the ferryl heme form following the same exposure (15).…”

Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

“…Once these homo-and heterocomplexes are formed, the subunits are stable enough to quantitatively reassemble into the holo-HbA tetramer, which is an effectively irreversible process. The formation, spectral properties, and structure of AHSP⅐bishistidyl met-␣ complexes have been examined extensively in vitro (18,21,22,28). The physiological function of this complex in vivo was examined in the current study by introducing endogenous Ahsp mutations that altered its rate of ␣O 2 autooxidation.…”

“…AHSP is comprised of three antiparallel ␣ helices with 11 unstructured amino acids at the carboxyl terminus (21)(22)(23). AHSP helix 1, helix 2, and the interconnecting loop interact with helices G and H of ␣-globin at a surface that overlaps with the interface for ␤-globin binding.…”

“…AHSP binding to ␣O 2 accelerates spontaneous heme iron oxidation (autooxidation) from an Fe 2ϩ (ferrous) to an Fe 3ϩ (or met) state. Autooxidation is followed immediately by internal coordination of His 58 (E7) to the hemin iron, resulting in a hexacoordinate bishistidyl or hemichrome structure (18,21,22,28). Bishistidyl hemoglobins occur as ␣-globin degradation products in ␤-thalassemia (4,6,29,30), during normal degradation of hemoglobin, and in some naturally occurring globins in mammalian tissues, plants, certain bacteria, and cold water fish (31)(32)(33)(34)(35)(36)(37)(38)(39)(40).…”

Heme degradation and oxidative stress in murine models for hemoglobinopathies: Thalassemia, sickle cell disease and hemoglobin C disease

“…The presence of γ or α mouse in heterotetramers may reduce the amount of membrane-associated, unstable β-chains. The low level of degradation products in NY1DD mice may, thus, be due to reduced polymer formation (the residual mouse α-chains inhibit polymer formation as efficiently as human γ) [26] or the more efficient interaction of murine α-globin with murine alpha Hb sparing protein (AHSP) [27]. Since NY1DD and NY1KO-γM mice have about the same percent β S , but very different levels of heme degradation products, we speculate that inefficient interaction of human α with murine AHSP may also contribute to the oxidative environment of murine RBCs that express exclusively human Hbs.…”