Mechanisms of Alpha Hemoglobin Stabilizing Protein (AHSP) Actions.

Alpha hemoglobin stabilizing protein (AHSP) is an abundant erythroid factor that specifically binds and stabilizes free α hemoglobin (Kihm et al., Nature 417:758, Gell et al., JBC, 277:40602). In mice, AHSP deficiency causes oxidative stress, mild anemia, and reticulocytosis. Furthermore, loss of AHSP exacerbates α globin precipitation and anemia in a murine model for β thalassemia. These data suggest that AHSP operates as a molecular chaperone to stabilize free α hemoglobin and inhibit its ability to catalyze redox chemistry. We investigated how AHSP associates with α hemoglobin and thereby reduces its oxidative capacity. By NMR and mutational analysis, we determined the structure of AHSP and defined its α globin-interacting surfaces. AHSP consists of three linked α helices. Key α globin-binding residues lie along helices 1 and 2 in a region that resembles the G and H helical fold of β globin. Hence, AHSP presents a surface resembling that of β hemoglobin and thus promotes the formation of a “pseudo α/β interface”. To investigate how formation of this complex affects α hemoglobin-mediated redox chemistry, we examined the ability of AHSP to inhibit peroxide-mediated oxidation of the redox-sensitive dye TMPD. Oxy-α hemoglobin alone catalyzed TMPD oxidation at a rate of 140 μMmin⁻¹; preincubation with AHSP reduced this rate significantly, to 65 μMmin⁻¹. Furthermore, AHSP reduced both heme loss from α hemoglobin and its ability to oxidize hemoglobin A. AHSP binding to oxy- α hemoglobin induced a spectral shift in the UV/visible range, suggesting that stabilization might be associated with structural alterations in the heme moiety. Specifically, the Soret peak was reduced and shifted leftward, while the 541 and 576 nm peaks of the visible region were reduced with concomitant rises at 500 nm and at the region above 580 nm. These alterations suggest that heme-bound iron is converted to a ferric (Fe³⁺) form that is liganded at all 6 coordinate positions, and therefore, can no longer catalyze redox chemistry. Consistent with this possibility, addition of KCN to the α hemoglobin-AHSP complex produced cyanomethemoglobin-AHSP, but at a slower rate than reaction of KCN with ferric-α hemoglobin alone. In addition, electron paramagnetic resonance and resonance raman spectroscopy showed that the heme iron exists in a low spin state within the α hemoglobin-AHSP complex. The oxidized complex was insensitive to pH alteration, indicating that iron was not bound by hydroxyl ion, which typically occurs at alkaline conditions. Together, our findings illustrate a potential mechanism whereby AHSP renders α hemoglobin chemically inert by inhibiting the reactivity of heme-bound iron. We propose that within the AHSP-α hemoglobin dimer, the oxidized heme is held within a bi-histidyl form, thus blocking its ability to act as a redox catalyst.