α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

alpha-Hemoglobin (alpha Hb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in alpha Hb heme coordination, but how these changes are facilitated by interactions at the alpha Hb center dot AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe alpha Hb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-alpha Hb and CO-alpha Hb center dot AHSP indicated that the seven helical elements of the native alpha Hb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-alpha Hb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 angstrom lengthening of the Fe-O-2 bond, explaining previous reports that AHSP decreases alpha Hb O-2 affinity roughly 4-fold and promotes autooxidation due primarily to a 3-4-fold increase in the rate of O-2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O-2 dissociation rate and equilibrium constants, and reduced O-2-alpha Hb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote alpha Hb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of alpha Hb into Hb when beta Hb is abundant but diverts alpha Hb to a redox resistant holding state when beta Hb is limiting.