Abstract
α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb 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-αHb 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 Å lengthening of the Fe-O2 bond, explaining previous reports that AHSP decreases αHb O2 affinity roughly 4-fold and promotes autooxidation due primarily to a 3-4-fold increase in the rate of O2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O2 dissociation rate and equilibrium constants, and reduced O2-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.
Highlights
Adult human hemoglobin (Hb)2 is a tetramer of two ␣Hb and two Hb subunits
To understand how promoting ␣Hb autooxidation is compatible with reduced reactive oxygen species production in erythroid cell, it is important to appreciate that repeated movement between different iron oxidation states can occur for a single heme group, and this redox cycling increases the probability of irreversible changes in the globin and other cellular components
A Proline Residue Is Precisely Positioned in Loop 1 of AHSP to Promote Autooxidation of ␣Hb—The Asp-29 –Pro-30 peptide bond in loop 1 of AHSP undergoes cis-trans isomerization that is coupled with small changes in the packing of the three ␣-helices in AHSP [1, 46]
Summary
Hemoglobin; AHSP, ␣-hemoglobin stabilizing protein; AHSPP30A, AHSP with a Pro-30 to Ala substitution; EXAFS, extended X-ray absorption fine structure; r.m.s., root mean square; e.s.d., estimated standard deviation; SEC, size exclusion chromatography; MS, multiple scattering; pMB, p-hydroxymercuribenzoate; XANES, x-ray absorption near edge structure; HSQC, heteronuclear single quantum correlation. As well as inducing changes in the structure of Fe(III) ␣Hb, AHSP binding causes a Ͼ10-fold increase in the autooxidation rate of Fe(II) ␣Hb [11, 20, 25, 28, 29], resulting in formation of bis-histidyl Fe(III) ␣Hb. To understand how promoting ␣Hb autooxidation is compatible with reduced reactive oxygen species production in erythroid cell, it is important to appreciate that repeated movement between different iron oxidation states can occur for a single heme group, and this redox cycling increases the probability of irreversible changes in the globin and other cellular components. A previous x-ray crystallographic study suggested that the strained
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