-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy--hemoglobin and Weakens the Iron-Oxygen Bond * □ S Received for publication, November 18, 2012, and in revised form, May 20, 2013 Published, JBC Papers in Press, May 21, 2013, DOI 10.1074/jbc.M112.437509 Claire F. Dickson ‡ , Anne M. Rich § , William M. H. D’Avigdor ¶ , Daniel A. T. Collins ‡ , Jason A. Lowry ¶ , Todd L. Mollan  , Eugene Khandros**, John S. Olson  , Mitchell J. Weiss**, Joel P. Mackay ¶ , Peter A. Lay § , and David A. Gell ‡1 From the ‡ Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000, Australia, § School of Chemistry, University of Sydney, NSW 2006, Australia, ¶ School of Molecular Bioscience, University of Sydney, NSW 2006, Australia,  Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, and **Cell and Molecular Biology Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Background: -Hemoglobin stabilizing protein (AHSP) is a hemoglobin chaperone. Results: AHSP causes a subtle perturbation of the proximal heme pocket of O 2 --hemoglobin, lengthening the Fe-O 2 bond and enhancing O 2 dissociation. Conclusion: Pro-30 in wild-type AHSP promotes Hb autooxidation by introducing strain into the proximal heme pocket. Significance: HbAHSP complexes are intermediates in Hb assembly and achieve Hb detoxification. -Hemoglobin (Hb)-stabilizing protein (AHSP) is a molec- ular chaperone that assists hemoglobin assembly. AHSP induces changes in Hb heme coordination, but how these changes are facilitated by interactions at the HbAHSP inter- face is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe Hb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-Hb and CO-HbAHSP indicated that the seven helical elements of the native Hb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. How- ever, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-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 Å lengthening of the Fe-O 2 bond, explaining previous reports that AHSP decreases 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 -Hb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote Hb autooxidation by introduc- ing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of Hb into Hb when Hb is abundant but diverts Hb to a redox resistant holding state when Hb is limiting. Adult human hemoglobin (Hb) 2 is a tetramer of two Hb and two Hb subunits. The Hb and Hb chains share a common fold, and each binds a single, iron-containing protoporphyrin IX (heme) molecule in a deep pocket, protected from solvent. The central iron atom in each heme is the site of physiological dioxygen (O 2 ) binding. -Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free Hb in a het- erodimeric complex (see Fig. 1A) (1–3) and is essential for nor- mal erythropoiesis (4 –12). One role of AHSP is to protect erythroid cells from oxidative damage arising from the action of the heme iron in free Hb (6). In aqueous solutions containing air-equilibrated buffer, the Fe(II) atoms of heme groups spontaneously oxidize to the ferric (Fe(III)) state, which results in the production of superoxide anions. This process is termed autooxidation. Autooxidation is inhibited by the physicochemical properties of the heme pocket that allow reversible oxygen binding. However, spontaneous oxidation is not blocked completely, and the superoxide anions generated undergo rapid enzymatic and non-enzymatic dispro- portionation to H 2 O 2 and O 2 .H 2 O 2 is a key mediator of oxida- tive stress that can react with either reduced or oxidized globins to produce highly reactive ferryl or oxo-ferryl (Fe(IV)) heme groups (13, 14), and subsequent reactions lead to destruction of Hb as well as production of potent pro-oxidants that damage membranes and other cellular components (15–17). Addition- ally, autooxidation to the ferric state renders Hb incapable of O 2 * This work was supported, in whole or in part, by National Institutes of Health (NIH) Grants HL47020, GM35649, and HL110900 (all to J. S. O.) and Institute of Biosciences and Bioengineering NIH Biotechnology Predoctoral Train- ing Grant GM008362 (to T. L. M.). This work was also supported by the Australian Research Council (ARC) (to P. A. L., J. P. M., and D. A. G.), an ARC Australian Professorial Fellowship (to P. A. L.), a National Health and Medi- cal Research Council Senior Research Fellowship (to J. P. M.), and Robert A. Welch Foundation Grant C-0612 (to J. S. O. and T. L. M.). □ S This article contains supplemental Tables 1–3. 1 To whom correspondence should be addressed: Menzies Research Institute Tasmania, University of Tasmania, 17 Liverpool St., Hobart, TAS 7000, Aus- tralia. Tel.: 61-3-62264608; E-mail: david.gell@utas.edu.au. 2 The abbreviations used are: Hb, hemoglobin; AHSP, -hemoglobin stabiliz- ing protein; AHSP P30A , 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 cor- relation THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 27, pp. 19986 –20001, July 5, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. 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