-hemoglobin-stabilizing protein (ahsp) perturbs the proximal

�-Hemoglobin-stabilizing Protein (AHSP) Perturbs theProximal Heme Pocket of Oxy-�-hemoglobin and Weakensthe 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 O2-�-hemoglobin, lengthening the Fe-O2 bond andenhancing O2 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. AHSPinduces changes in �Hb heme coordination, but how thesechanges are facilitated by interactions at the �Hb�AHSP inter-face is not well understood. To address this question we haveused NMR, x-ray absorption spectroscopy, and ligand bindingmeasurements to probe �Hb conformational changes inducedby AHSP binding. NMR chemical shift analyses of free CO-�Hband CO-�Hb�AHSP indicated that the seven helical elements ofthe 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,andH helices and the heme pocket of CO-�Hb bound to AHSP.Comparisons of iron-ligand geometry using extended x-rayabsorption fine structure spectroscopy showed that AHSPbinding induces a small 0.03 Å lengthening of the Fe-O2bond, explaining previous reports that AHSP decreases �HbO2 affinity roughly 4-fold and promotes autooxidation dueprimarily to a 3–4-fold increase in the rate of O2 dissociation.Pro-30 mutations diminished NMR chemical shift changes inthe proximal heme pocket, restored normal O2 dissociationrate and equilibrium constants, and reduced O2-�Hbautooxidation rates. Thus, the contacts mediated by Pro-30in 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 statewhen �Hb is limiting.

Adult humanhemoglobin (Hb)2 is a tetramer of two�Hbandtwo �Hb subunits. The �Hb and �Hb chains share a commonfold, and each binds a single, iron-containing protoporphyrinIX (heme) molecule in a deep pocket, protected from solvent.The central iron atom in each heme is the site of physiologicaldioxygen (O2) 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, theFe(II) atoms of heme groups spontaneously oxidize to the ferric(Fe(III)) state, which results in the production of superoxideanions. This process is termed autooxidation. Autooxidation isinhibited by the physicochemical properties of the heme pocketthat allow reversible oxygen binding. However, spontaneousoxidation is not blocked completely, and the superoxide anionsgenerated undergo rapid enzymatic and non-enzymatic dispro-portionation to H2O2 and O2. H2O2 is a key mediator of oxida-tive stress that can react with either reduced or oxidized globinsto produce highly reactive ferryl or oxo-ferryl (Fe(IV)) hemegroups (13, 14), and subsequent reactions lead to destruction ofHb as well as production of potent pro-oxidants that damagemembranes and other cellular components (15–17). Addition-ally, autooxidation to the ferric state rendersHb incapable ofO2

* 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 Instituteof Biosciences and Bioengineering NIH Biotechnology Predoctoral Train-ing Grant GM008362 (to T. L. M.). This work was also supported by theAustralian Research Council (ARC) (to P. A. L., J. P. M., and D. A. G.), an ARCAustralian 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: [email protected].

2 The abbreviations used are: Hb, hemoglobin; AHSP, �-hemoglobin stabiliz-ing 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-rayabsorption 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. Published in the U.S.A.

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transport and greatly enhances the rates of hemin loss and glo-bin denaturation.Compared with mature Hb, isolated �Hb and �Hb chains

produce higher levels of reactive oxygen species (18–20), pre-sumably linked to a loss of structural integrity of the hemepocket. Oxidative damage occurs in erythroid cells of �-thalas-saemic mice that contain elevated levels of unpaired �Hb (21–24). Kong et al. (6) showed similar increases in reactive oxygenspecies in AHSP�/� mice, suggesting that AHSP has a role inlimiting reactive oxygen species production during normalerythropoiesis. In a purified system AHSP inhibits the reactionof Fe(III) �Hbwith H2O2 (2, 20), and recentlyMollan et al. (20)demonstrated that AHSP binding prevents the production ofFe(IV)AO heme and associated protein radicals when Fe(III)�Hb is exposed to H2O2.

The low reactivity of Fe(III) �Hb in the presence of AHSPcorrelates with a change in �Hb heme pocket structure. Innative Hb, the �Hb heme group is coordinated through a singleHis-87 side chain, termed the proximal histidine (see Fig. 1B),allowing exogenous ligands in the distal heme pocket to inter-act with iron. In the ferric�Hb�AHSP complex, Fe(III) becomescoordinated by both the proximal and distal histidine sidechains (bis-histidyl heme coordination; see Fig. 1C) (1, 2, 11,25–27). Coordination of low-spin Fe(III) with six strong ligandsblocks the binding of exogenous ligands and associated electrontransfer reactions involving oxygen species. The switch fromnative to bis-histidyl heme coordination is accompanied by achange to a non-native �Hb tertiary structure (r.m.s deviationof 3.3 Å over 135 C� atoms), which dissociates from AHSP�100-fold slower than Fe(II) �Hb (20, 25, 27).

As well as inducing changes in the structure of Fe(III) �Hb,AHSP binding causes a �10-fold increase in the autooxidationrate of Fe(II)�Hb (11, 20, 25, 28, 29), also resulting in formationof bis-histidyl Fe(III) �Hb. To understand how promoting �Hbautooxidation is compatible with reduced reactive oxygen spe-cies production in erythroid cell, it is important to appreciatethat repeated movement between different iron oxidationstates can occur for a single heme group, and this redox cyclingincreases the probability of irreversible changes in the globinand other cellular components. In contrast, autooxidation of�Hb to the bis-histidyl �Hb structure dramatically lowers theFe(III)/Fe(II) redox potential (20) and inhibits interactions withexogenous ligands, thus inhibiting redox cycling. However,the structural mechanism by which AHSP accelerates �Hbautooxidation and, more generally, how AHSP promotes theprotective bis-histidyl structure is not well understood.Previous work from our group has suggested that AHSP

induces strain into the O2-�Hb structure that is relieved uponautooxidation by reorganization of the �Hb protein fold (25).Mutation of Pro-30 to Ala (AHSPP30A) in loop 1 of AHSP (Fig.1A) leads to an �4-fold reduction in �Hb�AHSP autooxidationrates, suggesting that Pro-30 contributes to the strained confor-mation. However, how contacts at the �Hb�AHSP interface aretransmitted to the �Hb heme pocket and the nature of thestrainedO2-�Hb structure are unknown. Difficulties in charac-terizing theO2-�Hb�AHSPcomplex arise from its propensity tospontaneously convert to a bis-histidyl structure. A previousx-ray crystallographic study suggested that the strained

O2-�Hb structure involved large-scale changes in the globinpolypeptide structure and heme coordination geometry (1).To explore these issues, we have used NMR and EXAFS to

probe the complexes of CO- and O2-liganded �Hb with AHSPin solution. The results show that the tertiary structure ofCO/O2-�Hb in these complexes closely resembles that ofCO/O2-�Hb in the native HbA tetramer and, therefore, thatour previous model of O2-�Hb�AHSP (1) is unlikely to repre-sent the initial AHSP binding event in solution. We show thatmutation of Pro-30 in AHSP leads to NMR chemical shift per-turbations in the F-helical region of CO-�Hb, and the concom-itant changes inO2 andCObinding reactivity help identify howthis strain introduced into the proximal portion of the hemepocket promotes autooxidation of O2-�Hb.

EXPERIMENTAL PROCEDURES

Protein Production—The structured region, residues 1–90 ofhuman AHSP, and mutants thereof were expressed and puri-fied as described previously (25). CO-�Hb was purified fromhuman blood (3). O2-�Hb was generated by gently blowingoxygen across the surface of CO-�Hb under a concentrated10-watt halogen light source. The procedure was carried out onice, and the formation of the O2-�Hb was monitored by UV-visible spectroscopy (Shimadzu UV-1601). Isotopically labeled�Hb for NMR studies was co-expressed with �Hb andmethio-nine aminopeptidase in Escherichia coli from the previouslydescribed plasmid pHE7 (30). Expression was carried out in theE. coli strain JM109. One liter of overnight culture in DM-4minimal media as described by Looker et al. (31) containingD-glucose (5 g liter�1), NH4Cl (0.7 g liter�1), thiamine (30 mgliter�1), ampicillin (100 mg liter�1), and trace metal solution(32) was inoculated into a final volume of 3.5 liters of DM-4medium containing thiamine (30 mg liter�1), ampicillin (100mg liter�1), tracemetal solution, yeast extract (50�l of 10%w/vsolution), and antifoam (Sigma) in a 5-liter fermentor vessel(New Brunswick BioFlo). Growth was maintained at 37 °C, pH

E

F

E

F

B

C

His-87 (F8)

His-58 (E7)

His-87 (F8)

His-58 (E7)

Heme

Heme

APro-30

Heme

Helix 1Helix 2

Helix 3

Loop 1

E

F

H

G

A

B

AHSP

αHb

FIGURE 1. Structure of the bis-histidyl �Hb�AHSP complex. A, shown is aschematic of the �Hb�AHSP complex (PDB 3IA3). Loop 1 and Pro-30 (spheres)of AHSP are positioned at the interface with �Hb. B, shown are the E-F helicesof �Hb in the native conformation, as seen in the deoxy Hb complex (PDB2DN2) (53), with His-87 (His F8) coordinating the heme iron. His-58 (His E7) sitsaway from the iron site and regulates the binding of diatomic ligands. C, in acomplex formed from AHSP and met-�Hb (PDB 3IA3), His-87 and His-58simultaneously coordinate the Fe(III) heme iron (bis-histidyl hemecoordination).

AHSP Perturbs the Proximal Heme Pocket of O2-�-Hemoglobin

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6.8, with aeration andmonitored in real time based ondissolvedO2 concentration. The culture exhausted the nitrogen supply atan absorbance of �1 (600 nm) and was provided with 2 g of15NH4Cl. The culture exhausted the glucose supply at anabsorbance of�3, and 1.5 g of D-[U(99%)-13C]glucose was pro-vided, and the temperaturewas shifted to 32 °C. The addition ofa further 2 g of 15NH4Cl, 1.5 g of D-[U(99%)-13C]glucoseoccurred at an absorbance of�3.5, and expressionwas inducedwith 0.2 mM isopropyl �-D-1-thiogalactopyranoside and 15 �M

hemin. Cells were collected by centrifugation after 5 h, and theresulting cell pellets were washed once in 10 mM Tris-HCl, 0.5mM EDTA, pH 8.0. Cell pellets were resuspended in the samebuffer and saturated with CO before lysis by sonication (Ultra-sonic Processor 500 W, Sonics & Materials Inc.). After clarifi-cation by centrifugation, recombinant Hb was purified as pre-viously described (33). TheHb chains were split by the additionof a 2-foldmolar excess (per cysteine) ofp-hydroxymercuriben-zoate (pMB) and purified as previously described (3).Size Exclusion Chromatography (SEC) and In-line Light

Scattering—SEC chromatography was carried out on a 24-mlSuperose 12 column (GE Healthcare) in 0.1 M sodium phos-phate, pH 7.0 (21 °C). Protein concentrations were determinedbased on UV-visible absorbance. The concentrations of AHSPand mutants thereof were obtained after unfolding in 6 M gua-nidine-HCl using molar extinction coefficients calculatedbased on the amino acid compositions (34). CO/O2-�Hb con-centrations were determined based on absorption peaks arisingfrom � 3 �* transitions in the porphyrin ring using molarextinction coefficients for the corresponding CO/O2 forms ofHb (35). Theweight-averagemolecularweight (Mw) of particlesin solutionwas determineddirectly frommeasurements of lightscattering and protein concentration (36). In-line light scatter-ing measurements were made at 41.5°, 90.0°, and 138.5° withrespect to the incident beam using a mini-DAWNwith 690 nmlaser (Wyatt Technology Corp., Santa Barbara CA), calibratedagainst toluene. Protein concentrations were determined basedon the refractive index measured by an Optilab differentialrefractometer (Wyatt Technology Corp.) calibrated againstNaCl standards.Molecularweight calculationswere performedfor each 50-�l fraction across the elution peaks using theASTRA software (Wyatt Technology Corp.) with the assump-tion of a standard value for the specific refractive index incre-ment with respect to sample concentration (dn/dC) of 0.19 mlg�1. The presence of the heme and pMB groups upon the valueof dn/dC was neglected in this case.NMR Spectroscopy—All NMR spectroscopic measurements

were conducted using a Bruker Avance II 600 MHz spectrom-eter equipped with a triple-resonance TCI cryoprobe (Bruker,Karlsruhe, Germany), then processed using Topspin 2.1(Bruker), and analyzed using SPARKY 3.1 (37). Samples wereformulated as described, with the addition of 20 �M 4,4-di-methyl-4-silapentane-1-sulfonic acid (DSS) and 5%D2O. Back-bone assignments were made using HNCA, HNCACB, CBCA-(CO)NH, and 15N-edited NOESY according to standardassignment strategies. His side chain resonances were assignedbased on 15N-HSQC, 15N-NOESY-HSQC, and two-dimen-sional TOCSY (two-dimensional total correlation spectrosco-py)/NOESY spectra. Tautomer/charge state was inferred for

His side chains from cross-peak intensities in the 15N-HSQC(38). Spectra were recorded at sample temperatures of 15 and25 °C for �Hb and �Hb�AHSP complexes, respectively, to opti-mize spectral quality.EXAFS—Samples for x-ray absorption spectroscopy were

prepared in 20 mM sodium phosphate buffer, pH 7.0 (21 °C),with 30% sucrose (0.88 M) as the cryoprotectant. Sucrose wasthe preferred cryoprotectant, as it caused minimal changes inthe UV-visible spectrum. The O2-�Hb�AHSP complex wasformed by mixing O2-�Hb with a 1.1 molar excess of AHSP.Final protein concentrations were 6 and 2.5 mM for O2-�Hband O2-�Hb�AHSP samples, respectively. The solutions wereinjected into 140-�l Lucite XAS cells (23 � 2 � 3 mm) with63.5-�m Kapton tape windows. This operation was conductedin a nitrogen-filled glove bag. Immediately after their prepara-tions, the samples were frozen in a liquid-nitrogen/n-hexaneslurry to prevent autooxidation. X-ray absorption spectra wererecorded at the Stanford Synchrotron Radiation Lightsource(SSRL) on beamline 9-3 (3 GeV, 100–50 mA) using a Si(220)double-crystalmonochromator. Beamline 9-3 had an upstreamcollimatingmirror and a downstream sagitally focusingmirror;both were rhodium-coated and also provided harmonic rejec-tion. Data were collected as fluorescence spectra using a 30-el-ement germanium array detector (39). For each sample, 10–21scans were recorded from 6785 to 8257 eV. The energy wascalibrated using an iron foil standard in which the first inflec-tion point was assigned to an energy of 7111.2 eV (40). Datascans were averaged, background-subtracted, and normalizedas described previously (41).EXAFS Data Analysis—The model-fitting calculations were

performed by means of the program XFIT (42). The values ofthe parameters that were varied to optimize the agreementbetween the observed and calculated EXAFS were the coordi-nates x, y, and z of all atoms in the model in relation to anarbitrary set of Cartesian axes, the Debye-Waller factors, �2, ascale factor, S02, and E0. The value of S02 was initially restrainedto be 0.9 with an estimated standard deviation (e.s.d.) 0.1 forO2-�Hb and 0.92 (e.s.d. 0.02) for O2-�Hb�AHSP (i.e. to typicalvalues for low-spin O2-myoglobin and O2-leghemoglobin).Both the observed and calculated EXAFS were Fourier-filtered(42). The k windows used for the EXAFS analyses are shown inthe figures. The goodness-of-fit parameter R was calculated asdescribed by Ellis and Freeman (42). The model used in theXFIT analyses is that described previously (41). The sixth coor-dination site was a dioxygen molecule where the terminal oxy-genwas arbitrarily constrained to lie in the plane perpendicularto the heme and passing through themeso-Cs. The O1-O2 dis-tance was restrained to be 1.22 Å (e.s.d. 0.01 Å), consistent withthat reported by Jameson et al. (43). The constraints andrestraints used for the bond lengths and bond angles in theporphyrin ring and the imidazole ligand and for the Debye-Waller factors were as described previously (41). Constraintsand restraints and multiple-scattering (MS) paths used in thefitting calculations are given in supplemental Tables.O2/CO Binding Measurements—Rate constants for O2 bind-

ing to and dissociation from ferrous �Hb�AHSP complexes aswell as rate constants for CO binding were measured usingmethods developed for relatively unstable Fe(II) O2-Hb com-




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plexes as described by Birukou et al. (45). In these measure-ments, stable CO-�Hb complexes are injected into laser pho-tolysis cuvettes containing mixtures of buffers equilibratedwith 1 atm O2 or 1 atm CO. The mixtures in % were O2:CO �100:0, 75:25, 50:50, 25:75, and 0:100. The CO-�Hb sampleswere photolysedwith a 0.5-�s, 577-nmdye laser pulse. Absorb-ance time courses for O2 and CO rebinding to deoxy-�Hbweremeasured on �s time scales at 436 nm, and then time coursesfor replacement of the transiently bound O2 by CO were mea-sured on millisecond to seconds time scales at 424 nm (see Fig.1 in Ref. 45). This method prevents significant autooxidationduring the measurements of O2 binding because the sampleremains in the Fe(II)-CO form in between laser photolysispulses. The dependence of the pseudo first order rates for thefast and slowphases onO2 andCOwere fitted globally to obtainvalues for the association rate constants for O2 and CO bindingto �Hb, k�O2 and k�CO, and the rate of O2 dissociation, kO2, asdescribed in Birukou et al. (45). Our measurements were madeusing 100 mM potassium phosphate buffer, pH 7.0, at 20 °C.Similar kinetic measurements of O2 and CO binding to native

and recombinant wild-type Fe(II)-�Hb�AHSP complexes weremade by Vasseur-Godbillon et al. (28).

RESULTS

A Proline Residue Is Precisely Positioned in Loop 1 of AHSP toPromote Autooxidation of �Hb—The Asp-29–Pro-30 peptidebond in loop 1 of AHSP undergoes cis-trans isomerization thatis coupled with small changes in the packing of the three �-hel-ices in AHSP (1, 46). Mutation of AHSP Pro-30 to a range ofother residue types abolishes cis-trans peptidyl isomerizationof AHSP and leads to a consistent 4-fold reduction in the rate ofO2-�Hb autooxidation (11, 25). We previously proposed thatcis-peptidyl Pro-30 makes physical contact with O2-�Hb thatpromotes autooxidation (25). However, it is possible that otherchanges in AHSP conformation related to cis-trans isomerizationmight explain the role of Pro-30 in �Hb autooxidation. To inves-tigate this possibility, we introduced proline mutations intoAHSPP30A at position 28 (AHSPP30A/N28P), 29 (AHSPP30A/D29P),31 (AHSPP30A/L31P), or 33 (AHSPP30A/S33P) as shown in Fig. 2A.Weargued thatproline residues in thesepositionsmight still allow

124

120

116

7.8 7.4 7.0

87

23

90

40

7788

89

24

81

25

55

AHSP

55

124

120

116

7.8 7.4 7.0

87

23

90

40

7788

89

24

81

25

AHSPP30A

124

120

116

7.8 7.4 7.0

AHSPP30A/L31P

124

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7.8 7.4 7.0

AHSPP30A/N28P

124

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7.8 7.4 7.0

AHSPP30A/D29P

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7.8 7.4 7.0

AHSPP30A/S33P

Helix 1 Helix 2Pro-30

AHSP KANKDLISAGLKEFSVLLNQQVFNDPLVSEEDMVTVVEDWMNFYINYYR

P30A -------------------------A-----------------------P30A/N28P -----------------------P-A-----------------------P30A/D29P ------------------------PA-----------------------P30A/L31P -------------------------AP----------------------P30A/S33P -------------------------A--P--------------------

5... 10........20........30........40........50...

A

D29A ------------------------A------------------------P30

B

1H chemical shift (ppm)

15N

che

mic

al s

hift

(ppm

)

C

D

AHSP αHb

H

GS33

N28D29

L31

E

F

G

H

FIGURE 2. Conformational exchange in AHSP is dependent on the position of proline residues in loop 1. A, shown is the sequence of the first two �-helicesand intervening loop 1 of wild-type human AHSP and AHSP mutants used in this study. Conserved positions relative to the wild-type sequence are marked withdashes. B, shown is the structure of the bis-histidyl �Hb�AHSP interface (white, PDB 3IA3; the Pro-30 side chain is shown in space-fill) with the AHSPP30A structureoverlaid (black, PDB 1Z8U). The positions of side chains mutated to proline in AHSPP30A are shown in space-fill. C and D, portions of the assigned 15N-HSQCspectra of AHSP (800 �M) and AHSPP30A (900 �M). E–H, portions of the 15N-HSQC spectra of AHSPP30A/N28P (400 �M), AHSPP30A/D29P (550 �M), AHSPP30A/L31P (900�M), and AHSPP30A/S33P (530 �M) are shown. All 15N-HSQC spectra were recorded at 15 °C in 20 mM sodium phosphate buffer, pH 6.9 –7.0.




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AHSP to undergo conformational exchange, but the proline sidechainswould presumably not preserve contactswith�Hb that aremadeby thenativePro-30.The locationsof the introducedprolinemutationsat the�Hb�AHSP interfaceare shown inFig. 2B.Wedidnot mutate Val-32 because this side chain packs into the hydro-phobic core between the AHSP helices.The propensity of the mutant AHSP proteins to undergo

cis-trans isomerization was assessed from 15N-HSQC NMRspectra. A portion of the 15N-HSQC spectrum of wild-typeAHSP is shown in Fig. 2C, with peaks arising fromN-HNgroupsin the same residue in cis-prolyl and trans-prolyl conformers ofAHSP labeled and joined by a dashed line. In the AHSPP30Aspectrum, a single N-HN peak was observed for each residue,consistent with a single AHSP conformer (Fig. 2D and Table 1)(1, 46). The spectrum of AHSPP30A/N28P contained an addi-tional set of weaker HSQC signals compared with AHSPP30A(Fig. 2E, arrows, and Table 1) that was consistent with �20% ofmolecules adopting an alternative conformation, most likelydue to isomerization of the Phe-27–Pro-28 peptide bond. The15N-HSQC spectra of AHSPP30A/D29P, AHSPP30A/L31P, andAHSPP30A/S33P (Fig. 2, F–H) were all consistent with a singleprotein conformation, which indicated that prolines at posi-tions 29, 31, and 33 do not result in measurable cis/transisomerization. Chemical shift analyses were consistent withminor conformational changes in AHSP proximal to loop 1 andindicated that none of themutations conferred gross structuralchanges (not shown). These results suggest that only cis-pep-tide bonds before Pro-30 or Pro-28 result in a significantamount of cis/trans isomerization.The effect of AHSP prolinemutations on �Hb autooxidation

is shown in Fig. 3. Autooxidation ofO2-�Hb to bis-histidyl�Hbwas measured by changes in the visible absorption spectrum of�Hb (Fig. 3,A and B). The absorbance time courses fit to singleexponential expressions and yielded first-order rate constantsfor autooxidation (kautoox; Fig. 3C and Table 1). AHSPP30Ashowed a 4-fold reduction in the value of the rate constantcompared with that of wild-type AHSP, as previously described(11, 25). All introduced-proline mutants had the same lowactivity as the AHSPP30A (Fig. 3C and Table 1). A control muta-tion AHSPD29A had the same activity as wild-type AHSP. Theresults indicate that the positioning of Pro-30 in loop 1 iscritical for function. In addition, the observation that freeAHSPP30A/N28P showed cis/trans isomers in solution, yet con-

ferred the same slowed rate of O2-�Hb autooxidation asAHSPP30A, suggests that the propensity of free AHSP toundergo conformational changes does not in itself contributeto O2-�Hb�AHSP autooxidation activity. We have shown pre-viously that stabilization of the cis-peptidyl Asp-29–Pro-30AHSP conformation does correlate with increased autooxida-tion activity (25). Together the above data are consistent withthe hypothesis that the effects of Pro-30 are mediated throughdirect contact between loop 1, containing the Pro-30 residue,and �Hb.Free Liganded �Hb Monomer Adopts Native-like Secondary

Structure—We next used NMR spectroscopy to probe forstructural differences between �Hb in the native Hb tetramerand free �Hb in solution. The aim of these studies was to probefor structural changes in�Hb that could explain themore rapid

TABLE 1�Hb-interaction parameters for AHSP mutants

Protein Kautoox, pH 7.0, 30 °C Residues N-HN peaksa

h�1

AHSP 0.59 � 0.07 90b 121bAHSPP30A 0.14 � 0.01 90 80AHSPD29A 0.53 � 0.02 90 134bAHSPP30A/N28P 0.13 � 0.02 90 113AHSPP30A/D29P 0.14 � 0.03 90 84AHSPP30A/L31P 0.14 � 0.05 90 85AHSPP30A/S33P 0.14 � 0.03 90 81

a Spin systems with characteristic chemical shifts for Asn, Gln, Arg, and Trp sidechains were excluded.

b The AHSP construct used comprises residues 1–90 with the C-terminal unstruc-tured residues removed (1). Approximately 50% of residues gave rise to two setsof signals in triple resonance NMR spectra, corresponding to the cis/trans AHSPconformers. Differences in signal overlap in HSQCs results in a total of 121 re-solved N-HN peaks for WT AHSP and 134 resolved peaks for AHSPD29A.

0

0.2

0.4

0.6

0.8

D29AWT P30AN28P D29P L31P S33P

0 5 10 15

0.08

0.1

0.12

0.14

0.16

Time (hours)

AHSP variant

kau

toox

(h

–1)

Abs

at 5

75.5

nm

450 500 550 600 650 700

Abs

orba

nce

Wavelength (nm)

0.05

0.1

0.15

0

575.5 nmA

B

C

0.06 AHSP

AHSPP30A

AHSPP30A/D29P

AHSPD29A

O2-αHb + AHSP

P30A

FIGURE 3. The position of proline in AHSP loop 1 is important for function.A, conversion of O2-�Hb�AHSP to bis-histidyl �Hb�AHSP was monitored by adecrease in intensity of the absorption band at 575.5 nm. B, time courses forconversion to bis-histidyl �Hb in the presence of AHSP or selected AHSPmutants are shown, with fits to an exponential function. C, rate constants forO2-�Hb conversion reactions using AHSP and AHSP mutants shown in Fig. 1Bare shown (average � S.D. for 3–9 repeat assays). Mutation of Pro-30 to Ala(AHSPP30A) reduces the rate constants by a factor of 4. The control mutation(AHSPD29A) had no significant effect. Introducing Pro at position 28, 29, 31,and 33 failed to rescue any activity.




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autooxidation observed in vitro andprovide a suitable referencepoint for subsequent NMR studies of �Hb�AHSP complexes.We found that sequential backbone assignments could not beobtained for residues 104–126 (corresponding to helices G andH), as also reported previously by Martineau and Craescu (47).The absence of NMR signals corresponding to these residuessuggests that theymight be in intermediate chemical exchange,perhaps as a result of �Hb self-association.In line with that possibility, we noted a marked decrease in

SEC peak elution timewith increasing�Hb load concentration.This behavior is typical of protein self-association and is con-sistent with previous studies of �Hb (48, 49). We reasoned thattreatment of �Hb with pMB, which covalently modifies Cys-104 of �Hb and inhibits Hb chain homo (48) and hetero inter-actions (50), would allow more complete NMR data to beobtained. It has previously been shown that pMB treatment hasno effect on theO2 and CO binding parameters of isolated �Hb(51). Light scattering was used to monitor the self-associationstatus of CO-�Hb before and after pMB treatment. We foundthat untreated CO-�Hb eluted from SECwith a light scatteringdata profile characteristic of reversible self-association, with amarked increase inMw toward the center (i.e. the highest con-centration) of the elution peak (Fig. 4A, eachMwmeasurementis indicated with a red �). In contrast, pMB treated CO-�Hb(CO-�HbPMB) eluted with a constant Mw of 16.7 � 1.3 acrossthe peak (Fig. 4A, black dots), indicating that CO-�HbPMB ispredominantly monomeric (formula weight of 16,053).The 15N-HSQC spectrum of CO-�HbPMB (Fig. 4B, black)

contained numerous additional peaks (labeled) that were notpresent in the untreated CO-�Hb spectrum (Fig. 4B, red) andsequential backbone assignments could now be obtained for130/134 non-proline residues of CO-�HbPMB. Comparison ofthe CO-�HbPMB and CO-�Hb backbone chemical shifts indi-cated that only the modified Cys-104 and immediately sur-

rounding residues were strongly influenced (not shown), con-firming that pMB modification does not substantially alter thetertiary structure of �Hb, consistent with functional measuresof O2 and CO binding (51). The �Hb self-association interfaceis clearly identified by theNMRdata (Fig. 4C, red/orange) and isthe same face that interacts with �Hb (Fig. 4C, surface) to formthe �Hb1�Hb1 heterodimer.

To obtain information about the secondary structure ofmonomeric CO-�HbPMB, we used TALOS� (52) to derive �and � backbone angle predictions from NMR chemical shiftdata. Seven helical secondary structure elements could beclearly identified corresponding to the A-C and E-H helices(Fig. 5). The angles derived by TALOS� were very similar toangles determined from crystal structures of native CO-Hb(PDB 2DN3) (53), with correlation coefficients of 0.82 (�) and0.87 (�) over 127 residues. One difference was that TALOS�consistently assigned the C helix region as �-helix rather thanthe 310-helix that is identified by the program DSSP (54) basedon crystal structure coordinates (PDB 2DN3).To obtain information about the heme pocket, we assigned

His side chain resonances for CO-�HbPMB (Fig. 6 and Table 2).His-58 andHis-87 side chain resonances displayed large upfielddeviations from random coil chemical shifts consistent withpositions above or below the plane of the heme ring. The chem-ical shifts were very similar to values previously obtained forCO-�Hb at pH 5.3–5.7, 36 °C, (55, 56), and the �Hb subunit ofintact CO-Hb at pH 6.9, 29 °C (30, 57), strongly supporting theconclusion that free �Hb adopts a heme pocket structure sim-ilar to that in Hb tetramers, with the heme iron bound throughN�2 of His-87. In summary, preparation of monomeric �Hbthrough treatment with pMB allowed us to obtain backboneand His side chain chemical shift data suitable for comparativeNMR studies with �Hb�AHSP complexes.

14 15 160.0

0.5

1.0

Elution volumeA

bs (

280

nm)

Mw (

kDa)

10

15

20

25A

CO-αHbCO-αHbPMB

9 8 7

125

120

115

F117

Y24

T108

F33

V107

M32

L125L105

D126

L109

H103

H122A123

C104R31,

F36

A110

F98A111

E30H20S102

T118S35H112

L113

L100A115

C


15N

che

mic

al s

hift

(ppm

)

B

G

H

B

C

A

E

B

F

βHb/αHb

αHb

CO-αHb (red) CO-αHbPMB (black)

FIGURE 4. Identification of the �Hb homodimerization interface. A, self-association of �Hb was inhibited by treatment with pMB. SEC traces are shown forCO-�Hb (red) or CO-�HbPMB after reaction with a 4-fold molar excess of pMB (black). Molecular weight from light scattering measurements is plotted acrosseach peak (red/black symbols). B, portions of the 15N-HSQC for CO-�Hb (red) and CO-�HbPMB (black) were recorded at 15 °C in 20 mM sodium phosphate, pH 6.9.Assignments are shown for new peaks in the CO-�HbPMB spectrum that are not present in the CO-�Hb spectrum. C, shown are a ribbon (�Hb) and surface (�Hb)representation of the �Hb/�Hb dimer from CO-Hb (PDB 2DN3). Residues missing in the CO-�Hb spectrum (red) or experiencing significant chemical shiftdifference between CO-�Hb and CO-�HbPMB (orange) map to the tight �Hb-interaction face, which is, therefore, also identified as the �Hb homodimerizationface.




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CO-�HbRetains Near-native Secondary Structure andHemeLigation in Complex with AHSP—To understand how AHSPpromotes autooxidation of �Hb, we examined chemical shiftdifferences between free CO-�HbPMB and the CO-�Hb�AHSPcomplex. Widespread changes in the 15N-HSQC of CO-�Hbwere observed upon binding of AHSP (Fig. 7A) despite thecomplex remaining in the CO-liganded form, as confirmed byUV-visible spectroscopy (not shown). A plot of backbonechemical shift differences between CO-�HbPMB and CO-�Hb�AHSP reveals that the B-C corner and the F, G, and Hhelices of �Hb are most affected (Fig. 7B).

The B-C corner, helix G, and helix H comprise the AHSPbinding face of �Hb (1, 2, 25), and chemical shift changes are

expected here as a result of the interface forming (Fig. 7B, graybackground shading). In contrast, chemical shift changes inhelix F cannot be explained by proximity to the AHSP surface,which implies that they arise froman induced structural changein �Hb. However, NMR signals arising from the F helix regionhad similar line widths to signals from other regions of the �Hbsubunit, and TALOS� derived � and � angles indicated thatthe F helix region remains well ordered with similar secondarystructure in the absence and presence of AHSP (Fig. 5).His-87 side chain resonances remained strongly shifted in

the complex with AHSP (Fig. 8A, red), with similar chemicalshift values to the free CO-�HbPMB form (Fig. 8A, black). Thisprovides strong evidence that His-87 remains the heme-ligat-ing residue in CO-�Hb�AHSP. In summary, AHSP binding toCO-�Hb results in small but significant conformationalchanges in helix F even though the �-helical character andheme binding function are retained. As such, these findingscontrast with a previous x-ray crystallographic study in whichwe reported thatO2-�Hb�AHSPP30A has a highly unusual heme

A B C E F G H

90

–90

0

CO-αHb:AHSP

90

–90

0

CO-αHbPMB

90

–90

0

9010 20 100 110 12030 40 50 60 70 80 130 140αHb residue number

CO-αHb:AHSPP30A

ψ

ψ

ψ

φ

φ

φ

90

–90

0

90

–90

0

90

–90

0

CO-αHb:AHSP

CO-αHbPMB

CO-αHb:AHSPP30A

FIGURE 5. NMR chemical shift-derived � angles for �Hb in the absence and presence of AHSP. Backbone � and � angle predictions were calculated fromHN, 15N, 13C�, and 13C� chemical shifts using the program TALOS� (52). Predictions for CO-�HbPMB, CO-�Hb�AHSP, and CO-�Hb�AHSPP30A are displayed as barswith a height �1 S.D. The � and � angles from the �Hb subunit of a CO-HbA crystal structure (PDB 2DN3) are superimposed (filled circles) with �-helix (blackbars) and 310 helix (white bars) secondary structure elements as assigned by DSSP (54).

8.0 7.5 7.0

220

200

180

50

20

72

112

45

89


15N

che

mic

al s

hift

(ppm

)

A8 47 19


15N

che

mic

al s

hift

(ppm

)

B

His-87His-58

240

220

200

180

160

FIGURE 6. Histidine region of the 15N-HSQC spectrum of CO-�HbPMB. A,shown is a portion of 15N-HSQC spectrum of CO-�HbPMB (0.8 mM) in 20 mM

sodium phosphate, pH 7.0, at 15 °C. The heme pocket His-58 and His-87 spinsystems contain highly shifted resonances (linked by solid lines). Note thataxes are discontinuous to remove large empty regions of the spectrum. B,shown is the central region of the spectrum shown in A with peaks assignedto surface histidine side chains.

TABLE 2NMR resonances of His side chains for CO-�HbPMB and CO-�Hb�AHSPResonances for CO-�HbPMB in 10 mM sodium phosphate, pH 7.0, at 288 K andCO-�Hb�AHSP in 10 mM sodium phosphate, pH 7.0, at 298 K.

His residue N�1 N�2 H�2 H�1 H�1

20 191.0 182.7 7.20 8.2145 229.0 175.1 7.27 7.9950 201.4 181.2 7.06 8.0758 250.6 153.8 4.47 8.3072 188.1 182.8 7.44 8.3887 163.3 218.2 1.02 1.47 9.4089 218.0 182.0 6.92 7.74112 187.3 179.1 6.76 8.3858 (with AHSP) 250.1 153.8 4.48 8.0587 (with AHSP) 163.2 218.6 1.10 1.43 9.31




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site geometry wherein the F helix is disordered, and heme iscoordinated by His-58 in helix E (1). It is possible that theunusual crystal structure is characteristic of theO2- and not the

CO-�Hb�AHSP complex but, as described below, that nowseems unlikely at least for the O2-�Hb�AHSP complex insolution.AHSP Pro-30 Perturbs the Proximal Heme Pocket of �Hb—

Because mutation of AHSP Pro-30 to Ala or other residue typesleads to a 4-fold decrease in the rate of O2-�Hb�AHSP autooxida-tion, we reasoned that CO-�Hb�AHSP and CO-�Hb�AHSPP30Acomplexes would display structural differences that give insightinto the mechanism underlying AHSP-mediated �Hb autooxida-tion.We, therefore, obtainedbackbone resonanceassignments forCO-�Hb in complex with AHSPP30A using standard triple reso-nance methods. Compared with wild-type AHSP, the AHSPP30Amutant inducedmore limited perturbations of CO-�Hb (Fig. 7C).A direct comparison of CO-�Hb chemical shifts in the CO-�Hb�AHSP and CO-�Hb�AHSPP30A complexes shows that the wild-typeAHSP causes significantly larger chemical shift perturbationsto theN-terminal fragment of �Hb helix G and the whole of helixF (Fig. 7D), which suggests that AHSPPro-30makes contacts thatare transmitted through the �Hb structure and lead to perturba-tions of helix F.Interestingly, we could not obtain backbone assignments for

residues 135–138 ofCO-�Hb in complexwithwild-typeAHSP,and signals for these residues appeared to be absent from theNMR spectra. In NMR spectra of CO-�Hb�AHSPP30A, residues135–138 were assigned and predicted as the regular �-helix byTALOS� (Fig. 5). Residues 135–138 of native �Hb are in aregion of helix H that contacts helix F (Fig. 7E), which suggeststhat loss of NMR signals could reflect a destabilization of helix

A

CO-αHbPMB versus CO-αHb:AHSP

0

0.2

0.4

∆δC

α,N

,HN

(ppm

)

10 20 30 40 50 60 70 80 90 100 110 120 130 140Residue number

0

0.6

0.8

CO-αHb:AHSP versus CO-αHb:AHSPP30A

0

0.2

0.4

10 20 30 40 50 60 70 80 90 100 110 120 130 140Residue number

0

0.6

0.8

CO-αHbPMB versus CO-αHb:AHSPP30A

0

0.2

0.4

10 20 30 40 50 60 70 80 90 100 110 120 130 140Residue number

0

0.6

0.8

F G HA B C EB

C

D

10 9 8 7130

125

120

115

110

105CO-αHb(black)CO-αHb:AHSP(red)


15N

che

mic

al s

hift

(ppm

)

AHSP-binding face

AAAAAAAAAAAAAAAAAAAAAAAHeme

F

G

E

AHSP

Pro-30

αHb

H B

CLoop 1

A

E

∆δC

α,N

,HN

(ppm

)∆δ

Cα

,N,H

N (p

pm)

aa135–138

FIGURE 7. AHSP binding perturbs helix F of CO-�HbPMB. A, shown are 15N-HSQC spectra of CO-�Hb and CO-�Hb�AHSP recorded at 298 K in 20 mM sodiumphosphate, pH 7.0. B, shown are combined, weighted, chemical shift differences (N,HN,C�) between CO-�HbPMB (15 °C) and CO-�Hb�AHSP (25 °C). C, shownare chemical shift differences between CO-�HbPMB (15 °C) and CO-�Hb�AHSPP30A (25 °C). D, shown are �Hb chemical shift differences between the CO-�Hb�AHSP and CO-�Hb�AHSPP30A complexes (both at 25 °C). Horizontal dotted and dashed lines indicate the average and increments of 1 S.D., respectively. E,shown is a molecular model of the O2-�Hb�AHSP complex generated by substituting O2-�Hb (PDB 2DN1) for the bis-histidyl �Hb subunit in the bis-histidyl�Hb�AHSP crystal structure (PDB 3IA3). Residues in �Hb experiencing a chemical shift difference (N,HN,C�) �0.2 ppm between the AHSP and AHSPP30A

complexes are colored red (residues missing assignments in one or both complexes are black).

His-58His-87

αHbPMB (black) αHb:AHSP (red)

8.6 7.8

240

220

200

180

160

15N

che

mic

al s

hift

(ppm

)


1.6 0.8

FIGURE 8. His-87 is the heme ligand in O2-�Hb�AHSP. Overlay of portion ofthe 15N-HSQCs of CO-�HbPMB (20 mM sodium phosphate, pH 7.0, 15 °C; black)and CO-�Hb�AHSP (20 mM sodium phosphate, pH 7.0, 25 °C; red) show thatthe His-58 and His-87 side chains experience only minor chemical shift per-turbations upon AHSP binding.




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H in �Hb�AHSP related to changes in structure of the F helix.Overall the results here indicate that AHSP Pro-30 makes con-tacts that perturb helices F, G, and H of �Hb, which togetherconstitute the proximal heme pocket of �Hb (Fig. 7E).Comparison of the Iron-Ligand Structures in O2-�Hb and

O2-�Hb�AHSP—NMRspectroscopicdata indicated thatHis-87 isretained as the iron binding His residue in CO-�Hb�AHSP, butthese data do not provide detailed information about the exactgeometries of the axial iron ligands. In addition, we could notdetermineNMR structures for theO2-�Hb�AHSP complexes dueto autooxidation duringNMRdata collection. To probe the elec-tronic structure and coordination environment around theheme iron more directly, we acquired x-ray absorption spectraat the iron K-edge for the physiologically more relevantO2-�Hb andO2-�Hb�AHSP complexes. Spectra were recordedat 10K tominimize photo-damage and increase the importanceof multiple-scattering contributions to the EXAFS (58, 59).Samples were prepared in buffer containing 0.88 M sucrose,which allowed the formation of a homogeneous glass uponfreezing (59). This procedure permitted measurements on

the unstable O2-�Hb�AHSP complex to be performed overextended time periods without conversion to the bis-histidylferric �Hb form. The x-ray absorption near edge structure(XANES) spectra of O2-�Hb and O2-�Hb�AHSP (not shown)show no irradiation damage, and the similarity to spectra of thelow-spin Fe(III) proteins metleghemoglobin, oxymyoglobin,and oxyleghemoglobin (60) suggest that all have a low-spinFe(III) center, which is consistent with an Fe(III)�-O2

. species(61). This classification is also supported by the diagnostic oxi-dation- and spin-state resonance Raman marker bands (1, 62).The slightly lower edge energy of O2-�Hb�AHSP comparedwith free O2-�Hb suggests a lengthening of the Fe-O2 bond inthe AHSP complex.EXAFSdata obtained fromO2-�HbandO2-�Hb�AHSPwere

fitted to models for the iron sites using restrained and con-strained multiple-scattering models (58, 63–65). The observedand calculated EXAFS and the corresponding Fourier trans-forms are shown forO2-�Hb andO2-�Hb�AHSP in Fig. 9,A–D,and the derived molecular structures for O2-�Hb andO2-�Hb�AHSP are shown in Fig. 9, E and F, respectively. The

A

B

C

D

0k (Å-1)

4 8 12 16

-8

χ(k)

× k

3

0

-4

8

4

0k (Å-1)

4 8 12 16

-8

χ(k)

× k

3

0

-4

8

4

Fou

rier-

Tran

sfor

m M

agni

tiude

-2

2

0

6

4

8

12

10

20 1 43 5Distance (Å)

20 1 43 5Distance (Å)

Fou

rier-

Tran

sfor

m M

agni

tiude

-2

2

0

6

4

8

12

10

O2-αHb O2-αHb:AHSP

E F

Fe–Np = 2.00 Å

Fe–Nε = 2.04 Å

Fe–O1 = 1.83 Å

Fe–O1–O2 = 130˚

O2-αHb O2-αHb:AHSP

Nε

Np

O1O2

Np

Np

Np

Fe–Np = 2.01 Å

Fe–Nε = 2.08 Å

Fe–O1 = 1.86 Å

Fe–O1–O2 = 135˚Nε

Np

O1O2

Np

Np

NpFe Fe

FIGURE 9. EXAFS analyses of the heme sites of O2-�Hb and O2-�Hb�AHSP. Shown are EXAFS (A) and Fourier-transform amplitude (B) of EXAFS of O2-�Hb.Shown are EXAFS (C) and Fourier-transform amplitude (D) of EXAFS of O2-�Hb�AHSP. Shown are the observed data (solid line), data calculated from the best fitto the model (dashed line), residual data (dotted line), and window used in the Fourier filter (dot-dash). Molecular structures and Fe-ligand bond lengths andFe-O–O bond angle for O2-�Hb (E) and O2-�Hb�AHSP (F) were obtained from EXAFS data.




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bond length/angle and other fitted parameters obtained arelisted in Table 3.The errors in the iron-ligand bond lengths determined by

EXAFS were estimated as the r.m.s. of contributions from bothrandom and systematic errors. The random (statistical) errorsdue to noise in the data were estimated by Monte-Carlo calcu-lations (42), and the systematic errors were assigned a conserv-ative consensus value, 0.02 Å (44). For O2-�Hb, the calculatedstatistical errors were 0.002 Å for Fe-Np, 0.008 Å for Fe-N�, and0.005 Å for Fe-O2. For O2-�Hb�AHSP, Monte-Carlo calcula-tions yielded r.m.s. errors of 0.003 Å for Fe-Np, 0.012 Å forFe-N�, and 0.006Å for Fe-O2. These statistical errorswere com-bined with the maximum systematic error to obtain the esti-mated maximum r.m.s. errors in the reported bond lengths(Table 3). Monte-Carlo calculations yielded an r.m.s. error of1.7° and 2.3° for the Fe-O-O bond angle in O2-�Hb andO2-�Hb�AHSP, respectively. These errors were combined withthe respective r.m.s. errors of 2.1° and 3.2°, resulting from vary-ing the O-O distance by �0.03 Å (Table 4) to give respectiveestimated maximum r.m.s. errors of 2.7° and 3.9° for O2-�HbandO2-�Hb�AHSP.Therefore, the error in this bond angle esti-mated by two independent methods is �3–4°. The resultingFe-O-O angles determined from restraining the starting anglebetween 115° and 175° and allowing the fit to go to convergencethen removing the restraint were 130° (for 6 minima and 143°for 1 minimum; the latter was a local minimumwith a higher Rvalue and a low So2 value for a heme protein) in O2-�Hb (Table5) and 135° (for 6 minima and 101° for 1 minimum) inO2-�Hb�AHSP (Table 6). The fit obtained for the Fe-O-O bondangle of 101°, when the angle was initially restrained to be 115°and then this restraint was removed and the fit allowed to go toconvergence, is likely to be a falseminimum.This is because it isnot chemically reasonable and is not consistent with the simi-larity in the shape of theXANES ofO2-�Hb andO2-�Hb�AHSP(not shown), as XANES is sensitive to bond angles. These anal-yses show that the bond lengths are independent of all of thesechanges in starting models and constraints/restraints, and thebond angles relax to the non-constrained values shown inTable3. These detailed analyses of parameter space provide confi-dence that the EXAFS fits are robust and are not trapped in falseminima. Similarly, varying the tilt or rotation of the proximalimidazole did not significantly alter the fitted bond lengthparameters (not shown).

Overall the iron-site geometry of O2-�Hb and O2-�Hb�AHSP is similar to that derived from high resolution (1.25 Å)crystal structures of O2-HbA (53). However, the Fe-N� andFe-O bond lengths were shorter in O2-�Hb than in O2-�Hb�AHSP (2.04 cf. 2.08 Å and 1.83 cf. 1.86 Å). Although these dif-ferences arewithin the conservative estimatesof accuracygiven inTable3 (0.02Å), thesystematicerrorcontributionsareexpected tobe the same for bothproteins and, hence, the errors obtained fromMonte Carlo calculations are a better indication of the relativeerrors. For theFe-O2bond these errorswere 0.005 and0.006Å inO2-�Hb and O2-�Hb�AHSP, respectively, indicating that the0.03 Å lengthening of Fe-O2 in O2-�Hb�AHSP is significant. Itshould be emphasized that the Fe-O2 bonds are substantiallyshorter than those of the other heme-ligand bonds, which iswhy the precision is so high. This is due to the rapid increase inthe importance of single and multiple-scattering contributionsas the bond length decreases.The O2-�Hb�AHSP data were also fitted to a model where

the iron-ligand bond lengths and Fe-O-O bond angle were con-strained to be that in the O2-�Hb�AHSPP30A structure (1), andE0, S02, and the Debye-Waller factors were fitted (Table 3, bot-tom row). The resulting temperature factors were unacceptablyhigh (0.059 Å2 for the proximal imidazole N�, 0.079 Å2 for O1,and 0.080 Å2 for O2) with an R value of 32.5%. Clearly, thismodel does not fit the solution structure.The ability of this methodology to determine precise bond

length and angle information on axial ligands has been estab-lished in studies of porphyrin model complexes and by thedetermination of precise bonding parameters in the heme envi-ronments of met- and deoxymyoglobin (41, 66) before the pub-lication of the high resolution protein crystallographic struc-tures (67, 68). Even taking the small differences betweenO2-�Hb�AHSP and O2-�Hb into account, it is clear that theinitial solution complex of O2-�Hb�AHSP has a “normal” coor-dination geometry similar to that in free O2-�Hb chains.AHSP Reduces Reactivity of �Hb with O2 and CO—In 2006,

Vasseur-Godbillon et al. (28) reported that AHSP bindingreduces the O2 and CO affinities of �Hb roughly 3–5-fold. Werepeated these ligand binding experiments with wild type andtwo Pro-30 mutants of AHSP. Table 7 presents a summary ofthe rate constants forO2 andCOassociation (k�O2 and k�CO) to,and O2 dissociation (kO2) from, �Hb in the presence and

TABLE 3A comparison of Fe-ligand geometry from EXAFS and X-ray crystallographic analyses

Species MethodFe-Ligand distances Fe-O-O Debye-Waller factors, �2 Other fit parameters

Np N� O1 O1-O2 angle (°) Np N� O1 O2 E0 S02 R

Å Å eV %O2-�Hb Crystala 2.02 (8) 2.07 (8) 1.82 (8) 1.23 (8) 124

MSb 2.00 (2) 2.04 (2) 1.83 (2) 1.21 (2) 130 (3) 0.002 0.001 0.003 0.004 7125.1 (2) 0.92 (2) 15.9O2-�Hb�AHSP Crystalc 2.0 (5) 2.1 (5) 2.7 (5) 1.3 (5) 143

MSd 2.01 (2) 2.08 (2) 1.86 (2) 1.22 (2) 135 (3) 0.001 0.002 0.002 0.008 7125.4 (3) 0.915 (3)e 16.8MSf 2.03f 2.12f 2.74f 1.25f 143f 0.002 0.059 0.079 0.080 7131.2 0.932 32.5

a PDB 2DN1 data at 1.25 Å resolution. Bond length errors are Cruickshank diffraction-data precision indicator (DPI) as calculated by REFMAC multiplied by 21/2 to convertfrom average coordinate error to average bond length error.

b This work; 12-scan average. Error estimates in parentheses are calculated as described under “Comparison of the Iron-Ligand Structures.”c PDB 1Y01 data at 2.8 Å resolution. Bond length errors are 21/2 � DPI based on Rfree factor.d This work; 21-scan average. Error estimates in parentheses are calculated as described under “Comparison of the Iron-Ligand Structures.”e Restraint on S02 was tightened to be 0.92(2) Å2; i.e., similar to O2-�Hb.f Iron�ligand bond lengths and Fe-O-O bond angle were constrained to be those in the O2-�Hb�AHSPA30 structure (PDB 1Y01).




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absence of AHSP variants. The ratio of k�O2/kO2 provides theO2 association equilibrium or affinity constant (KO2). Asreported by Vasseur-Godbillon et al. (28), AHSP binding toO2-�Hb causes a small decrease in the rate of O2 binding and alarger 3–4-fold increase in the rate of O2 dissociation. The netresult is an �4-fold decrease in O2 affinity (Table 7). Theseresults support the XAFS data and provide direct evidence thatAHSP binding leads to weakening of the Fe-O2 bond, which in

turn leads to a significant increase in the susceptibility ofO2-�Hb to autooxidation.

AHSP binding to �Hb also slows the rate of bimolecular CObinding by a factor of�3–4-fold, as also reported previously byVasseur-Godbillon et al. (28) (Table 7). CO binding is limitedprimarily by the rate of internal bond formation and, therefore,provides a sensitive measure of iron reactivity that is primarilydetermined by interactions between the heme iron and the

TABLE 4Effect of varying the O-O bond distance restraint on the Fe-O-O angle in human O2-�Hb and O2-�Hb�AHSPThe quantities enclosed by the brackets ({}) are the values of �res used by XFIT, which is analogous to e.s.d.

SpeciesRestraint on O-O bond Fe-Ligand distances

Fe-O-O angleDebye-Waller Factors, �2

S02 R{�res} Np N� O1 O2 Np N� O1

Å Å ° Å2 %O2-�Hb 1.19 {0.01} 2.00 2.04 1.83 1.18 131.4 0.001 0.001 0.003 0.920 14.8

1.25 {0.01} 2.00 2.04 1.83 1.25 133.5 0.001 0.001 0.003 0.906 15.41.22 {0.03} 2.00 2.04 1.83 1.21 130 0.002 0.001 0.003 0.92 15.9

O2-�Hb�AHSP 1.19 {0.01} 2.00 2.08 1.86 1.19 137.6 0.001 0.002 0.002 0.916 16.81.25 {0.01} 2.01 2.08 1.86 1.25 134.0 0.001 0.002 0.002 0.915 16.81.22 {0.03} 2.01 2.08 1.86 1.22 135 0.001 0.002 0.002 0.915 16.8

TABLE 5Effect of restraining the Fe-O-O bond angle in human O2-�Hb on the MS XAFS analysisThe quantities enclosed by the brackets ({}) are the values of �res used by XFIT, which is analogous to e.s.d.

Restraint on Fe-O-O bond angle Fe-Ligand distancesFe-O-O angle (°)

Debye-Waller factors, �2

S02{�res} Np N� O1 O2 Np N� O1 O2 R

Å Å %115° {1°} 2.00 2.05 1.83 1.22 115.0 0.001 0.001 0.003 0.080 0.893 15.9125° {1°} 2.00 2.04 1.83 1.22 126.6 0.001 0.001 0.003 0.004 0.902 15.2135° {1°} 2.00 2.05 1.83 1.21 134.9 0.001 0.001 0.003 0.009 0.909 15.3145° {1°} 2.00 2.05 1.83 1.22 144.6 0.001 0.001 0.003 0.004 0.886 15.6155° {1°} 2.00 2.05 1.83 1.22 155.0 0.001 0.001 0.003 0.080 0.895 15.8165° {1°} 2.00 2.05 1.83 1.22 165.0 0.001 0.001 0.003 0.080 0.894 15.9175° {1°} 2.00 2.05 1.83 1.22 175.0 0.001 0.001 0.003 0.080 0.894 15.9No constraint 2.00 2.05 1.83 1.21 129 0.001 0.001 0.003 0.004 0.919 14.1115°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0125°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0135°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0145°b 2.00 2.06 1.84 1.22 143.4 0.001 0.001 0.003 0.004 0.884 15.6155°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0165°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0175°b 2.00 2.04 1.83 1.21 129.7 0.002 0.001 0.003 0.004 0.922 15.0

a The original fit where Fe-O-O is unrestrained is included for comparison.b Fits were performed in which the Fe-O-O angles restrained the starting angle between 115° and 175° and then allowed the fit to go to convergence, then removing therestraint.

TABLE 6Effect of restraining the Fe-O-O bond angle in human O2-�Hb�AHSP on the MS XAFS analysisThe quantities enclosed by the brackets ({}) are the values of �res used by XFIT, which is analogous to e.s.d.

Restraint on Fe-O-O Bond angle Fe-Ligand distancesFe-O-O angle

Debye-Waller factors, �2

S02 R{�res} Np N� O1 O2 Np N� O1 O2

Å ° Å2 %115° {1°} 2.01 2.08 1.86 1.22 115.0 0.001 0.003 0.002 0.080 0.914 17.4125° {1°} 2.01 2.08 1.86 1.23 126.7 0.001 0.002 0.002 0.010 0.915 17.3135° {1°} 2.01 2.08 1.86 1.22 135.1 0.001 0.002 0.002 0.008 0.915 16.8145° {1°} 2.01 2.07 1.86 1.22 144.6 0.001 0.003 0.002 0.029 0.914 17.3155° {1°} 2.01 2.08 1.86 1.22 155.0 0.001 0.003 0.002 0.080 0.914 17.4165° {1°} 2.01 2.08 1.86 1.22 165.0 0.001 0.003 0.002 0.080 0.914 17.4175° {1°} 2.01 2.08 1.86 1.22 175.0 0.001 0.003 0.002 0.080 0.914 17.4No constraint 2.01 2.08 1.86 1.22 135 0.001 0.002 0.002 0.008 0.915 16.8115°b 2.01 2.07 1.86 1.22 101 0.001 0.004 0.002 0.004 0.914 14.7125°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8135°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8145°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8155°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8165°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8175°b 2.01 2.08 1.86 1.22 135.4 0.001 0.002 0.002 0.008 0.915 16.8

a The original fit where Fe-O-O is unrestrained is included for comparison.b Fe-O-O angles were initially restrained between 115° and 175° and the fit allowed to go to convergence, then the restrain was removed and the fit allowed to go toconvergence.




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proximal histidine. The �4-fold decrease in k�CO and O2 affin-ity are consistent with conformational strain introduced on theproximal side of the heme pocket of �Hb�AHSP due to interac-tions with AHSP Pro-30. This interpretation is strongly sup-ported by the effects of Pro-30 mutations on ligand binding. Asshown in Table 7, AHSPP30A and AHSPP30Wmutations dimin-ish the effects of AHSP binding on both O2 affinity and the rateof CO association, and in the case of AHSPP30W, the observedligand binding parameters are similar to those of free �Hb.Thus, all our structural, spectroscopic, and functional data sug-gest that structural changes in the proximal heme pocket areinduced by AHSP Pro-30, causing a weakening of the Fe-O2bond and an increase in the rate of autooxidation.

DISCUSSION

AHSP promotes autooxidation of O2-�Hb to prevent it fromreacting with H2O2 under conditions of oxidative stress. Theresultant bis-histidyl heme does not react with H2O2 and,therefore, cannot generate reactive ferryl species and proteinradicals (2, 20). The bis-histidyl �Hb�AHSP structure has beenwell characterized by x-ray crystallographic and spectroscopicmethods (1, 2, 25–27), but the formation of this structure fromO2-�Hbhas been lesswell understood. The data presented hereprovide key insights as to howAHSP facilitates formation of thebis-histidyl state by making specific interactions at the�Hb�AHSP interface and fit a model, explained below, in which�Hb�AHSP complexes can adopt one of two well ordered con-formations that are intermediates in Hb assembly and achieve�Hb detoxification (Fig. 10).Structure of �Hb—By reacting pMBwith Cys-104 and block-

ing �Hb self-association, we obtained chemical shift-derivedbackbone dihedral angles and chemical shift analysis of His sidechains, which together show that the conformation of free �Hbis very similar to its structure in the native Hb tetramer. Thisconclusion is consistent with previous findings that hemepocket structure is well preserved in isolated subunits, based onfar-UV (69, 70) and near-UV CD (69–72) and homonuclearNMR studies of ferrous and ferric�Hb (47, 55, 56, 73, 74). Noneof these data provide any hints as towhy free�Hb ismore proneto oxidation and subsequent rapid loss of the heme moiety andaggregation. Similar observations have been made in other sys-tems. For example, themonomeric pointmutants of the carrierprotein transthyretin display identical x-ray crystal structuresto the wild-type protein and yet have substantially reducedthermodynamic stabilities (75). In such cases, a native-like con-formation still represents the lowest energy state available;however, the free energy well in which this conformer sits isshallower due to a reduction in the number of stabilizing inter-

actions. In the case of�Hb, stabilization is provided bymultiplesubunit interactions in the tetramer.CO- and O2-Hb Have Native-like Conformations in Complex

with AHSP—A previous x-ray crystallographic study from ourgroup suggested that O2-�Hb underwent large conformationalchanges upon binding AHSP (1), and it was assumed that thesechanges were responsible for the accelerated autooxidation. Inthat study residues Asp-74–Leu-91 of �Hb (helix F) appeareddisordered (as judged from a lack of interpretable electron den-sity), and the heme-ligating residue was switched from theusual His-87 in helix F to His-58 in helix E. However, NMR lineshape and TALOS� chemical shift data presented here showthat, in the solution state, helix F retains an ordered �-helicalstructure in CO-�Hb�AHSP. In addition, analysis of His sidechain resonances strongly suggests that His-87 retains theheme binding role. In the crystal structure, the Fe-O2 bond isunusually long (2.7 Å) (1). Such a long Fe-O2 bond is inconsis-tent with visible absorption and XANES and EXAFS spectro-scopic data (Table 3), which indicate that the iron coordination

TABLE 7AHSP��Hb ligand binding kinetic parameters at pH 7, 20 °C

Protein k�O2 kO2 k�CO KA,O2

�M�1 s�1 s�1 �M�1 s�1

�Hb 37 � 9 (40 � 2)a (50)b 29 � 7 (22 � 6)a (40)b 5.3 � 0.3 (5.2 � 0.5)a (6)b 1.3 � 0.5 (1.8 � 0.5)a (1.2)bAHSP��Hbc 23 (35)b 73 (130)b 1.4 (2)b 0.30 (0.35)bAHSPP30A��Hb 35 47 2.2 0.7AHSPP30W��Hb 28 25 3.8 1.1

a Rate parameters were taken from Birukou et al. (45).b These values were measured at pH 7.0, 25 °C by Vasseur-Godbillon et al. (28).c The values of the kinetic parameters for AHSP��Hb represent the average of two independent determinations.

AHSP

Fe(III) αHb

αHb

Fe(II) / O2-αHb

FAST

SLOW

O2 –

βHb limiting/ oxidative stress

Red

ucta

se +

O2

Red

ucta

se

P30contact

Native Hb

Hb with Fe(III)bishis-αHbBis-histidyl heme

Nativeheme site

FIGURE 10. Model for �Hb�AHSP intermediates in Hb assembly. Free �Hb(left) has a native-like conformation (gray color). Pathways for incorporation of�Hb into Hb are shown as black arrows. Deoxy Fe(II) �Hb or O2-�Hb bindAHSP (top row) with a near-native structure and are rapidly combined withavailable �Hb to produce native Hb. Fe(III) �Hb binds AHSP in the high affinitybis-histidyl structure (orange, bottom row), inhibiting release of Fe(III) �Hb toHb. Methemoglobin reductase reduces bis-histidyl �Hb to the native �Hbstructure. In Fe(II)/O2-�Hb�AHSP, interaction between AHSP Pro-30 and helixG of �Hb (star) introduces strain in the proximal heme pocket (orange shad-ing). Under conditions of limiting �Hb or elevated redox stress, autooxidationof Fe(II)/O2-�Hb to the inert bis-histidyl complex occurs (red arrows).




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geometries of O2-�Hb andO2-�Hb�AHSP are very similar. TheEXAFS data do indicate a slight lengthening of the Fe-O2 bondfrom 1.83 Å in O2-�Hb to 1.86 Å in O2-�Hb�AHSP, providingstructural evidence that AHSP promotes superoxide dissocia-tion and a decrease in O2 affinity (Table 7).

Several possible explanations exist for the differencesbetween the crystal structure described previously and thesolution structure reported here. A detergent molecule locatedclose to the heme group in the crystalmight have influenced thestructure; however, the detergent makes very few direct con-tacts with the protein. Perhaps more significantly, the crystalswere grown under a standard atmosphere making it likely thatpartial autooxidation of �Hb would occur. In addition, theintense x-ray beams used for protein crystallography can pro-mote reduction of ferric proteins (59). Amixture of heme statesarising from these processes would introduce static disorder inthe crystal, complicating analysis of the x-ray data. Neverthe-less, the crystal structure does provide evidence that helix F of�Hb is susceptible to conformational distortion in the presenceof AHSP.AHSPPro-30 Promotes Changes in the ProximalHeme Pocket

of O2-�Hb That Enhance Autooxidation—We have previouslyshown that mutation of Pro-30 to a range of other residuestypes leads to a 4-fold reduction in autooxidation of �Hb. Herewe show that introducing Pro at other positions in loop 1 didnot restore native O2-�Hb autooxidation and, although theAHSPP30A/N28P may undergo cis/trans isomerization, O2-�Hbautooxidation was not enhanced above the rate observed forAHSPP30A, which is only 2–3-fold greater than that of freeO2-�Hb (27). These data emphasize that the position of Pro-30in AHSP loop 1 is critical to enhancing autooxidation.AHSP Pro-30 is positioned adjacent to the beginning of helix

G in the bis-histidyl �Hb�AHSP complex. However, in theabsence of crystallographic data, the degree of interactionbetween Pro-30 and helix G in O2-�Hb�AHSP is unknown.Based on mutation and binding data, which show that Pro-30destabilizes theO2-�Hb�AHSP interface but not the bis-histidyl�Hb�AHSP interface (11, 25, 27), we previously proposed thatPro-30 introduces a steric clash at theO2-�Hb�AHSP interface,which is relieved upon transition to the bis-histidyl �Hb�AHSPstructure. Here we show that AHSP binding induces NMRchemical shift perturbations in F, G, and H helices of CO-�Hb,which constitute the proximal heme pocket, and that mutationof Pro-30 markedly reduces these disturbances. These findingsare consistent with a steric interaction between AHSP Pro-30and �Hb helix G that is propagated through packing interac-tions of the F, G, and H helices (represented as orange shadingof these helices in Fig. 10, top row). Bonding interactionsbetween the heme iron and proximal His-87 in helix F and thegeometry of the proximal pocket are important factors influ-encing ligand reactivity of the iron (76, 77). It is, therefore, sig-nificant that AHSP decreases O2 affinity and CO associationrates of �Hb and that mutation of Pro-30 restores ligand reac-tivity of the heme iron to near-normal levels, which suggestsstrongly that Pro-30-induced disturbance of the proximal hemepocket contributes to enhanced O2-�Hb autooxidation andlower ligand reactivity in the presence of wild-type AHSP.

O2-�Hb�AHSP and Bis-histidyl�AHSP Intermediates in HbAssembly—Our data suggest a model in which AHSP can bind�Hb in one of two well ordered conformations depending onthe oxidation/ligand state of the heme (Fig. 10). O2-�Hb isbound in a near-native conformation (Fig. 10, top), whereasFe(III) �Hb adopts a non-native globin conformation and bis-histidyl heme coordination (Fig. 10, bottom). We expect thatFe(II) deoxy �Hb and CO-�Hb will also bind to AHSP withnear-native protein conformations on the basis that theseforms of �Hb all show the same affinity for AHSP as O2-�Hb,which is 100-fold weaker than the interaction of AHSPwith thenon-native bis-histidyl Fe(III) �Hb conformation (20, 25, 27).Notably, deoxy-Fe(II) �Hb does not adopt bis-histidyl hemecoordination (26) despite the availability of an open iron axialcoordination site. These studies emphasize that specific coor-dination between the distal His-58 and the Fe(III) iron centerand the structural changes in the �Hb polypeptide chain occurin a concerted fashion during oxidation of the�Hb�AHSP com-plex and do not occur when the iron atom is reduced.Based on kinetic data, Khandros et al. (11) and Mollan et al.

(27) proposed that �Hb synthesized in the Fe(III) form in thepresence of AHSP (78) proceeds through a bis-histidyl�Hb�AHSP folding intermediate before heme reduction andincorporation into Hb (Fig. 10, solid black arrows). Bis-histidylferric �Hb can in principle be transferred to �Hb and subse-quently reduced (Fig. 10, dashed arrows); however, the rate offerric �Hb�AHSP dissociation is probably too slow to be phys-iologically relevant, and reduction almost certainly occursmore rapidly (27). Based on autooxidation rates measured invitro (11, 20, 25, 28, 29), newly reduced Fe(II)/O2-�Hb shouldbe rapidly incorporated intoHbbefore autooxidation can occurif the concentration of free �Hb is high. However, under con-ditions of limiting �Hb that are known to occur during normalerythropoiesis (79, 80), the likelihood increases for the conver-sion of Fe(II)/O2-�Hb�AHSP species to bis-histidyl ferric com-plexes (Fig. 10, red arrows). The more rapid autooxidation of�Hb in the presence of AHSP may be advantageous, as it pro-vides ameans to trap �Hb in a relatively unreactive Fe(III) statethat inhibits redox cycling and reactions with reactive oxygenspecies that could otherwise occur with free �Hb. In addition,both free O2-�Hb and met-Hb subunits are more prone toH2O2-induced ferryl and protein radical formation and precip-itation (20, 81), making it advantageous to remove freeO2-�Hband stabilize it as bis-histylmet-�Hb�AHSPunder conditions ofoxidative stress.To investigate the biological importance of autooxidation

rate, Khandros et al. (11) performed gene knock-in experimentsin mice. Mutations of AHSP Pro-30 to Ala or Trp did not dis-turb erythropoiesis (11), which suggested that a 4-fold decreasein �Hb�AHSP autooxidation rate is not sufficient to elicitchanges in erythropoiesis in the normal mouse model (11, 27).However, it is important to recognize that O2-�Hb�AHSPP30Astill autooxidizes more quickly than does free O2-�Hb (11, 25,27) and that other interactions across the �Hb�AHSP interface,in addition to those with Pro-30, are important for autooxida-tion and hemichrome formation.A transition between two metastable �Hb�AHSP structures,

as outlined in Fig. 10, avoids persistent disordered states that




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would favor unregulated reactions with an exposed heme andrapid heme dissociation. In this regard, it is interesting thatmutations of Pro-30 to Ala or Trp decreaseO2-�Hb autooxida-tion rates but enhance heme dissociation rates (11). In conclu-sion, it appears that the AHSP sequence has evolved to achievea fine balance of distorting�Hb sufficiently to promote changesin heme coordination that inhibit reactions with H2O2 withoutinducing an unacceptably high risk of heme loss or other irre-versible denaturation events.A Physiological Role for O2-�Hb�AHSP beyond the Red Blood

Cell—Recently Straub et al. (82) reported that �Hb and AHSP(but not �Hb) are expressed in arterial endothelial cells at siteswhere the endothelial cellsmake direct contact with underlyingsmooth muscle (myoendothelial junctions) and that �Hbexpression regulates NO signaling from the endothelium tosmoothmuscle. NO signaling is important formaintaining vas-cular tone and modulating the response of smooth muscle tovasodilator and vasoconstrictor messengers. Importantly, theeffect of heme proteins on NO signaling is dependent on theiron oxidation and ligand state. Fe(III) heme interacts only veryweakly with NO, providing no barrier to NO diffusion. In con-trast, O2-heme reacts rapidly with NO to form nitrate (NO3

�)and Fe(III) heme, thus quenching NO signaling (83–85). In theendothelium, the reaction of NO with O2-�Hb�AHSP isexpected to generate bis-histidyl �Hb�AHSP. Therefore, inendothelial cells the balance between O2-�Hb�AHSP andFe(III) bis-histidyl �Hb�AHSP will impose restrictive/permis-sive states for NO signaling, respectively. In this regard,autooxidation and the interaction of Fe(III) bis-histidyl�Hb�AHSP with reductase systems in endothelial cells are crit-ical processes. Understanding these processes is now necessaryfor a full understanding of NO signaling in the vasculature.

Acknowledgments—We thank Dr. Chien Ho for the kind gift of thepHE7 plasmid and Drs. Aviva Levina, Hugh Harris, MatthewLatimer, and Allyson Soo Hoo for assistance at Stanford SynchrotronRadiation Lightsource. This work was performed with support fromthe Australian Synchrotron Research Program, which was funded bythe Commonwealth of Australia under the Major National ResearchFacilities Program. Portions of this research were carried out at theStanford Synchrotron Radiation Lightsource. The Stanford Synchro-tron Radiation Lightsource Structural Molecular Biology Program issupported by the Department of Energy, Office of Biological and Envi-ronmental Research, and by the National Institutes of Health,National Center for Research Resources, Biomedical TechnologyProgram.

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Joel P. Mackay, Peter A. Lay and David A. GellJason A. Lowry, Todd L. Mollan, Eugene Khandros, John S. Olson, Mitchell J. Weiss,

Claire F. Dickson, Anne M. Rich, William M. H. D'Avigdor, Daniel A. T. Collins,-hemoglobin and Weakens the Iron-Oxygen BondαOxy-

-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket ofα

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