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

Background: α-Hemoglobin stabilizing protein (AHSP) is a hemoglobin chaperone. Results: AHSP causes a subtle perturbation of the proximal heme pocket of O2-α-hemoglobin, lengthening the Fe-O2 bond and enhancing O2 dissociation. Conclusion: Pro-30 in wild-type AHSP promotes αHb autooxidation by introducing strain into the proximal heme pocket. Significance: αHb·AHSP complexes are intermediates in Hb assembly and achieve αHb detoxification. 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.

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 heterodimeric complex (see Fig. 1A) (1)(2)(3) and is essential for normal 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 disproportionation to H 2 O 2 and O 2 . H 2 O 2 is a key mediator of oxidative 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)(16)(17). Additionally, autooxidation to the ferric state renders Hb incapable of O 2 transport and greatly enhances the rates of hemin loss and globin denaturation.
Compared with mature Hb, isolated ␣Hb and ␤Hb chains produce higher levels of reactive oxygen species (18 -20), presumably linked to a loss of structural integrity of the heme pocket. Oxidative damage occurs in erythroid cells of ␤-thalassaemic mice that contain elevated levels of unpaired ␣Hb (21)(22)(23)(24). Kong et al. (6) showed similar increases in reactive oxygen species in AHSP Ϫ/Ϫ mice, suggesting that AHSP has a role in limiting reactive oxygen species production during normal erythropoiesis. In a purified system AHSP inhibits the reaction of Fe(III) ␣Hb with H 2 O 2 (2,20), and recently Mollan et al. (20) demonstrated that AHSP binding prevents the production of Fe(IV)AO heme and associated protein radicals when Fe(III) ␣Hb is exposed to H 2 O 2 .
The low reactivity of Fe(III) ␣Hb in the presence of AHSP correlates with a change in ␣Hb heme pocket structure. In native Hb, the ␣Hb heme group is coordinated through a single His-87 side chain, termed the proximal histidine (see Fig. 1B), allowing exogenous ligands in the distal heme pocket to interact with iron. In the ferric ␣Hb⅐AHSP complex, Fe(III) becomes coordinated by both the proximal and distal histidine side chains (bis-histidyl heme coordination; see Fig. 1C) (1,2,11,(25)(26)(27). Coordination of low-spin Fe(III) with six strong ligands blocks the binding of exogenous ligands and associated electron transfer reactions involving oxygen species. The switch from native to bis-histidyl heme coordination is accompanied by a change to a non-native ␣Hb tertiary structure (r.m.s deviation of 3.3 Å over 135 C ␣ atoms), which dissociates from AHSP ϳ100-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 Ͼ10-fold increase in the autooxidation rate of Fe(II) ␣Hb (11,20,25,28,29), also 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. In contrast, autooxidation of ␣Hb to the bis-histidyl ␣Hb structure dramatically lowers the Fe(III)/Fe(II) redox potential (20) and inhibits interactions with exogenous ligands, thus inhibiting redox cycling. However, the structural mechanism by which AHSP accelerates ␣Hb autooxidation and, more generally, how AHSP promotes the protective bis-histidyl structure is not well understood.
Previous work from our group has suggested that AHSP induces strain into the O 2 -␣Hb structure that is relieved upon autooxidation by reorganization of the ␣Hb protein fold (25). Mutation of Pro-30 to Ala (AHSP P30A ) in loop 1 of AHSP (Fig.  1A) leads to an ϳ4-fold reduction in ␣Hb⅐AHSP autooxidation rates, suggesting that Pro-30 contributes to the strained conformation. However, how contacts at the ␣Hb⅐AHSP interface are transmitted to the ␣Hb heme pocket and the nature of the strained O 2 -␣Hb structure are unknown. Difficulties in characterizing the O 2 -␣Hb⅐AHSP complex arise from its propensity to spontaneously convert to a bis-histidyl structure. A previous x-ray crystallographic study suggested that the strained O 2 -␣Hb structure involved large-scale changes in the globin polypeptide structure and heme coordination geometry (1).
To explore these issues, we have used NMR and EXAFS to probe the complexes of CO-and O 2 -liganded ␣Hb with AHSP in solution. The results show that the tertiary structure of CO/O 2 -␣Hb in these complexes closely resembles that of CO/O 2 -␣Hb in the native HbA tetramer and, therefore, that our previous model of O 2 -␣Hb⅐AHSP (1) is unlikely to represent the initial AHSP binding event in solution. We show that mutation of Pro-30 in AHSP leads to NMR chemical shift perturbations in the F-helical region of CO-␣Hb, and the concomitant changes in O 2 and CO binding reactivity help identify how this strain introduced into the proximal portion of the heme pocket promotes autooxidation of O 2 -␣Hb.

EXPERIMENTAL PROCEDURES
Protein Production-The structured region, residues 1-90 of human AHSP, and mutants thereof were expressed and purified as described previously (25). CO-␣Hb was purified from human blood (3). O 2 -␣Hb was generated by gently blowing oxygen across the surface of CO-␣Hb under a concentrated 10-watt halogen light source. The procedure was carried out on ice, and the formation of the O 2 -␣Hb was monitored by UVvisible spectroscopy (Shimadzu UV-1601). Isotopically labeled ␣Hb for NMR studies was co-expressed with ␤Hb and methionine aminopeptidase in Escherichia coli from the previously described plasmid pHE7 (30). Expression was carried out in the E. coli strain JM109. One liter of overnight culture in DM-4 minimal media as described by Looker et al. (31) containing D-glucose (5 g liter Ϫ1 ), NH 4 Cl (0.7 g liter Ϫ1 ), thiamine (30 mg liter Ϫ1 ), ampicillin (100 mg liter Ϫ1 ), and trace metal solution (32)  6.8, with aeration and monitored in real time based on dissolved O 2 concentration. The culture exhausted the nitrogen supply at an absorbance of ϳ1 (600 nm) and was provided with 2 g of 15 NH 4 Cl. The culture exhausted the glucose supply at an absorbance of ϳ3, and 1.5 g of D-[U(99%)-13 C]glucose was provided, and the temperature was shifted to 32°C. The addition of a further 2 g of 15 NH 4 Cl, 1.5 g of D-[U(99%)-13 C]glucose occurred at an absorbance of ϳ3.5, and expression was induced with 0.2 mM isopropyl ␤-D-1-thiogalactopyranoside and 15 M hemin. Cells were collected by centrifugation after 5 h, and the resulting cell pellets were washed once in 10 mM Tris-HCl, 0.5 mM EDTA, pH 8.0. Cell pellets were resuspended in the same buffer and saturated with CO before lysis by sonication (Ultrasonic Processor 500 W, Sonics & Materials Inc.). After clarification by centrifugation, recombinant Hb was purified as previously described (33). The Hb chains were split by the addition of a 2-fold molar excess (per cysteine) of p-hydroxymercuribenzoate (pMB) and purified as previously described (3).

Size Exclusion Chromatography (SEC) and
In-line Light Scattering-SEC chromatography was carried out on a 24-ml Superose 12 column (GE Healthcare) in 0.1 M sodium phosphate, pH 7.0 (21°C). Protein concentrations were determined based on UV-visible absorbance. The concentrations of AHSP and mutants thereof were obtained after unfolding in 6 M guanidine-HCl using molar extinction coefficients calculated based on the amino acid compositions (34). CO/O 2 -␣Hb concentrations were determined based on absorption peaks arising from 3 * transitions in the porphyrin ring using molar extinction coefficients for the corresponding CO/O 2 forms of Hb (35). The weight-average molecular weight (M w ) of particles in solution was determined directly from measurements of light scattering and protein concentration (36). In-line light scattering measurements were made at 41.5°, 90.0°, and 138.5°with respect to the incident beam using a mini-DAWN with 690 nm laser (Wyatt Technology Corp., Santa Barbara CA), calibrated against toluene. Protein concentrations were determined based on the refractive index measured by an Optilab differential refractometer (Wyatt Technology Corp.) calibrated against NaCl standards. Molecular weight calculations were performed for each 50-l fraction across the elution peaks using the ASTRA software (Wyatt Technology Corp.) with the assumption of a standard value for the specific refractive index increment with respect to sample concentration (dn/dC) of 0.19 ml g Ϫ1 . The presence of the heme and pMB groups upon the value of dn/dC was neglected in this case.
NMR Spectroscopy-All NMR spectroscopic measurements were conducted using a Bruker Avance II 600 MHz spectrometer 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 were formulated as described, with the addition of 20 M 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and 5% D 2 O. Backbone assignments were made using HNCA, HNCACB, CBCA-(CO)NH, and 15 N-edited NOESY according to standard assignment strategies. His side chain resonances were assigned based on 15 N-HSQC, 15 N-NOESY-HSQC, and two-dimensional TOCSY (two-dimensional total correlation spectroscopy)/NOESY spectra. Tautomer/charge state was inferred for His side chains from cross-peak intensities in the 15 N-HSQC (38). Spectra were recorded at sample temperatures of 15 and 25°C for ␣Hb and ␣Hb⅐AHSP complexes, respectively, to optimize 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 was the preferred cryoprotectant, as it caused minimal changes in the UV-visible spectrum. The O 2 -␣Hb⅐AHSP complex was formed by mixing O 2 -␣Hb with a 1.1 molar excess of AHSP. Final protein concentrations were 6 and 2.5 mM for O 2 -␣Hb and O 2 -␣Hb⅐AHSP samples, respectively. The solutions were injected into 140-l Lucite XAS cells (23 ϫ 2 ϫ 3 mm) with 63.5-m Kapton tape windows. This operation was conducted in a nitrogen-filled glove bag. Immediately after their preparations, the samples were frozen in a liquid-nitrogen/n-hexane slurry to prevent autooxidation. X-ray absorption spectra were recorded at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 9-3 (3 GeV, 100 -50 mA) using a Si(220) double-crystal monochromator. Beamline 9-3 had an upstream collimating mirror and a downstream sagitally focusing mirror; both were rhodium-coated and also provided harmonic rejection. Data were collected as fluorescence spectra using a 30-element germanium array detector (39). For each sample, 10 -21 scans were recorded from 6785 to 8257 eV. The energy was calibrated using an iron foil standard in which the first inflection point was assigned to an energy of 7111.2 eV (40). Data scans were averaged, background-subtracted, and normalized as described previously (41).
EXAFS Data Analysis-The model-fitting calculations were performed by means of the program XFIT (42). The values of the parameters that were varied to optimize the agreement between the observed and calculated EXAFS were the coordinates x, y, and z of all atoms in the model in relation to an arbitrary set of Cartesian axes, the Debye-Waller factors, 2 , a scale factor, S 0 2 , and E 0 . The value of S 0 2 was initially restrained to be 0.9 with an estimated standard deviation (e.s.d.) 0.1 for O 2 -␣Hb and 0.92 (e.s.d. 0.02) for O 2 -␣Hb⅐AHSP (i.e. to typical values for low-spin O 2 -myoglobin and O 2 -leghemoglobin). Both the observed and calculated EXAFS were Fourier-filtered (42). The k windows used for the EXAFS analyses are shown in the figures. The goodness-of-fit parameter R was calculated as described by Ellis and Freeman (42). The model used in the XFIT analyses is that described previously (41). The sixth coordination site was a dioxygen molecule where the terminal oxygen was arbitrarily constrained to lie in the plane perpendicular to the heme and passing through the meso-Cs. The O 1 -O 2 distance was restrained to be 1.22 Å (e.s.d. 0.01 Å), consistent with that reported by Jameson et al. (43). The constraints and restraints used for the bond lengths and bond angles in the porphyrin ring and the imidazole ligand and for the Debye-Waller factors were as described previously (41). Constraints and restraints and multiple-scattering (MS) paths used in the fitting calculations are given in supplemental Tables.
O 2 /CO Binding Measurements-Rate constants for O 2 binding to and dissociation from ferrous ␣Hb⅐AHSP complexes as well as rate constants for CO binding were measured using methods developed for relatively unstable Fe(II) O 2 -Hb com-plexes as described by Birukou et al. (45). In these measurements, stable CO-␣Hb complexes are injected into laser photolysis cuvettes containing mixtures of buffers equilibrated with 1 atm O 2 or 1 atm CO. The mixtures in % were O 2 :CO ϭ 100:0, 75:25, 50:50, 25:75, and 0:100. The CO-␣Hb samples were photolysed with a 0.5-s, 577-nm dye laser pulse. Absorbance time courses for O 2 and CO rebinding to deoxy-␣Hb were measured on s time scales at 436 nm, and then time courses for replacement of the transiently bound O 2 by CO were measured on millisecond to seconds time scales at 424 nm (see Fig.  1 in Ref. 45). This method prevents significant autooxidation during the measurements of O 2 binding because the sample remains in the Fe(II)-CO form in between laser photolysis pulses. The dependence of the pseudo first order rates for the fast and slow phases on O 2 and CO were fitted globally to obtain values for the association rate constants for O 2 and CO binding to ␣Hb, kЈ O2 and kЈ CO , and the rate of O 2 dissociation, k O2 , as described in Birukou et al. (45). Our measurements were made using 100 mM potassium phosphate buffer, pH 7.0, at 20°C. Similar kinetic measurements of O 2 and CO binding to native and recombinant wild-type Fe(II)-␣Hb⅐AHSP complexes were made by Vasseur-Godbillon et al. (28).

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). Mutation of AHSP Pro-30 to a range of other residue types abolishes cis-trans peptidyl isomerization of AHSP and leads to a consistent 4-fold reduction in the rate of O 2 -␣Hb autooxidation (11,25). We previously proposed that cis-peptidyl Pro-30 makes physical contact with O 2 -␣Hb that promotes autooxidation (25). However, it is possible that other changes in AHSP conformation related to cis-trans isomerization might explain the role of Pro-30 in ␣Hb autooxidation. To investigate this possibility, we introduced proline mutations into AHSP P30A at position 28 (AHSP P30A/N28P ), 29 (AHSP P30A/D29P ), 31 (AHSP P30A/L31P ), or 33 (AHSP P30A/S33P ) as shown in Fig. 2A. We argued that proline residues in these positions might still allow Pro-30  AHSP to undergo conformational exchange, but the proline side chains would presumably not preserve contacts with ␣Hb that are made by the native Pro-30. The locations of the introduced proline mutations at the ␣Hb⅐AHSP interface are shown in Fig. 2B. We did not mutate Val-32 because this side chain packs into the hydrophobic core between the AHSP helices. The propensity of the mutant AHSP proteins to undergo cis-trans isomerization was assessed from 15 N-HSQC NMR spectra. A portion of the 15 N-HSQC spectrum of wild-type AHSP is shown in Fig. 2C, with peaks arising from N-H N groups in the same residue in cis-prolyl and trans-prolyl conformers of AHSP labeled and joined by a dashed line. In the AHSP P30A spectrum, a single N-H N peak was observed for each residue, consistent with a single AHSP conformer ( Fig. 2D and Table 1) (1,46). The spectrum of AHSP P30A/N28P contained an additional set of weaker HSQC signals compared with AHSP P30A (Fig. 2E, arrows, and Table 1) that was consistent with ϳ20% of molecules adopting an alternative conformation, most likely due to isomerization of the Phe-27-Pro-28 peptide bond. The 15 N-HSQC spectra of AHSP P30A/D29P , AHSP P30A/L31P , and AHSP P30A/S33P (Fig. 2, F-H) were all consistent with a single protein conformation, which indicated that prolines at positions 29, 31, and 33 do not result in measurable cis/trans isomerization. Chemical shift analyses were consistent with minor conformational changes in AHSP proximal to loop 1 and indicated that none of the mutations conferred gross structural changes (not shown). These results suggest that only cis-peptide bonds before Pro-30 or Pro-28 result in a significant amount of cis/trans isomerization.

D29A ------------------------A------------------------
The effect of AHSP proline mutations on ␣Hb autooxidation is shown in Fig. 3. Autooxidation of O 2 -␣Hb to bis-histidyl ␣Hb was measured by changes in the visible absorption spectrum of ␣Hb (Fig. 3, A and B). The absorbance time courses fit to single exponential expressions and yielded first-order rate constants for autooxidation (k autoox ; Fig. 3C and Table 1). AHSP P30A showed a 4-fold reduction in the value of the rate constant compared with that of wild-type AHSP, as previously described (11,25). All introduced-proline mutants had the same low activity as the AHSP P30A ( Fig. 3C and Table 1). A control mutation AHSP D29A had the same activity as wild-type AHSP. The results indicate that the positioning of Pro-30 in loop 1 is critical for function. In addition, the observation that free AHSP P30A/N28P showed cis/trans isomers in solution, yet con-ferred the same slowed rate of O 2 -␣Hb autooxidation as AHSP P30A , suggests that the propensity of free AHSP to undergo conformational changes does not in itself contribute to O 2 -␣Hb⅐AHSP autooxidation activity. We have shown previously that stabilization of the cis-peptidyl Asp-29 -Pro-30 AHSP conformation does correlate with increased autooxidation activity (25). Together the above data are consistent with the hypothesis that the effects of Pro-30 are mediated through direct 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 for structural differences between ␣Hb in the native Hb tetramer and free ␣Hb in solution. The aim of these studies was to probe for structural changes in ␣Hb that could explain the more rapid  autooxidation observed in vitro and provide a suitable reference point for subsequent NMR studies of ␣Hb⅐AHSP complexes. We found that sequential backbone assignments could not be obtained for residues 104 -126 (corresponding to helices G and H), as also reported previously by Martineau and Craescu (47). The absence of NMR signals corresponding to these residues suggests that they might 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 time with increasing ␣Hb load concentration. This behavior is typical of protein self-association and is consistent with previous studies of ␣Hb (48,49). We reasoned that treatment of ␣Hb with pMB, which covalently modifies Cys-104 of ␣Hb and inhibits Hb chain homo (48) and hetero interactions (50), would allow more complete NMR data to be obtained. It has previously been shown that pMB treatment has no effect on the O 2 and CO binding parameters of isolated ␣Hb (51). Light scattering was used to monitor the self-association status of CO-␣Hb before and after pMB treatment. We found that untreated CO-␣Hb eluted from SEC with a light scattering data profile characteristic of reversible self-association, with a marked increase in M w toward the center (i.e. the highest concentration) of the elution peak (Fig. 4A, each M w measurement is indicated with a red ϩ). In contrast, pMB treated CO-␣Hb (CO-␣Hb PMB ) eluted with a constant M w of 16.7 Ϯ 1.3 across the peak (Fig. 4A, black dots), indicating that CO-␣Hb PMB is predominantly monomeric (formula weight of 16,053).
The 15 N-HSQC spectrum of CO-␣Hb PMB (Fig. 4B, black) contained numerous additional peaks (labeled) that were not present in the untreated CO-␣Hb spectrum (Fig. 4B, red) and sequential backbone assignments could now be obtained for 130/134 non-proline residues of CO-␣Hb PMB . Comparison of the CO-␣Hb PMB and CO-␣Hb backbone chemical shifts indicated that only the modified Cys-104 and immediately sur-rounding residues were strongly influenced (not shown), confirming that pMB modification does not substantially alter the tertiary structure of ␣Hb, consistent with functional measures of O 2 and CO binding (51). The ␣Hb self-association interface is clearly identified by the NMR data (Fig. 4C, red/orange) and is the same face that interacts with ␤Hb (Fig. 4C, surface) to form the ␣Hb 1 ␤Hb 1 heterodimer.
To obtain information about the secondary structure of monomeric CO-␣Hb PMB , we used TALOSϩ (52) to derive and backbone angle predictions from NMR chemical shift data. Seven helical secondary structure elements could be clearly identified corresponding to the A-C and E-H helices (Fig. 5). The angles derived by TALOSϩ were very similar to angles determined from crystal structures of native CO-Hb (PDB 2DN3) (53), with correlation coefficients of 0.82 () and 0.87 () over 127 residues. One difference was that TALOSϩ consistently assigned the C helix region as ␣-helix rather than the 3 10 -helix that is identified by the program DSSP (54) based on crystal structure coordinates (PDB 2DN3).
To obtain information about the heme pocket, we assigned His side chain resonances for CO-␣Hb PMB (Fig. 6 and Table 2). His-58 and His-87 side chain resonances displayed large upfield deviations from random coil chemical shifts consistent with positions above or below the plane of the heme ring. The chemical shifts were very similar to values previously obtained for CO-␣Hb at pH 5.3-5.7, 36°C, (55,56), and the ␣Hb subunit of intact CO-Hb at pH 6.9, 29°C (30,57), strongly supporting the conclusion that free ␣Hb adopts a heme pocket structure similar to that in Hb tetramers, with the heme iron bound through N ⑀2 of His-87. In summary, preparation of monomeric ␣Hb through treatment with pMB allowed us to obtain backbone and His side chain chemical shift data suitable for comparative NMR studies with ␣Hb⅐AHSP complexes.  . Identification of the ␣Hb homodimerization interface. A, self-association of ␣Hb was inhibited by treatment with pMB. SEC traces are shown for CO-␣Hb (red) or CO-␣Hb PMB after reaction with a 4-fold molar excess of pMB (black). Molecular weight from light scattering measurements is plotted across each peak (red/black symbols). B, portions of the 15 N-HSQC for CO-␣Hb (red) and CO-␣Hb PMB (black) were recorded at 15°C in 20 mM sodium phosphate, pH 6.9. Assignments are shown for new peaks in the CO-␣Hb PMB 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 shift difference between CO-␣Hb and CO-␣Hb PMB (orange) map to the tight ␤Hb-interaction face, which is, therefore, also identified as the ␣Hb homodimerization face.

CO-␣Hb Retains Near-native Secondary Structure and Heme
Ligation in Complex with AHSP-To understand how AHSP promotes autooxidation of ␣Hb, we examined chemical shift differences between free CO-␣Hb PMB and the CO-␣Hb⅐AHSP complex. Widespread changes in the 15 N-HSQC of CO-␣Hb were observed upon binding of AHSP (Fig. 7A) despite the complex remaining in the CO-liganded form, as confirmed by UV-visible spectroscopy (not shown). A plot of backbone chemical shift differences between CO-␣Hb PMB and CO-␣Hb⅐AHSP reveals that the B-C corner and the F, G, and H helices of ␣Hb are most affected (Fig. 7B).
The B-C corner, helix G, and helix H comprise the AHSP binding face of ␣Hb (1, 2, 25), and chemical shift changes are expected here as a result of the interface forming (Fig. 7B, gray background shading). In contrast, chemical shift changes in helix F cannot be explained by proximity to the AHSP surface, which implies that they arise from an induced structural change in ␣Hb. However, NMR signals arising from the F helix region had similar line widths to signals from other regions of the ␣Hb subunit, and TALOSϩ derived and angles indicated that the F helix region remains well ordered with similar secondary structure 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 chemical shift values to the free CO-␣Hb PMB form (Fig. 8A, black). This provides strong evidence that His-87 remains the heme-ligating residue in CO-␣Hb⅐AHSP. In summary, AHSP binding to CO-␣Hb results in small but significant conformational changes in helix F even though the ␣-helical character and heme binding function are retained. As such, these findings contrast with a previous x-ray crystallographic study in which we reported that O 2 -␣Hb⅐AHSP P30A has a highly unusual heme

TABLE 2 NMR resonances of His side chains for CO-␣Hb PMB and CO-␣Hb⅐AHSP
Resonances for CO-␣Hb PMB in 10 mM sodium phosphate, pH 7.0, at 288 K and CO-␣Hb⅐AHSP in 10 mM sodium phosphate, pH 7.0, at 298 K. site geometry wherein the F helix is disordered, and heme is coordinated by His-58 in helix E (1). It is possible that the unusual crystal structure is characteristic of the O 2 -and not the CO-␣Hb⅐AHSP complex but, as described below, that now seems unlikely at least for the O 2 -␣Hb⅐AHSP complex in solution.

His
AHSP Pro-30 Perturbs the Proximal Heme Pocket of ␣Hb-Because mutation of AHSP Pro-30 to Ala or other residue types leads to a 4-fold decrease in the rate of O 2 -␣Hb⅐AHSP autooxidation, we reasoned that CO-␣Hb⅐AHSP and CO-␣Hb⅐AHSP P30A complexes would display structural differences that give insight into the mechanism underlying AHSP-mediated ␣Hb autooxidation. We, therefore, obtained backbone resonance assignments for CO-␣Hb in complex with AHSP P30A using standard triple resonance methods. Compared with wild-type AHSP, the AHSP P30A mutant induced more limited perturbations of CO-␣Hb (Fig. 7C). A direct comparison of CO-␣Hb chemical shifts in the CO-␣Hb⅐ AHSP and CO-␣Hb⅐AHSP P30A complexes shows that the wildtype AHSP causes significantly larger chemical shift perturbations to the N-terminal fragment of ␣Hb helix G and the whole of helix F (Fig. 7D), which suggests that AHSP Pro-30 makes contacts that are transmitted through the ␣Hb structure and lead to perturbations of helix F.
Interestingly, we could not obtain backbone assignments for residues 135-138 of CO-␣Hb in complex with wild-type AHSP, and signals for these residues appeared to be absent from the NMR spectra. In NMR spectra of CO-␣Hb⅐AHSP P30A , residues 135-138 were assigned and predicted as the regular ␣-helix by TALOSϩ (Fig. 5). Residues 135-138 of native ␣Hb are in a region of helix H that contacts helix F (Fig. 7E), which suggests that loss of NMR signals could reflect a destabilization of helix  H in ␣Hb⅐AHSP related to changes in structure of the F helix.

AHSP-binding face A A A A A A A A A A A A A A A A A A A A A A A
Overall the results here indicate that AHSP Pro-30 makes contacts that perturb helices F, G, and H of ␣Hb, which together constitute the proximal heme pocket of ␣Hb (Fig. 7E).

Comparison of the Iron-Ligand Structures in O 2 -␣Hb and O 2 -␣Hb⅐AHSP-NMR spectroscopic data indicated that
His-87 is retained as the iron binding His residue in CO-␣Hb⅐AHSP, but these data do not provide detailed information about the exact geometries of the axial iron ligands. In addition, we could not determine NMR structures for the O 2 -␣Hb⅐AHSP complexes due to autooxidation during NMR data collection. To probe the electronic structure and coordination environment around the heme iron more directly, we acquired x-ray absorption spectra at the iron K-edge for the physiologically more relevant O 2 -␣Hb and O 2 -␣Hb⅐AHSP complexes. Spectra were recorded at 10 K to minimize photo-damage and increase the importance of 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 upon freezing (59). This procedure permitted measurements on the unstable O 2 -␣Hb⅐AHSP complex to be performed over extended time periods without conversion to the bis-histidyl ferric ␣Hb form. The x-ray absorption near edge structure (XANES) spectra of O 2 -␣Hb and O 2 -␣Hb⅐AHSP (not shown) show no irradiation damage, and the similarity to spectra of the low-spin Fe(III) proteins metleghemoglobin, oxymyoglobin, and oxyleghemoglobin (60) suggest that all have a low-spin Fe(III) center, which is consistent with an Fe(III) ϩ -O 2 . species (61). This classification is also supported by the diagnostic oxidation-and spin-state resonance Raman marker bands (1,62). The slightly lower edge energy of O 2 -␣Hb⅐AHSP compared with free O 2 -␣Hb suggests a lengthening of the Fe-O 2 bond in the AHSP complex. EXAFS data obtained from O 2 -␣Hb and O 2 -␣Hb⅐AHSP were fitted to models for the iron sites using restrained and constrained multiple-scattering models (58,(63)(64)(65). The observed and calculated EXAFS and the corresponding Fourier transforms are shown for O 2 -␣Hb and O 2 -␣Hb⅐AHSP in Fig. 9, A-D, and the derived molecular structures for O 2 -␣Hb and O 2 -␣Hb⅐AHSP are shown in Fig. 9, E and F, respectively. The   bond length/angle and other fitted parameters obtained are listed in Table 3.
The errors in the iron-ligand bond lengths determined by EXAFS were estimated as the r.m.s. of contributions from both random and systematic errors. The random (statistical) errors due to noise in the data were estimated by Monte-Carlo calculations (42), and the systematic errors were assigned a conservative consensus value, 0.02 Å (44).  (Table 4) to give respective estimated maximum r.m.s. errors of 2.7°and 3.9°for O 2 -␣Hb and O 2 -␣Hb⅐AHSP. Therefore, the error in this bond angle estimated by two independent methods is ϳ3-4°. The resulting Fe-O-O angles determined from restraining the starting angle between 115°and 175°and allowing the fit to go to convergence then removing the restraint were 130°(for 6 minima and 143°f or 1 minimum; the latter was a local minimum with a higher R value and a low S o 2 value for a heme protein) in O 2 -␣Hb ( Table  5) and 135°(for 6 minima and 101°for 1 minimum) in O 2 -␣Hb⅐AHSP ( Table 6). The fit obtained for the Fe-O-O bond angle of 101°, when the angle was initially restrained to be 115°a nd then this restraint was removed and the fit allowed to go to convergence, is likely to be a false minimum. This is because it is not chemically reasonable and is not consistent with the similarity in the shape of the XANES of O 2 -␣Hb and O 2 -␣Hb⅐AHSP (not shown), as XANES is sensitive to bond angles. These analyses show that the bond lengths are independent of all of these changes in starting models and constraints/restraints, and the bond angles relax to the non-constrained values shown in Table  3. These detailed analyses of parameter space provide confidence that the EXAFS fits are robust and are not trapped in false minima. Similarly, varying the tilt or rotation of the proximal imidazole did not significantly alter the fitted bond length parameters (not shown).  Table 3 (0.02 Å), the systematic error contributions are expected to be the same for both proteins and, hence, the errors obtained from Monte Carlo calculations are a better indication of the relative errors. For the Fe-O 2 bond these errors were 0.005 and 0.006 Å in O 2 -␣Hb and O 2 -␣Hb⅐AHSP, respectively, indicating that the 0.03 Å lengthening of Fe-O 2 in O 2 -␣Hb⅐AHSP is significant. It should be emphasized that the Fe-O 2 bonds are substantially shorter than those of the other heme-ligand bonds, which is why the precision is so high. This is due to the rapid increase in the importance of single and multiple-scattering contributions as the bond length decreases.
The O 2 -␣Hb⅐AHSP data were also fitted to a model where the iron-ligand bond lengths and Fe-O-O bond angle were constrained to be that in the O 2 -␣Hb⅐AHSP P30A structure (1), and E 0 , S 0 2 , and the Debye-Waller factors were fitted (Table 3, bottom row). The resulting temperature factors were unacceptably high (0.059 Å 2 for the proximal imidazole N ⑀ , 0.079 Å 2 for O 1 , and 0.080 Å 2 for O 2 ) with an R value of 32.5%. Clearly, this model does not fit the solution structure.
The ability of this methodology to determine precise bond length and angle information on axial ligands has been established in studies of porphyrin model complexes and by the determination of precise bonding parameters in the heme environments of met-and deoxymyoglobin (41,66) before the publication of the high resolution protein crystallographic structures (67,68). Even taking the small differences between O 2 -␣Hb⅐AHSP and O 2 -␣Hb into account, it is clear that the initial solution complex of O 2 -␣Hb⅐AHSP has a "normal" coordination geometry similar to that in free O 2 -␣Hb chains.
AHSP Reduces Reactivity of ␣Hb with O 2 and CO-In 2006, Vasseur-Godbillon et al. (28) reported that AHSP binding reduces the O 2 and CO affinities of ␣Hb roughly 3-5-fold. We repeated these ligand binding experiments with wild type and two Pro-30 mutants of AHSP. Table 7 presents a summary of the rate constants for O 2 and CO association (kЈ O2 and kЈ CO ) to, and O 2 dissociation (k O2 ) from, ␣Hb in the presence and

Fe-Ligand distances Fe-O-O Debye-Waller factors, 2
Other fit parameters absence of AHSP variants. The ratio of kЈ O2 /k O2 provides the O 2 association equilibrium or affinity constant (K O2 ). As reported by Vasseur-Godbillon et al. (28), AHSP binding to O 2 -␣Hb causes a small decrease in the rate of O 2 binding and a larger 3-4-fold increase in the rate of O 2 dissociation. The net result is an ϳ4-fold decrease in O 2 affinity (Table 7). These results support the XAFS data and provide direct evidence that AHSP binding leads to weakening of the Fe-O 2 bond, which in turn leads to a significant increase in the susceptibility of O 2 -␣Hb to autooxidation.
AHSP binding to ␣Hb also slows the rate of bimolecular CO binding by a factor of ϳ3-4-fold, as also reported previously by Vasseur-Godbillon et al. (28) (Table 7). CO binding is limited primarily by the rate of internal bond formation and, therefore, provides a sensitive measure of iron reactivity that is primarily determined by interactions between the heme iron and the

Effect of varying the O-O bond distance restraint on the Fe-O-O angle in human O 2 -␣Hb and O 2 -␣Hb⅐AHSP
The quantities enclosed by the brackets ({}) are the values of res used by XFIT, which is analogous to e.s.d.

TABLE 5 Effect of restraining the Fe-O-O bond angle in human O 2 -␣Hb on the MS XAFS analysis
The quantities enclosed by the brackets ({}) are the values of res used by XFIT, which is analogous to e.s.d.

TABLE 6 Effect of restraining the Fe-O-O bond angle in human O 2 -␣Hb⅐AHSP on the MS XAFS analysis
The quantities enclosed by the brackets ({}) are the values of res used by XFIT, which is analogous to e.s.d.

Fe-O-O angle
Debye-Waller factors, 2 proximal histidine. The ϳ4-fold decrease in kЈ CO and O 2 affinity are consistent with conformational strain introduced on the proximal side of the heme pocket of ␣Hb⅐AHSP due to interactions with AHSP Pro-30. This interpretation is strongly supported by the effects of Pro-30 mutations on ligand binding. As shown in Table 7, AHSP P30A and AHSP P30W mutations diminish the effects of AHSP binding on both O 2 affinity and the rate of CO association, and in the case of AHSP P30W , the observed ligand binding parameters are similar to those of free ␣Hb. Thus, all our structural, spectroscopic, and functional data suggest that structural changes in the proximal heme pocket are induced by AHSP Pro-30, causing a weakening of the Fe-O 2 bond and an increase in the rate of autooxidation.

DISCUSSION
AHSP promotes autooxidation of O 2 -␣Hb to prevent it from reacting with H 2 O 2 under conditions of oxidative stress. The resultant bis-histidyl heme does not react with H 2 O 2 and, therefore, cannot generate reactive ferryl species and protein radicals (2,20). The bis-histidyl ␣Hb⅐AHSP structure has been well characterized by x-ray crystallographic and spectroscopic methods (1,2,(25)(26)(27), but the formation of this structure from O 2 -␣Hb has been less well understood. The data presented here provide key insights as to how AHSP facilitates formation of the bis-histidyl state by making specific interactions at the ␣Hb⅐AHSP interface and fit a model, explained below, in which ␣Hb⅐AHSP complexes can adopt one of two well ordered conformations that are intermediates in Hb assembly and achieve ␣Hb detoxification (Fig. 10).
Structure of ␣Hb-By reacting pMB with Cys-104 and blocking ␣Hb self-association, we obtained chemical shift-derived backbone dihedral angles and chemical shift analysis of His side chains, which together show that the conformation of free ␣Hb is very similar to its structure in the native Hb tetramer. This conclusion is consistent with previous findings that heme pocket structure is well preserved in isolated subunits, based on far-UV (69,70) and near-UV CD (69 -72) and homonuclear NMR studies of ferrous and ferric ␣Hb (47,55,56,73,74). None of these data provide any hints as to why free ␣Hb is more prone to oxidation and subsequent rapid loss of the heme moiety and aggregation. Similar observations have been made in other systems. For example, the monomeric point mutants of the carrier protein transthyretin display identical x-ray crystal structures to the wild-type protein and yet have substantially reduced thermodynamic stabilities (75). In such cases, a native-like conformation still represents the lowest energy state available; however, the free energy well in which this conformer sits is shallower due to a reduction in the number of stabilizing inter-actions. In the case of ␣Hb, stabilization is provided by multiple subunit interactions in the tetramer.

CO-and O 2 -Hb Have Native-like Conformations in Complex with AHSP-
A previous x-ray crystallographic study from our group suggested that O 2 -␣Hb underwent large conformational changes upon binding AHSP (1), and it was assumed that these changes were responsible for the accelerated autooxidation. In that study residues Asp-74 -Leu-91 of ␣Hb (helix F) appeared disordered (as judged from a lack of interpretable electron density), and the heme-ligating residue was switched from the usual His-87 in helix F to His-58 in helix E. However, NMR line shape and TALOSϩ chemical shift data presented here show that, in the solution state, helix F retains an ordered ␣-helical structure in CO-␣Hb⅐AHSP. In addition, analysis of His side chain resonances strongly suggests that His-87 retains the heme binding role. In the crystal structure, the Fe-O 2 bond is unusually long (2.7 Å) (1). Such a long Fe-O 2 bond is inconsistent with visible absorption and XANES and EXAFS spectroscopic data (Table 3), which indicate that the iron coordination    (Table 7).
Several possible explanations exist for the differences between the crystal structure described previously and the solution structure reported here. A detergent molecule located close to the heme group in the crystal might have influenced the structure; however, the detergent makes very few direct contacts with the protein. Perhaps more significantly, the crystals were grown under a standard atmosphere making it likely that partial autooxidation of ␣Hb would occur. In addition, the intense x-ray beams used for protein crystallography can promote reduction of ferric proteins (59). A mixture of heme states arising from these processes would introduce static disorder in the crystal, complicating analysis of the x-ray data. Nevertheless, the crystal structure does provide evidence that helix F of ␣Hb is susceptible to conformational distortion in the presence of AHSP.
AHSP Pro-30 Promotes Changes in the Proximal Heme Pocket of O 2 -␣Hb That Enhance Autooxidation-We have previously shown that mutation of Pro-30 to a range of other residues types leads to a 4-fold reduction in autooxidation of ␣Hb. Here we show that introducing Pro at other positions in loop 1 did not restore native O 2 -␣Hb autooxidation and, although the AHSP P30A/N28P may undergo cis/trans isomerization, O 2 -␣Hb autooxidation was not enhanced above the rate observed for AHSP P30A , which is only 2-3-fold greater than that of free O 2 -␣Hb (27). These data emphasize that the position of Pro-30 in AHSP loop 1 is critical to enhancing autooxidation.
AHSP Pro-30 is positioned adjacent to the beginning of helix G in the bis-histidyl ␣Hb⅐AHSP complex. However, in the absence of crystallographic data, the degree of interaction between Pro-30 and helix G in O 2 -␣Hb⅐AHSP is unknown. Based on mutation and binding data, which show that Pro-30 destabilizes the O 2 -␣Hb⅐AHSP interface but not the bis-histidyl ␣Hb⅐AHSP interface (11,25,27), we previously proposed that Pro-30 introduces a steric clash at the O 2 -␣Hb⅐AHSP interface, which is relieved upon transition to the bis-histidyl ␣Hb⅐AHSP structure. Here we show that AHSP binding induces NMR chemical shift perturbations in F, G, and H helices of CO-␣Hb, which constitute the proximal heme pocket, and that mutation of Pro-30 markedly reduces these disturbances. These findings are consistent with a steric interaction between AHSP Pro-30 and ␣Hb helix G that is propagated through packing interactions of the F, G, and H helices (represented as orange shading of these helices in Fig. 10, top row). Bonding interactions between the heme iron and proximal His-87 in helix F and the geometry of the proximal pocket are important factors influencing ligand reactivity of the iron (76,77). It is, therefore, significant that AHSP decreases O 2 affinity and CO association rates of ␣Hb and that mutation of Pro-30 restores ligand reactivity of the heme iron to near-normal levels, which suggests strongly that Pro-30-induced disturbance of the proximal heme pocket contributes to enhanced O 2 -␣Hb autooxidation and lower ligand reactivity in the presence of wild-type AHSP.

O 2 -␣Hb⅐AHSP and Bis-histidyl⅐AHSP Intermediates in Hb
Assembly-Our data suggest a model in which AHSP can bind ␣Hb in one of two well ordered conformations depending on the oxidation/ligand state of the heme (Fig. 10). O 2 -␣Hb is bound in a near-native conformation (Fig. 10, top), whereas Fe(III) ␣Hb adopts a non-native globin conformation and bishistidyl heme coordination (Fig. 10, bottom). We expect that Fe(II) deoxy ␣Hb and CO-␣Hb will also bind to AHSP with near-native protein conformations on the basis that these forms of ␣Hb all show the same affinity for AHSP as O 2 -␣Hb, which is 100-fold weaker than the interaction of AHSP with the non-native bis-histidyl Fe(III) ␣Hb conformation (20,25,27). Notably, deoxy-Fe(II) ␣Hb does not adopt bis-histidyl heme coordination (26) despite the availability of an open iron axial coordination site. These studies emphasize that specific coordination between the distal His-58 and the Fe(III) iron center and the structural changes in the ␣Hb polypeptide chain occur in a concerted fashion during oxidation of the ␣Hb⅐AHSP complex 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 the presence of AHSP (78) proceeds through a bis-histidyl ␣Hb⅐AHSP folding intermediate before heme reduction and incorporation into Hb (Fig. 10, solid black arrows). Bis-histidyl ferric ␣Hb can in principle be transferred to ␤Hb and subsequently reduced (Fig. 10, dashed arrows); however, the rate of ferric ␣Hb⅐AHSP dissociation is probably too slow to be physiologically relevant, and reduction almost certainly occurs more rapidly (27). Based on autooxidation rates measured in vitro (11,20,25,28,29), newly reduced Fe(II)/O 2 -␣Hb should be rapidly incorporated into Hb before autooxidation can occur if the concentration of free ␤Hb is high. However, under conditions of limiting ␤Hb that are known to occur during normal erythropoiesis (79,80), the likelihood increases for the conversion of Fe(II)/O 2 -␣Hb⅐AHSP species to bis-histidyl ferric complexes (Fig. 10, red arrows). The more rapid autooxidation of ␣Hb in the presence of AHSP may be advantageous, as it provides a means to trap ␣Hb in a relatively unreactive Fe(III) state that inhibits redox cycling and reactions with reactive oxygen species that could otherwise occur with free ␣Hb. In addition, both free O 2 -␣Hb and met-Hb subunits are more prone to H 2 O 2 -induced ferryl and protein radical formation and precipitation (20,81), making it advantageous to remove free O 2 -␣Hb and stabilize it as bis-histyl met-␣Hb⅐AHSP under conditions of oxidative stress.
To investigate the biological importance of autooxidation rate, Khandros et al. (11) performed gene knock-in experiments in mice. Mutations of AHSP Pro-30 to Ala or Trp did not disturb erythropoiesis (11), which suggested that a 4-fold decrease in ␣Hb⅐AHSP autooxidation rate is not sufficient to elicit changes in erythropoiesis in the normal mouse model (11,27). However, it is important to recognize that O 2 -␣Hb⅐AHSP P30A still autooxidizes more quickly than does free O 2 -␣Hb (11,25,27) and that other interactions across the ␣Hb⅐AHSP interface, in addition to those with Pro-30, are important for autooxidation and hemichrome formation.
A transition between two metastable ␣Hb⅐AHSP structures, as outlined in Fig. 10, avoids persistent disordered states that would favor unregulated reactions with an exposed heme and rapid heme dissociation. In this regard, it is interesting that mutations of Pro-30 to Ala or Trp decrease O 2 -␣Hb autooxidation rates but enhance heme dissociation rates (11). In conclusion, it appears that the AHSP sequence has evolved to achieve a fine balance of distorting ␣Hb sufficiently to promote changes in heme coordination that inhibit reactions with H 2 O 2 without inducing an unacceptably high risk of heme loss or other irreversible denaturation events.
A Physiological Role for O 2 -␣Hb⅐AHSP 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 sites where the endothelial cells make direct contact with underlying smooth muscle (myoendothelial junctions) and that ␣Hb expression regulates NO signaling from the endothelium to smooth muscle. NO signaling is important for maintaining vascular tone and modulating the response of smooth muscle to vasodilator and vasoconstrictor messengers. Importantly, the effect of heme proteins on NO signaling is dependent on the iron oxidation and ligand state. Fe(III) heme interacts only very weakly with NO, providing no barrier to NO diffusion. In contrast, O 2 -heme reacts rapidly with NO to form nitrate (NO 3 Ϫ ) and Fe(III) heme, thus quenching NO signaling (83)(84)(85). In the endothelium, the reaction of NO with O 2 -␣Hb⅐AHSP is expected to generate bis-histidyl ␣Hb⅐AHSP. Therefore, in endothelial cells the balance between O 2 -␣Hb⅐AHSP and Fe(III) bis-histidyl ␣Hb⅐AHSP will impose restrictive/permissive states for NO signaling, respectively. In this regard, autooxidation and the interaction of Fe(III) bis-histidyl ␣Hb⅐AHSP with reductase systems in endothelial cells are critical processes. Understanding these processes is now necessary for a full understanding of NO signaling in the vasculature.