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Originally published In Press as doi:10.1074/jbc.M703795200 on December 27, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5960-5970, February 29, 2008
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Deciphering the Structural Role of Histidine 83 for Heme Binding in Hemophore HasA*Formula

Célia Caillet-Saguy1, Paola Turano, Mario Piccioli, Gudrun S. Lukat-Rodgers, Mirjam Czjzek, Bruno Guigliarelli, Nadia Izadi-Pruneyre, Kenton R. Rodgers, Muriel Delepierre, and Anne Lecroisey

From the Unité de RMN des Biomolécules, CNRS URA 2185, Institut Pasteur, 75015 Paris, France

Received for publication, May 8, 2007 , and in revised form, November 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of bacteria to cause diseases depends on many parameters allowing the pathogen to invade the vertebrate host. One of them is its skill to scavenge the host iron ion. Indeed, iron is an essential metal for nearly all living organisms. However, its oxidized iron(III) form is hardly soluble, and its reduced iron(II) form is highly toxic. Therefore, in biological fluids, iron mostly exists as a complex with iron-binding proteins or in heme carrier proteins and is scarcely available. Hence, Gram-negative bacteria have developed multiple iron/heme acquisition systems to survive. Most of them rely on outer membrane receptors either specific for exogenous iron/heme sources present in the biotope or for molecules called siderophores or hemophores. These molecules are synthesized and released by bacteria into the extracellular medium (1). Their function is to capture iron or heme from the host and to return it to the specific receptor.

The first hemophore was discovered in Serratia marcescens (2). Since then, several orthologs have been found in Pseudomonas aeruginosa (3), Pseudomonas fluorescens (4), Yersinia pestis (5), Yersinia enterocolytica (Sanger Institute) and Yersinia pseudotuberculosis (6). HasA (heme acquisition system) hemophores form an independent family of highly conserved hemecarrier proteins that are not homologous to any other known proteins. They bind free heme, or extract heme from proteins such as hemoglobin and hemopexin, and deliver it to hemophore-specific outer membrane receptors, termed HasR (1), which in turn release it into the bacteria (2, 7).

S. marcescens wild-type HasA (HasAWT) is a 19-kDa monomeric protein that binds b-heme with a very high affinity (Kd = 1.9 x 10-11 M) and a 1:1 stoichiometry (8, 9). In the heme loaded hemophore holoHasAWT, the iron ion is in the oxidized form with the lowest redox potential (-550 mV) reported so far for heme-binding proteins (9). The x-ray structure of holo-HasAWT shows an unprecedented heme-binding site (10). Heme is held by two extended loops that connect the {alpha} and β faces of the protein and is rather exposed to solvent (186 Å2) (Fig. 1). The heme iron ion is bound by an unusual pair of ligands: a tyrosine (Tyr75) and a histidine (His32) (10). This ligand pair has only been observed in a very few proteins, e.g. some hemoglobins from invertebrates (11-13), the reduced cytochrome c maturation protein CcmE (14), and the oxidized cytochrome cd1 nitrite reductase (15). Some abnormal human hemoglobins (16) and mutated heme proteins also present this type of coordination (17). X-ray and NMR data previously showed that, in holoHasAWT, the Tyr75 oxygen forms a tight hydrogen bond with the N{delta}1 of a neighboring histidine, His83, that increases the nucleophilic character of Tyr75 and strengthens the Tyr75-iron coordination bond (18, 19). The role of His83 in heme binding was highlighted by alanine mutagenesis of the two iron axial ligands and of His83, followed by the determination of the Kd for heme in the mutant proteins. Consistently, heme affinity in H83A is 265 times smaller (Kd = 5.0 x 10-9 M) than in HasAWT, whereas the mutation of the axial ligand induces a 400-fold loss of affinity for Y75A and only a 5-fold loss for H32A. Moreover, His83 has been proposed as an alternative iron ligand in the absence of Tyr75 or of both His32 and Tyr75 (20). Noteworthy, in contrast to His32, the Tyr75-His83 pair is conserved in all the hemophores, which suggests that the Tyr75-His83 hydrogen bond plays a central and recurrent role for the activity of these heme carrier proteins.

To decipher the structural role of His83 in HasA, we characterized the electronic properties and the iron coordination of holoHasAWT and holoH83A in the oxidized form as a function of pH, using a panel of techniques such as NMR, EPR, x-ray crystallography, resonance Raman, and UV-visible spectroscopies. Although holoHasAWT possesses a unique coordination as a function of pH, substitution of His83 with an alanine residue gives rise to pH-dependent coordination forms with different axial ligation. The role of the Tyr75-His83 hydrogen bond for the stabilization of the Tyr75-iron axial coordination was unraveled. Disruption of this hydrogen bond at physiological pH results in a five-coordinate species that most probably represents an intermediate of the heme transfer mechanism to the receptor.


Figure 1
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  		FIGURE 1.
Ribbon representation of the holoHasAWT structure (Protein Data Bank code 1B2V). The heme is in blue, and the two axial ligands, His32 and Tyr75, are in red. Residue His83, which is H-bonded to Tyr75, is in green.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of HasA Proteins—Wild-type hemophore HasAWT from S. marcescens, and mutant H83A were obtained and cloned as previously reported (2, 20). Uniformly 15N- and 15N/13C-labeled proteins were produced at 303 K in M9 minimal medium containing 15NH4Cl and 13C-glycerol as the sole nitrogen and carbon sources and were purified as described previously (9, 21). The purity of the proteins was checked by SDS-PAGE. The protein concentrations were calculated using the previously determined {epsilon}277 nm values of 19500 M-1 cm-1 for HasAWT and 18500 M-1 for H83A mutant. Cleavage of the last nine residues (180-188) of HasA proteins was performed using the S. marcescens protease PrtSM to prevent sequential proteolysis of the C-terminal amino acids (22). Unless otherwise specified, the samples were dissolved in 20 mM phosphate buffer. Heme loading was performed as previously described (19).

The cyano-complexes were prepared by adding a 15-fold excess of KCN to the proteins (100 mM KCN stock solution in 20 mM phosphate, pH 7).

Absorption Spectroscopy—The absorption measurements were achieved in a Perkin-Elmer Lambda 2 spectrophotometer at ambient temperature using 1- or 0.2-cm path length cells.

Resonance Raman Spectroscopy—Resonance Raman (rR)2 spectra were recorded from protein samples that were 25-80 µM, contained in 5-mm NMR tubes spinning at ~20 Hz. Raman scattering was excited using either 413.1-nm emission from a Kr+ laser (15-20 milliwatt) or 514.1 nm emission from an Ar laser (140 milliwatt). UV-visible absorbance spectroscopy was used to check sample integrity before and after the rR spectra were recorded. The spectra were recorded at ambient temperature as previously described (23). The spectra were calibrated using the Raman bands of toluene and dimethylformamide. The samples of HasA-H83A examined for isotopic sensitivity were prepared from a 0.3 mM stock protein solution. A 9-µl aliquot was diluted into 50 mM CAPS buffer at pH 10.0 prepared in D2O (99.9% 2H) or H218O (96% 18O). The final protein concentration was 25 µM.

NMR Spectroscopy—NMR experiments were performed on protein samples with concentration ranging from 0.8 to 2.5 mM in 20 mM phosphate buffer adjusted at the requested pH. Onedimensional 1H NMR spectra were acquired with 8192 data points either at 16.4 T with a spectral width of 240 ppm or at 11.7 T with a spectral width of 200 ppm. During the recycle delay of 150 or 30 ms, respectively, water suppression was achieved by presaturation. One-dimensional 15N NMR spectra were acquired at 9.4 T with a 30-µs 90° pulse, a spectral width of 450 ppm, 2048 data points, and a recycle delay of 10 ms. Onedimensional 13C NMR spectra were acquired at 16.4 T with a 12.4-µs 90° pulse, a spectral width of 500 ppm, and 4096 data points. Several experiments were performed with carriers at -200, 150, 567, and 850 ppm to identify signals over wide spectral regions. Recycle delays were 10 or 200 ms. 13C T1 values were measured via the inversion recovery sequence. Several series were performed using 10 and 100 ms as recycle delays with the 13C-carrier frequency at different positions to properly invert and excite all 13C resonances. Direct detected 13C-13C COSY experiments were acquired at 16.4 T with 2048 x 256 data points, 1024 scans, and a recycle delay of 300 ms for holo-HasAWT and 4096 x 512 data points, 512 scans, and a recycle delay of 250 ms for holoH83A. Spectral widths of 220 ppm in both dimensions with the carriers at 150 ppm for the indirect dimension and at 110 ppm for the direct dimension were used. Unless otherwise specified, NMR spectra were recorded at 303 K.

EPR Spectroscopy—EPR spectra were recorded with a Bruker ESP300E spectrometer fitted to an Oxford Instrument ESR900 helium flow cryostat. The experiments were carried out on protein samples with concentration ranging from 0.1 to 0.5 mM in 50 mM sodium acetate buffer, pH 5.5, 50 mM phosphate buffer, pH 7, and 50 mM Tricine, pH 9. Spin quantitations of the low and high spin heme signals were performed in nonsaturating conditions as previously described (23).

X-ray Crystallography—The crystallization experiments were performed using the hanging drop vapor diffusion method. The drops were prepared by mixing 2 µl of the protein solution (4.0 mg/ml in 50 mM phosphate buffer, pH 7, 0.02% NaN3) with 1 µl of the reservoir solution (100 mM sodium caco-dylate, pH 6.5, 200 mM Zn(OAc)2, 10% (v/v) 2-propanol). The brownish crystals grew to 0.2 x 0.4 x 0.08 mm3 over a period of 2 months at 20 °C.


Figure 2
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  		FIGURE 2.
Final electron density of the heme region of H83A (Protein Data Bank code 2UYD) surrounded at a 1{sigma} level, showing the two axial ligands Tyr75 and His32. The diffraction data have been refined with the two heme group orientations related by 180° rotation along the {alpha}-{gamma} meso axis as identified in holoHasAWT (19, 26). The low resolution of the electron density might be due to high flexibility and/or the presence of several conformations.

 
X-ray diffraction data were collected at 100 K at the ESRF (Grenoble, France) beamline ID14-EH1 using an ADSC Quantum 4R CCD detector. The wavelength of the synchrotron x-rays was 0.933 Å. The crystal was rotated through 90° with a 1.0° oscillation range/frame. The raw data were processed and merged with MOSFLM (24) and SCALA (25). The crystals of holoH83A (space group P3221) diffracted to a maximum resolution of 2.6 Å. Molecular replacement with holoHasAWT (Protein Data Bank code 1B2V) as model gave a unique solution with a correlation coefficient of 0.72 and a R factor of 35.4%. Refinement was performed using REFMAC5, part of the CCP4 package. The electron density clearly indicated the presence of the heme group but was diffuse and featureless (Fig. 2). Therefore, the geometry of the heme group was tightly constrained to standard values and refined in the two heme orientations related by 180° rotation along the {alpha}-{gamma} meso axis identified in holoHasAWT (19, 26). Water molecules were added with CCP4/wARP (27). The stereochemistry of the final structure was evaluated using PROCHECK (28). The refinement statistics and final R factors are listed in Table 1.


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  		TABLE 1
Data collection and refinement statistics

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Visible Absorbance Spectra of Holoproteins
Fig. 3 shows the absorbance spectra of holoHasAWT and holoH83A. The spectrum of holoHasAWT at pH 5.6 (Fig. 3A) shows maxima at 406 nm (Soret band), 494, 537, 568, and 618 nm that are consistent with a mixture of two species, one high spin (HS) and one low spin (LS). Indeed, whereas the charge transfer band at 618 nm is characteristic of a HS species, the presence of two Q-bands at 537 and 568 nm indicates the presence of a LS species. The absorbance spectrum did not change upon titration from pH 4.4 to 10.4, showing that the population of the two species is pH-insensitive. It is noteworthy that the addition of KCN to the protein at a 15-fold excess did not produce any spectroscopic changes, either at pH 7.3 or at pH 9.4 (data not shown).

In contrast to holoHasAWT, the absorbance spectrum of holoH83A is sensitive to pH. At acidic and neutral pH, the spectra exhibit a Soret maximum at 404 nm, Q-bands at 502 nm and 536 nm, and a CT band at 630 nm (Fig. 3B). This pattern is consistent with the presence of a HS species. When pH increases, bands at 502, 536, and 630 nm decrease, whereas new bands emerge at 490, 543, and 610 nm. The disappearance of the charge transfer band at 630 nm at the advantage of the 610-nm band indicates that the HS species observed at acidic and neutral pH progressively disappears when pH increases for the benefit of a new HS species. Moreover, a LS species appears, as shown by a new band at 576 nm. Thus, at alkaline pH, holoH83A exists as a mixture of a HS and a LS species, the HS species being different from the one observed at acidic pH. Like holoHasAWT, no detectable change was observed after the addition of a 15-fold excess of a KCN solution neither at pH 7.3, where a HS species is dominant, nor at pH 9.4, where two HS species and one LS species are present.

Resonance Raman Spectra of holoHasAWT and holoH83A Mutant
Consistent with previously reported NMR data, the rR spectra of holoHasAWT show a mixture of LS and HS species. The rR data further indicate that the HS component of holoHasAWT is a hexacoordinate species. The 1300-1700 cm-1 region of heme rR spectra comprises bands corresponding to porphyrin in-plane vibrational modes. They are diagnostic for oxidation state, coordination number, and spin state of the heme iron atom (44, 45). The spectrum of holoHasAWT (Fig. 4A) is indicative of a ferric species with its characteristic {nu}4 band at 1372 cm-1. The spin state marker bands indicate an equilibrium mixture of six coordinate high spin (6c HS) ({nu}2, 1561 cm-1; {nu}3, 1476 cm-1; {nu}10, under the 1624 cm-1 band) and six coordinate low spin (6c LS) ({nu}2, 1580 cm-1; {nu}3, 1503 cm-1; {nu}10, 1633 cm-1) states. The ratio of the two {nu}3 and two {nu}10 band intensities does not change between pH 5.2 and 9.2, whereas the LS bands increase in intensity at the expense of their HS counterparts as the temperature is lowered (data not shown). These observations suggest that the mixture of 6c HS and 6c LS hemes arises from either two pH independent axial ligand sets or a single axial ligand set characterized by a pH-insensitive spin state equilibrium.


Figure 3
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  		FIGURE 3.
UV-visible spectra of ferric holoHasAWT (A) at pH 7 and ferric holoH83A (B) at the indicated pH.

 
For hemoproteins, excitation into the tyrosinate-Fe(III) CT band (near 500 nm) can provide resonance enhancement of Raman scattering by vibrations characteristic of bound phenolate. No bands attributable to bound tyrosine vibrations were identified in the 514.5-nm excited rR spectra of HasAWT. However, most rR bands assigned to Fe-Tyr- stretching modes have been observed in spectra of 5c HS hemes (14, 29-33). Because the heme in HasAWT is clearly six coordinate, Raman scattering by these {nu}(Fe-Tyr-) and coordinated tyrosinate modes may be poorly enhanced with green excitation.

Spectra of holoH83A were recorded over the pH range of 5.2 to 10.0 with both B-band (Soret) and Q-band excitation at 413.1 (Fig. 4) and 514.5 nm (Fig. 5), respectively. Three pH-dependent forms, two HS and one LS, were detected, as suggested by the optical absorbance spectra and show their pH-dependent interconversion.

High frequency spectra recorded at pH 5.2 or 7.2 contain {nu}2, {nu}3 (Fig. 4A), {nu}10, {nu}11, and {nu}19 (Fig. 5) bands that reveal the presence of 5c and 6c HS hemes. They fall within frequency ranges characteristics of coordination number and spin state, as indicated in the figures. The {nu}3 band observed at 1477 cm-1 is consistent with a 6c HS heme. The second {nu}3 band at 1487 cm-1 falls at the low end of the frequency range expected for 5c HS proteins (29). The more intense {nu}2 band is consistent with the presence of a 6c LS species, although no {nu}3 band attributable to 6c LS heme can be observed in the Soret-excited spectra recorded at pH 5.2 and 7.2. The evidence of the 6c LS species is corroborated by pairs of {nu}10 and {nu}11 bands that arise from both HS and LS hemes in the Q-band excited spectra (Fig. 5). Both the 1477- and 1487-cm-1 {nu}3 bands corresponding to the 6c and 5c HS species persist at pH 9.0, whereas a new {nu}3 band grows at 1501 cm-1 characteristic of the 6c LS species. The 6c HS and 6c LS {nu}3 bands increase in intensity with increasing pH relative to the 1487 cm-1 band are consistent with a transition from 5c to 6c and with an equilibrium between 6c HS and 6c LS hemes at alkaline pH. The relative intensities of the HS and LS pairs of {nu}10 and {nu}11 bands with increasing pH corroborate the presence of this equilibrium (Fig. 5). Spin state equilibria of this nature have been reported for hydroxide complexes of various heme proteins (34-36). Persistence of bands attributable to 5c HS heme at pH 9 and 10 clearly shows that complete conversion to the hydroxide complex is not achieved under the conditions of the rR experiment.

The low frequency region rR spectra are useful in identifying metal-ligand vibrations and porphyrin deformations, many of which involve motion of peripheral substituents (37, 38). HoloH83A (Fig. 4B) exhibits bands in the 480-570-cm-1 range, where {nu}(Fe-OH) bands of HS and LS heme hydroxides occur. However, because the frequencies of these bands are insensitive to both D2O and 18OH2, they are assigned to porphyrin modes (44). The bands between 380 and 425 cm-1 are due to propionate and vinyl bending modes and do not vary significantly as a function of pH (Fig. 4B). The HasAWT spectrum exhibits two vinyl bending bands, consistent with the two vinyl-protein interactions. The spectra of holoH83A exhibit a single vinyl bending band in acidic and neutral solution and a broadened band in alkaline solution, suggesting that the structure of the WT heme pocket has been modified. At low pH, a pronounced band is observed at 196 cm-1 (Fig. 4B). Although this is in a region often associated with modes having axial iron-His stretching character, the only such modes that have been identified in ferric hemes are in those having the bis-His axial ligand set. Because this set of ligands is inaccessible in holoH83A, the 196 cm-1 band is concluded to arise from a porphyrin mode. Based on its greater intensity at low pH, where HS pentacoordinate iron dominates the heme speciation, the 196 cm-1 band is attributed to a mode having porphyrin out-of-plane character.


Figure 4
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  		FIGURE 4.
413.1-nm excited high frequency (A) and low frequency (B) rR spectra of holoHasAWT and holoH83A at the indicated pH.

 


Figure 5
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  		FIGURE 5.
514.1-nm excited low frequency rR spectra of holoH83A at the indicated pH.

 
Characterization of holoHasAWT and holoH83A Mutant by NMR
1H NMR Spectroscopy—The analysis of 1H-15N hetero-nuclear single-quantum correlation spectra of holoHasAWT and holoH83A indicate that for both systems the secondary structure and protein folding are maintained over the investigated pH range (supplemental Fig. S1 and Ref. 19).

One-dimensional experiments over large spectral windows were used to monitor the heme coordination sphere. We previously showed that, in HasAWT, HS and a LS species were in fast exchange on the NMR time scale (19). Indeed, the one-dimensional 1H NMR spectrum of the protein shows heme methyl resonances with chemical shift values intermediate between those expected for a purely HS S = 5/2 heme iron(III) and those for a purely LS S = 1/2 heme iron(III) (Fig. 6A). One major and one minor set of resonances are visible on the spectrum, corresponding to the two possible orientations of the heme along the {alpha}-{gamma} meso axis (10, 19). The chemical shifts and the nonselective T1 and T2 values for the hyperfine-shifted signals are in the 1-10-ms range, and the line widths are ~400 Hz at 16.4 T (supplemental Table S1). The relaxation times are close to those expected for a HS species (39). Two exchangeable, broad and fast relaxing signals with a 70:30 intensity ratio are observed at 65.8 and 73.2 ppm, respectively (Fig. 6A). Their line widths are ~1000 Hz, and their T1 values are shorter than 2 ms. They typically correspond to imidazole N{delta}1H of iron coordinated histidines (39-42) and are attributed to His32 N{delta}1H in the two heme orientations. Most of the hyperfine-shifted resonances in the 80 to -30 ppm region exhibit a pronounced temperature dependence of their chemical shift, with a nonlinear and anti-Curie behavior (supplemental Fig. S2A) for the heme methyls and His32 N{delta}1H, consistent with an equilibrium between a LS and a HS form (43-45). Hyperfine-shifted signals of holoHasAWT do not show significant shifts from pH 4.5 to 10.4, indicating that the equilibrium between the two spin states is not affected by pH. The addition of a 10-fold excess of cyanide failed to influence the position and intensity of any of the resonances and did not show any detectable signal indicative of a cyanide adduct.


Figure 6
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  		FIGURE 6.
1H NMR spectra, reported in the region 105/-40 ppm, of holo-HasAWT at pH 5.6 (A) and holoH83A at pH 5.2 (B), pH 7.1 (C), and pH 9.9 (D). The spectra were recorded at 11.7 T at 303 K in 20 mM phosphate buffer. The signals of the minor alkaline form are marked by asterisks. The inset in D shows the His32 N{delta}1 H signals.

 
The holoH83A 1H NMR spectrum obtained at acidic pH displays two sets of well resolved peaks in a 3:1 ratio with four distinct heme-methyl resonances between 60 and 80 ppm, typical of HS species (Fig. 6B) (39). They correspond to the two different heme orientations in the heme-binding pocket. A very broad, exchangeable, resonance associated with the high spin species is present at 102.3 ppm, in the spectra at 303 K and pH 5.6, with a T1 < 1 ms and a line width of ~1000 Hz. It is characteristic of the N{delta}1H of an axial histidine for a HS species (39, 46). Because no other histidine can coordinate the iron in holoH83A, it corresponds to His32 N{delta}1H. Longitudinal relaxation times are less than 1 ms for the four heme methyls and ~4-5 ms for all the other downfield shifted signals. As illustrated in supplemental Fig. S2B, hyperfine-shifted resonances exhibit a Curie behavior, indicative of a pure HS state (39). The meso 1H shifts are characteristically downfield (~40 ppm) for the hexacoordinate HS ferric complexes and upfield (-20 to -70 ppm) for the pentacoordinate high spin ferric complexes (39, 47). The 1H NMR spectrum of holoH83A clearly locates meso H-peak at ~-25 ppm. Therefore, at acidic pH values holoH83A is a pentacoordinate HS species. In addition to the two sets of resonances described above, the spectrum of holoH83A at acidic and neutral pH exhibits unresolved overlapping signals between 20 and 40 ppm (Fig. 6, B and C). With increasing pH, the acidic pentacoordinate HS species decreases in intensity, whereas the overlapping signals at 20-40 ppm increase. Additionally, a minor set of signals (~10%) appears in the region 40-60 ppm. They are overwhelmed by the acidic HS form and can be unambiguously identified only at pH > 9 (Fig. 6D). The two alkaline species undergo line narrowing at increasing pH, indicative of an exchange process in the quasislow regime. Line narrowing at higher pH allows the identification of several well resolved peaks in the 20-40-ppm region with T1 of ~3 ms and line widths of ~500 Hz. The temperature dependence of these signals is nonlinear (supplemental Fig. S2C). All of these findings are consistent with an hydroxide-bound form (36, 61-63). Two broad and exchangeable signals, missing at acidic pH, are observable at alkaline pH (a major one at 64.7 ppm and a minor one at 70.5 ppm at pH 9.9 and 303 K) (Fig. 6D, inset). These resonances, characteristics of the imidazole N{delta}1H of a heme iron-coordinated histidine (39-42), correspond to His32 N{delta}1H in the two heme orientations. Thus, His32 is a heme iron ligand in the hydroxide-bound species. The minor alkaline form (10%) is characterized by larger shifts with respect to the hydroxide form, accounting for a predominantly HS species.

No spectra changes were observed upon the addition of a 15-fold buffered KCN to holoH83A at pH 7.3 or 9.4. Thus, whatever the dominant species in solution, holoH83A does not bind cyanide at any pH.

15N NMR Spectroscopy—holoH83A spectra were recorded at various pH values at 303 K. The histidine resonances were assigned by comparison with the already available assignment of holoHasAWT and holoH83A at pH 5.5 and 7.0, at 317 K (19). The His32 N{delta}1 resonance is observed at 194.2 ppm, at pH 7 (Fig. 7). The His32 N{epsilon}2 signal is undectectable as in holoHasAWT, in agreement with its coordination to the heme iron. The spectrum recorded at pH 9 shows dramatic broadening of all resonances, thus preventing histidine identification at alkaline pH (data not shown).


Figure 7
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  		FIGURE 7.
1H -coupled 15N NMR spectrum of uniformly 15N-labeled holoH83A at 303 K, pH 7.0. The spectrum was recorded at 40 MHz 15N Larmor Frequency, on a 1.3 mM sample. Approximately 1 x 107 scans were collected.

 


Figure 8
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  		FIGURE 8.
13C NMR spectra at 303 K of holoHasAWT (1.6 mM) at pH 5.6 (A; taken from Ref. 19), holoH83A (2.1 mM) at pH 5.6 (B), and holoH83A (1.9 mM) at pH 8.3 (C). The expanded scale of trace C permits the identification of 13C signals (indicated with asterisks) attributed to the alkaline major form. The spectra were recorded at a 175 MHz 13C Larmor frequency.

 
13C NMR Spectroscopy—Resonances of the iron-coordinated residues can be observed using 13C direct detection in one-dimensional experiments (19). The comparison of holoHasAWT and holoH83A spectrum at pH 5.6 is shown in Fig. 8. The holoH83A spectra show eight hyperfine-shifted well resolved peaks in the downfield region (Fig. 8B, peaks A-H). Two additional signals were observed at lower temperature (283 K) (data not shown). On the basis of the relative intensity, five pairs corresponding to the two heme orientations could be identified, a set of major signals labeled A, C, D, F, and H and a set of minor signals. Four pairs have T1 values shorter than 1 ms, and one pair has T1 in the 10-13-ms range (Table 2). T1 values reported for holoHasAWT are of the same order of magnitude (19). Analogous with the findings for the 1H heme resonances, larger hyperfine shifts with respect to the wild-type and Curie behavior are observed in the 13C spectrum of the mutant. This behavior can be accounted for by the presence of a HS form. The number of hyperfine-shifted resonances is smaller than for holo-HasAWT, consistent with the presence of a single protein ligand. We therefore assigned them to the axial ligand His32.


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  		TABLE 2
Hyperfine-shifted 13C NMR signals of HasA-H83A at 303 K pH 5.6

 
At pH 8.3, a new set of weakly hyperfine-shifted signals is observed in the range +200/-50 ppm (Fig. 8C, inset). Their relative intensity with respect to that of the strongly hyperfine-shifted signals is the same as observed in the corresponding 1H spectrum between the 5c HS and hydroxide-bound forms. Resonances in a similar chemical shift range have been detected for the axial ligands in LS cytochrome c.3

The paramagnetic tailored 13C-13C COSY spectra in the aromatic region at acidic pH of holoHasAWT and holoH83A are essentially superimposable. However, in the mutant, a new cross-peak appears in the region diagnostic for Cβ-C{gamma} correlations of aromatic residues. This peak can only correspond to the Tyr75 Cβ-C{gamma} correlation because all the other aromatic residues have been already assigned in both spectra, which are similar in this region (Fig. 9, A and B). In the holoH83A spectrum at pH 8.3, the latter correlation is still present. Moreover, an additional peak appears that corresponds to the Tyr75 Cβ-C{gamma} correlation of the low spin form (Fig. 9C). 13C-13C COSY cross-peaks are detectable only if their line width is smaller than ~300 Hz (48). A coordination bond induces a strong electron spin delocalization from the metal to the ligand affecting both chemical shift and nuclear relaxation (49), thus making the signals of the ligands undetectable in the 13C-13C COSY. This means that Tyr75 is not a heme iron ligand in any of the two forms detected by NMR as a function of pH. In holoHasAWT 13C-13C COSY, Tyr75 Cβ-C{gamma} correlation was not observed (Fig. 9A).


Figure 9
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  		FIGURE 9.
Portions of two-dimensional, paramagnetic tailored, 13C-13C COSY spectra containing the Cβ-C{gamma} correlations of phenylalanine and tyrosine residues. A, holoHasAWT at pH 5.6. B, holoH83A at pH 5.6. C, holoH83A at pH 8.3.

 


Figure 10
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  		FIGURE 10.
15 K EPR spectra of holoHasAWT in solution at pH 7.0 (A) and pH 9 (B); holoHasAWT polycrystal sample (C); holoH83A in solution at pH 5.5 (D), pH 7.5 (E), and pH 9.0 (F). Experimental conditions were microwave power of 4 milliwatts at 9.42 GHz and modulation amplitude of 1 mT at 100 kHz.

 
Electron Paramagnetic Resonance Spectroscopy of the Ferric Proteins—At neutral pH, holoHasAWT exhibits a rhombic LS signal with g values at 2.80, 2.20, and 1.71 (Fig. 10A). A weak HS S = 5/2 signal with a slight rhombic character is also visible at g = 6.3 and 5.8. It represents less than 10% of the total spin intensity. The spectrum does not change at pH 5.5 (not shown), but at pH 9.0 some spectral heterogeneities are revealed by the appearance of a second LS component at g = 2.76, 2.20, and 1.76. However, the ratio of the HS to LS signals remains essentially unchanged at this pH (Fig. 10B). Thus, the EPR results indicate that the coordination sphere of the iron ion is not modified between pH 5.5 and 9.0.

To compare the local environment of the heme Fe3+ ion for the protein in solution and in the crystal, EPR experiments were also performed on a sample made of small HasAWT crystals spread in mother liquor at pH 8 and frozen. The spectrum of this polycrystalline sample shows a major LS signal at g = 2.8, 2.2, and 1.71 and a minor HS signal (Fig. 10C). Their close similarity with those given by the protein solution shows that the magnetic properties and the coordination of the heme Fe3+ ion are the same.

The EPR spectrum of holoH83A variant at acidic pH shows a HS signal with axial symmetry (Fig. 10D) at g{perp} = 5.8 and g// = 1.99. At neutral and basic pH (Fig. 10, E and F), a LS signal appears as the major component with g = 2.73, 2.19, and 1.78, and a slightly rhombic HS signal at g = 6.5 and 5.4 is also present. The relative proportion of the HS signal appeared to be preparation-dependent but remains lower than 20%.

X-ray Structure of Ferric holoH83A Mutant—One molecule was present in the asymmetric unit, giving a crystal volume/protein mass (VM) of 4.33 Å3 Da-1 and a solvent content of 72% by volume (50). The coordinates describing one protein molecule with one heme group per asymmetric unit were refined to a final R factor and Rfree of 23.6% and 27.3%, respectively. As for the structure of holoHasAWT, the last 14 C-terminal residues are undetectable.

Although holoH83A only diffracted to medium resolution, and the B-factors are substantially larger than those of holo-HasAWT (supplemental Fig. S3), comparison of both structures show that they are very similar (supplemental Fig. S4). Overall root mean square deviation is 0.51 Å. The most significant differences concern the two heme-binding loops, i.e. residues 32-44 and 73-81 that move away from the heme in holoH83A, with an apparent increase in the His32 N{epsilon}2-Fe and Tyr75 O{eta}-Fe bond lengths. However, the quality of the structure precludes to determine whether Tyr75 is coordinated to the heme iron in the crystal of holoH83A or there is no more an axial ligand, as established for holoH83A in solution. Indeed, in holoHasAWT, the tyrosinate form of Tyr75 is stabilized by a tight hydrogen bond between His83 N{delta}1 and Tyr75 O{eta} (1.86 Å). In holoH83A, the only potential candidate to form a hydrogen bond with the hydroxyl proton of Tyr75 is the Ala83 carbonyl oxygen. Although the distance between the two atoms, ~4 Å, does not preclude the formation of a weak hydrogen bond, the tyrosinate character of Tyr75 would remain poor.


Figure 11
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  		SCHEME 1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The heme iron spin state and the coordination sphere of both holoHasAWT and holoH83A have been defined over a wide range of pH values. All of the spectroscopic techniques indicate that the heme iron coordination in holoHasAWT does not undergo changes in the pH range 4.5-10.4. Resonance Raman shows that the sixth position in the high spin state is neither vacant nor occupied by a water molecule, supporting His32 and Tyr75 ligation. NMR data accounts for a thermal high spin-low spin equilibrium in fast exchange on the NMR time scale with a single set of axial ligands, His32 and Tyr75. The equilibrium is related to modulations in the strength of the coordination bond between the iron and Tyr75, which in turn depends on the strength of the hydrogen bond between Tyr75 O{eta} and His83 N{delta}1 and on the tyrosine protonation state. For holoH83A, the different techniques monitor changes in iron coordination number and spin state as a function of pH that are summarized in Table 3.


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  		TABLE 3
Coordination numbers and spin states for the different forms of holoH83A by the various spectroscopic techniques as a function of pH

 
In solution at acidic pH the major form of holoH83A is a high spin pentacoordinate species. The fifth ligand is unambiguously identified by NMR as His32. Resonance Raman reveals the presence of two additional six coordinated species with HS and LS state. Relative intensities of rR bands do not generally correlate with the relative populations because of differences in their resonance enhancement. NMR spectra at acidic pH show evidence of a minor species, characterized by broad features in the 20-40-ppm range, accounting for ~20% of total signal intensity and attributed to an OH- bound form. Signal broadening arises from an exchange process, quasi-slow on the NMR time scale, with another protein form, whose intensity is beyond detection at acidic pH and probably accounting for the 6c HS species observed via rR spectroscopy.

At increasing pH, all of the spectroscopic techniques monitor a decrease of the 5c HS species. UV-visible and EPR spectroscopies show the appearance of two new species, one HS and one LS species. They are consistent with the six-coordinated species detected by rRaman. Relative intensity of NMR signals shows that the six-coordinated LS form is the dominant species at alkaline pH values and that the HS spin species is ~10%. However, the nature of these species and their spin state need to be commented.

As outlined above, relaxation data and chemical shifts of the dominant alkaline species account for a His32/OH- axial coordination. As described in the literature for a variety of ferric heme proteins, this is not a purely low spin state as also confirmed by the temperature dependence of the chemical shifts (51-54).

Acid-base transitions taking place in ferric heme proteins with a H2O/OH- character are generally correlated to a change in the protonation status of a close residue in the heme pocket (55). This residue, which is a histidine in most cases, forms a hydrogen bond with the iron bound water (55). The histidine deprotonation causes ionization of the bound water, resulting in a predominantly low spin hydroxide form. In holoH83A, the most likely residue for deprotonation in close proximity to the bound water is Tyr75. Thus, we propose that, at increasing pH, the phenolic oxygen atom of Tyr75 forms a hydrogen bond with the water ligand, thereby stabilizing the hydroxide complex, as summarized in Scheme 1 and Table 3. The modulation of the H-bond strength involving H2O/OH- may broaden the HS and LS {nu}Fe-OH modes, such that they are not observed in the low frequency rR spectrum. The pKa of the transition from the five-coordinated form and the six-coordinated form is ~8.4 (Scheme 1). Transitions involving deprotonation of bound water have been reported to occur with a wide range of pKa values (56, 57). In the present case, exchange broadening prevents us from an accurate estimate of the pKa for the transition between the 6c HS and the 6c LS form (Scheme 1). However, their relative intensity does not vary at pH > 8. Estimates of rate constants for the two equilibria depicted in Scheme 1 are ~6 x 104 s-1 for the transition between the 6c HS and the 6c LS form and much lower than 5 x 104 s-1 for the transition between five-coordinated to six-coordinated species. Although the various spectroscopic techniques we employed here probe a wide range of time scales, they converge to a consistent view of pH-dependent heme speciation in solution.

Accessibility of heme to anionic ligands seems restricted in holoHasAWT and holoH83A, which do not bind cyanide, either at neutral or at basic pH, despite their spin state. Cyanide is usually considered to be a strong ligand for ferric heme. However, the nature of the residues in the heme active site heavily affects the binding properties of the cyanide anion. Reduced or even loss of affinity toward HS heme iron (III) has been found in the case of heme hydrophobic environment (58-61). In holo-HasAWT and holoH83A, the heme is surrounded by residues involved in both hydrophobic and stacking interactions. The only three polar residues in the vicinity of the heme are all on the His32 side. In holoH83A, the replacement of a histidine with an alanine mutant makes the Tyr75 side of the heme more accessible but also more hydrophobic.

His83 modulates the stability of hexacoordinate heme in holoHasAWT through hydrogen bonding with the coordinated side chain of Tyr75. The purified hemophore, HasAWT, has a much higher intrinsic affinity for heme (Kd = 1.9 x 1011 M) than its purified physiological target, HasR (Kd = 0.2 x 10-6 M) (23). As previously suggested, relative affinities should invert to accomplish heme transfer from the hemophore to the cell surface receptor (19). Removal of His83 is not sufficient to cause such an inversion but reduces heme affinity by approximately 2 orders of magnitude. The modulation of the iron-Tyr75 bond strength caused by the Tyr75-His83 hydrogen bond suggests a certain plasticity of the heme-binding pocket on the Tyr75 side of the heme that could play crucial roles in the mechanism of heme release. Mutagenesis studies have suggested that the binding of HasAWT to HasR involves two regions that make independent contacts with the receptor: the regions of residues 51-60 and 95-107 (62). Residues 51-53 are at the edge of the heme-binding pocket and very close to tyrosine 75 (C{alpha} Ser51-C{alpha} Tyr75, 5.8 Å). Conformational changes in holo-HasAWT that are induced by binding to HasR could propagate to the heme-binding loop bearing Tyr75, thereby triggering the elongation and breaking of the His83-Tyr75 hydrogen bond.

The results presented herein show that disruption of the His83-Tyr75 H-bond results in a 5c heme. Because transfer of heme to HasR requires scission of the Fe-HasA bonds, the present characterization of holoH83A at physiological pH provides a picture of a possible mechanistic intermediate in that heme transfer reaction.


   FOOTNOTES
 
* This work was supported by funds from Ministère de l'Education Nationale de la Recherche et de la Technologie and the Caisse Nationale du Régime Social des Indépendants (to C. C.-S.); Marie Curie Host Fellowship MEST-CT-2004-504391 from the European Union (to C. C.-S.); and NIAID, National Institutes of Health Grants 1R15AI072719-01 and NCRR P20RR15556 (to K. R. R.). This work was also supported in part by EU-NMR Contract RII3-026145 and by funds from Progetti di Ricerca di Rilevante Interesse Nationalé 2005, CNRS, Institut Pasteur, and the Egide-Galileo Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Figs. S1-S4. Back

1 To whom correspondence should be addressed: Unité de RMN des Biomolécules, CNRS URA 2185, Institut Pasteur, Paris, France. Tel.: 33-1-40-61-88-73; Fax: 33-1-45-68-89-29; E-mail: ccaillet{at}pasteur.fr or alecrois{at}pasteur.fr.

2 The abbreviations used are: rR, resonance Raman; CAPS, 3-(cyclohexylamino) propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HS, high spin; LS, low spin. Back

3 P. Turano, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank I. Bertini and C. Wandersman for discussions and critical reading. We are grateful to S. Létoffé and C. Simenel for technical help. We thank the European Synchrotron Radiation Facilities (Grenoble, France) beamline ID14-EH1 for beam time and technical assistance.



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