Heme binding by Corynebacterium diphtheriae HmuT: Function and heme environment

Abstract

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicate that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen-bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen-bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

Graphical Abstract

Iron is required for infection in essentially all bacterial pathogens (1). In vertebrate infections, the most abundant source of iron is heme (iron protoporphyrin IX), which comes primarily from hemoglobin. Bacteria have developed sophisticated approaches to transport heme into the cytoplasm. These pathways have been the focus of many recent reviews (1–11) with particular emphasis on the structures of heme transport proteins (12–14), Gram-positive bacteria (15), including Bacillus anthracis (16) and Staphylococcus aureus (17–20), and Gram-negative bacteria (21).

To date, pathogenic bacterial heme uptake pathways that have been characterized in detail have involved ATP-binding cassette (ABC) transporters. These transmembrane systems utilize the energy yield from ATP hydrolysis to pump various compounds across cellular membranes (22–24). An ABC transporter comprises two transmembrane modules and two ATPase subunits. Import ABC transporters are commonly found in prokaryotic systems and have an associated substrate binding protein [in this instance a heme binding protein (HBP)] that brings the substrate to the ABC transporter (14).

A number of HBP’s have been structurally characterized. Pseudomonas aeruginosa PhuT (25) and Shigella dysenteriae ShuT (25;26) both contain a conserved tyrosine that binds the heme in a pentacoordinate fashion. Yersinia pestis HmuT (YpHmuT) utilizes a His/Tyr ligation and binds heme both as a monomer and as a π-stacked dimer (27;28). S. aureus IsdE (29) and Streptococcus pyogenes SiaA/HtsA (30;31) both form hexacoordinate heme complexes having His/Met axial ligation.

A number of other membrane-bound proteins involved in the capture of host heme are known to have tyrosine axial ligands. The S. aureus Isd (Iron-regulated surface determinant) pathway employs a series of proteins for heme uptake and transfer including IsdA (29), IsdB (32), IsdC (33), and IsdH (34). Each of these proteins has one to three NEAT (near-iron transport) domains (17;35). The NEAT domains that bind heme [IsdA-N1 (29;36;37), IsdB-N2 (38), IsdC-N2 (33;37), and IsdH-N3 (34;38)] do so using tyrosine as an axial ligand (17;39;40). The x-ray structure of IsdB-N2 indicates that this protein can bind heme with an axial methionine in addition to the conserved tyrosine (32). X-ray structures of other NEAT proteins, such as IsdX1 and IsdX2-N5 from B. anthracis, also show the heme to be bound by an axial tyrosine (41–43). All of these Isd heme-binding NEAT domains have a second Tyr four residues downstream of the ligand on the same β-strand. The side chain of the second Tyr serves as a hydrogen-bond (H-bond) donor to the phenolate oxygen atom of the axial Tyr ligand, whose bent coordination geometry enforces a π-stacking interaction with the porphyrin ring. Recently, a NEAT domain, Hbp2-N2, has been described in which a tyrosine from the β-7 strand is the axial ligand, rather than one in the β-8 YXXXY motif (44).

Bis-histidine motifs are found in HmuY from Porphyromonas gingivalis (45) as well as in membrane-spanning heme receptors (7). Other motifs in the heme receptor class are also known; HmuR from Neisseria meningitidis has a five-coordinate tyrosine ligation (46) while PhuR from P. aeruginosa has been shown to have His/Tyr ligation (47). In still another variant on heme binding motifs in heme uptake pathways, Shp from S. pyogenes has bis-Met axial ligation (48).

The variety of heme binding motifs in heme uptake pathways leads to an interest in further characterization of other proteins associated with heme transfer. One such pathway is found in the pathogen Corynebacterium diphtheriae, a Gram-positive bacterium which is the causative agent of diphtheria (49–53), a well-known upper respiratory tract disease that carries a high mortality rate in humans (54). Diphtheria is still common in developing countries due to low vaccination rates (55;56). C. diphtheriae requires iron for survival and for virulence (49;57–59).

C. diphtheriae acquires heme via an ABC-type heme binding protein transporter system (57). A variety of heme sources can be used including hemoglobin (Hb), hemoglobin/haptoglobin, and myoglobin (Mb) (60). The heme utilization (hmu) operon includes hmuT (the HBP /substrate binding protein), hmuU (the permease) and hmuV (the ATPase), which form an ABC transport system (61;62). The htaA gene is located immediately upstream of the hmuTUV locus, and it is followed by a promoter region and the htaC gene. Downstream of the hmuV gene is a promoter region and the htaB gene. The hmuTUV and htaA genes form a single operon, while htaB and htaC are transcribed independently (63). HtaA and HtaB are proposed to be anchored to the cytoplasmic membrane through a C-terminal hydrophobic region. Both proteins are exposed to the bacterial surface, suggesting that these heme binding proteins may function as heme receptors (62). It has been shown that HtaA passes heme to HtaB (64). The next protein in the pathway is the HBP HmuT, which donates heme to the HmuUV transporter. Once the heme is brought into the cell, the heme oxygenase HmuO catalyzes the O₂–dependent degradation of the heme, which releases its iron for further use in cellular functions (65;66).

Sequence alignment studies of CdHmuT with other Corynebacterium species reveal two conserved tyrosines: Tyr235 and Tyr349 as well as a conserved histidine (His136) and methionine (Met292). Homology modeling shows that Tyr235 is probably an axial ligand and that either His136 or Met292 could also bind as a sixth ligand. CdHmuT is unique among its homologs in that the proposed axial His and Tyr ligands are reversed with respect to their positions in the structurally characterized proteins ShuT, PhuT and YpHmuT. That is, the tyrosines in all proteins in heme uptake pathways known to date come from the N-terminal part of the sequence, but CdHmuT is predicted to have the tyrosine from the C-terminal part of the sequence. As this prediction indicates a key role for tyrosine in heme transfer, it was important to establish the axial ligands in CdHmuT. Herein we report the biophysical characterization of CdHmuT and a number of its mutants. UV-visible, resonance Raman (rR), and magnetic circular dichroism (MCD) studies combine to give a picture of this novel heme uptake protein.

Materials and Methods

Bacterial strains and media

E. coli strains DH5α and TOP10 (Invitrogen) were used for routine cloning and plasmid maintenance, while XL-1 Gold (Stratagene) was used in the mutagenesis experiments. E. coli BL21(DE3) (Novagen) was used for protein expression. Corynebacterium ulcerans strain CU77 was previously described (Schmitt & Drazek, 2001) and carries a point mutation that results in premature termination of the hmuT gene (Schmitt, unpublished observation). Chromosomal DNA from C. diphtheriae strain 1737 (Popovic et al., 1996) was used as the source DNA for PCR. Luria-Bertani (LB) medium was used for culturing of E. coli, and Heart Infusion Broth containing 0.2% Tween 80 (HIBTW) was used for growth of C. ulcerans strains. Bacterial stocks were maintained in 20% glycerol at −80°C. Antibiotics were added to LB medium at 50 μg/ml for kanamycin and to HIBTW for C. ulcerans cultures at 2 μg/ml for chloramphenicol. HIBTW was made low iron by the addition of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) at 12 μg/ml. Modified PGT (mPGT) is a semi-defined low iron media that has been previously described (Tai et al., 1990). Antibiotics, EDDA, Tween 80, were obtained from Sigma Chemical Co. and hemoglobin (human) was purchased from MP BioMedical.

Plasmid construction

The HmuT expression construct was developed using the pET28a expression vector (Novagen). A PCR-derived DNA fragment containing the C. diphtheriae hmuT coding region was initially cloned into the pCR-Blunt II-TOPO vector (Invitrogen). The DNA fragment harboring the hmuT gene was subsequently ligated into the NcoI-EcoRI sites in pET28a and the expression plasmid was then transformed into BL21(DE3). The cloned hmuT gene in the pET28a vector lacked the 20 - amino acid N-terminal secretion signal and contained an N-terminal Strep-tag, which was used for protein purification. The following primers were used in the PCR: hmuTF; 5′-CC ATGGCA AGC TGG AGC CAC CCG CAG TTC GAA AAG GGT GTC CAG GGC ACA TAT-3′; hmuTR; 5′-GAATTC CTA TAC CTG TGG GTC ATAC-3′: underlined sequences indicate restriction sites and the sequence in italics encodes the 8-amino acid Strep-tag.

Site-directed mutagenesis and hemoglobin-iron utilization assays

Site directed mutants were made using the QuikChange Lightning kit (Stratagene) according to the manufacturer’s instructions. Briefly, 125 ng of each primer containing the targeted base change and 50 ng of plasmid template were used in the QuikChange reaction. Methylated template DNA was removed from the reaction by digestion with DpnI restriction endonuclease, and mutagenized DNA was recovered by transformation into XL1-Gold competent cells. The presence of the base changes was confirmed by sequence analysis. Plasmids used for site-directed mutagenesis were pET28a containing the cloned Strep-tag-hmuT gene, and plasmid pCD842, which harbors the hmuT gene on the E. coli-Corynebacterium shuttle vector pCM2.6 (67). The hemoglobin utilization assay has been described previously (66).

Expression and purification of CdHmuT

HmuT was expressed and purified from BL21DE3 (pEThmuT) cells. The N-terminal leader sequence was deleted and replaced with a Strep-tag. The native construct started at residue 21 (Gly) and extended through the native stop codon. The culture was prepared in a Luria-Bertini (LB) medium containing 50 μg/mL kanamycin. Inoculation was done with an overnight pre-culture and cells were grown at 37 °C with shaking at 220 rpm. When the OD₆₀₀ of the culture reached 0.5 – 0.6, protein expression was induced by adding isopropyl β-D-1- thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM. The culture was incubated for 3 h at 27 °C. Cells were harvested by centrifugation at 8000 x g. The cell pellet was resuspended in lysis solution (100 mM Tris-Cl, 150 mM NaCl, pH 8.0) containing a protease inhibitor cocktail (Roche Complete Mini, EDTA-free, following the manufacturer protocol). The cells were broken using a cell disrupter or sonication. The lysate was then centrifuged at 8000 x g, and the supernatant was syringe-filtered with a 0.45 μm filter.

All of the following purification steps were conducted at 4 °C using fast protein liquid chromatography and all buffer solutions were pH 8.0 unless specified otherwise. The protein sample was loaded onto a Strep-Tactin Superflow column (5 mL, IBA BioTAGnology) equilibrated with buffer A (100 mM Tris-Cl, 150 mM NaCl, pH 8.0). Unbound material was washed out with 5 column volumes (CV) of buffer A. HmuT was eluted with 10 CV of buffer B containing 100 mM Tris-Cl, 150 mM NaCl, 2.5 mM desthiobiotin, pH 8.0 applied via a linear gradient. The purities of the fractions were evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Native PAGE did not show dimers, indicating that the protein is monomeric in solution. Minor differences in the optical spectra were observed as a function of the buffer type.

Magnetic circular dichroism spectroscopy

Magnetic circular dichroism (MCD) spectra were measured with a magnetic field strength of 1.41 T by using a JASCO J815 spectrophotometer. This instrument was equipped with a JASCO MCD-1B electromagnet and interfaced with a Silicon Solutions PC through a JASCO IF-815-2 interface unit. Data acquisition and manipulation using Cary or Jasco software has been previously described (68). To ensure homogeneity of ferric oxidation for the various mutants, ferricyanide was used to fully oxidize the heme center, followed by desalting chromatography. The resulting spectra were compared to data from other heme-containing proteins with known binding site structures and optical spectra. All spectral measurements for all proteins were carried out with a 0.2 cm quartz cuvette at 4 °C in 50 mM phosphate buffer (either pH 6.5 or 10).

Resonance Raman spectroscopy

Resonance Raman (rR) spectra were collected using the 441.6-nm emission line from a HeCd laser or either 406.7 nm or 413.1 nm emission from a Kr⁺ laser. Spectra were recorded at ambient temperature using the 135° backscattering geometry with the laser be am focused to a line on a spinning 5 mm NMR tube. Toluene, DMSO, and CH₂Br₂ were used as external standards for spectral calibration. UV-visible spectra were recorded before and after rR experiments to verify that the samples were not altered by exposure to the laser beam. The final concentrations of all Fe(III) samples were between 25 and 80 μM protein in 100 mM buffer solution. The buffers used were CHES, pH 10.0, Tris-Cl, pH 8.8 or 8.0, sodium phosphate buffer, pH 7.0 or 5.8, MES, pH 5.1, and sodium acetate buffer, pH 5.0. The D₂Oand H₂¹⁸O samples of the Y235A mutant were made by diluting a concentrated sample of the protein into CHES buffer made with either D₂O or H₂¹⁸O at pD 10 or pH 10, respectively. Resonance Raman spectra were collected with laser powers between 9 and 12 mW at the sample.

The ferric fluoride adduct of Y235A HmuT was prepared by titration with 0.8 M NaF solution in 0.1 M sodium phosphate buffer at pH 5.8. The final protein and NaF concentrations were 80 μM and 330 mM, respectively. The laser power was 9.7 mW for the 406.7 nm Kr⁺ excitation and 4.6 mW for the 441.6 nm HeCd excitation.

Ferrous HmuT(Y235A) samples (36 μM) were prepared under anaerobic conditions in 0.1 M Tris-Cl pH 8.2 by adding an 86-fold excess of sodium dithionite in buffered solution. Laser power for the ferrous samples was 4 to 8 mW. Ferrous carbonyl adducts (36 to 75 μM) were made by reducing the proteins with a 70-to 180-fold excess of buffered sodium dithionite under an atmosphere of natural abundance CO or ¹³CO (99 atom % ¹³C) in 0.1 M Tris-Cl pH 8.8. Laser power for the heme carbonyl samples was held between 2 and 4.5 mW to minimize photolysis.

Results

Heme ligation in ferric HmuT

An alignment of the HmuT amino acid sequences of various Corynebacterium species showed that two tyrosine residues (Y235 and Y349) as well as H136 and M292 were highly conserved in this group of bacteria (Figure S1). A homology model of HmuT was created by I-TASSER(69) with YpHmuT as one of the templates (Figure 1). The two structures were similar overall with a root mean square difference (RMSD) between the model and YpHmuT of 0.673 Å. Alignment of the CdHmuT sequence with four HBP’s for which crystal structures have been solved: PhuT from P. aeruginosa (25), ShuT from S. dysenteriae (25), IsdE from S. aureus (70) and YpHmuT(27) indicated that it was probable that CdHmuT would have a Tyr as one axial ligand, and a Met or His as the second (Figure S2). However, CdHmuT was predicted to be unique in that the axial ligands would be reversed (N-terminal His and C-terminal Met or Tyr) with respect to structurally characterized homologous hexacoordinate heme proteins (N-terminal Tyr and C-terminal His for YpHmuT and N-terminal Met and C-terminal His for IsdE). Based on this homology model and sequence alignment, the H136A, Y235A, and M292A mutants were created to probe the heme axial ligation.

Figure 1.

I-TASSER homology modeling of CdHmuT created using PyMOL (119). Shown are the locations of H136, Y235, M292, and Y349.

Conserved residues and the biological function of HmuT

To determine if H136A, Y235A, and M292A are important for the biological function of HmuT, the ability of the cloned hmuT genes to complement a Hb-iron utilization defect was assessed. It was previously shown that the CU77 mutant strain of C. ulcerans HmuT (CuHmuT) was defective for HmuT activity (57). The cloned hmuT gene from either Corynebacterium species could fully restore the wild-type phenotype to this strain. Complementation studies with each of the H136A, Y235A, and M292A CdHmuT mutants revealed that only Y235A was unable to restore growth fully (Figure S3). Thus, Y235 is essential for the heme-iron utilization function of HmuT.

Spectroscopy of wild-type CdHmuT

The UV-visible spectrum of ferric wild-type (WT) CdHmuT showed a Soret peak at 407 nm and four peaks in the α,β region at 492, 546, 569, and 616 nm (Figure 2). The charge transfer band at 616 nm is characteristic of a high-spin species, and the two α,β bands at 569 and 546 nm are consistent with the presence of a low-spin species. Hence, these data suggest an equilibrium mixture of high-spin (HS) and low-spin (LS) WT CdHmuT. This UV-visible spectrum is strikingly similar to that observed for S. marcescens HasA (SmHasA) (406, 494, 537, 568 and 618 nm) which is known to have His/Tyr axial ligation and exists in a thermal spin equilibrium (71).

Figure 2.

UV-visible spectra of the Fe(III) forms of WT CdHmuT (solid line), H136A (dashed line), and Y235A (dotted line) normalized at the Soret. The samples were taken in 50 mM Tris-Cl at pH7.0.

Ligation of the heme was further probed by the use of MCD. Initial comparisons of the spectra of WT CdHmuT with models having His-only or His/Met axial ligation did not give entirely similar spectral profiles (data not shown). The UV-visible and MCD spectra were then compared with two five-coordinate Tyr-ligated proteins, bovine liver catalase (BLC) (72–74) and the H93Y mutant of Mb (75). Figure S4 shows agreement in the Soret region and moderate agreement in the α,β region of the UV-visible spectrum. However, the three species are different in the MCD spectrum. Towards the blue, Mb-H93Y and HmuT are similar, but BLC is quite different. Towards the red, the peaks and troughs observed for HmuT are similar to, but shifted from, Tyr-ligated BLC, and there is no agreement with Mb-H93Y. These results lead to the conclusion that WT CdHmuT does not adopt a five-coordinate, tyrosine-ligated geometry.

WT CdHmuT was then compared to a model for the His/Tyr ligand set: leghemoglobin a with exogenous phenol (76). This complex adopts a six-coordinate heme binding structure with a histidine axial ligand and a phenol in the transposition. Figure 3 shows that both the UV-visible and MCD spectra were in agreement with those of the WT CdHmuT supporting the conclusion that CdHmuT is a His/Tyr protein.

Figure 3.

The UV-visible and MCD comparison spectra for Fe(III) WT CdHmuT at pH 6.5 with Fe(III) phenol-bound leghemoglobin a. The samples were taken in 50 mM phosphate buffer. Spectra were slightly dependent on buffer conditions. The spectrum of phenol-bound leghemoglobin a was replotted from (76).

Soret-excited resonance Raman (rR) spectra of WT CdHmuT were recorded over the pH range of 5.0 to 10.0 (Figure S5). Two bands are observed in the 1470 – 1510 cm⁻¹ region of the rR spectrum. The ν₃ core size marker band frequencies observed in this range are indicative of heme spin state and coordination number. The pair of ν₃ bands observed at 1475 and 1504 cm⁻¹ indicates that the protein contains a mixture of six-coordinate high-spin (6cHS) and six-coordinate low-spin (6cLS) hemes. This is consistent with their UV-visible spectra shown in Figure 2. The rR spectra were independent of pH, i.e., the identities of the axial ligands and populations of the 6cHS and 6cLS hemes were pH independent. Thus, the HS/LS equilibrium is not governed by any acid-base speciation of the heme or the protein over this pH range.

Spectroscopy of M292A CdHmuT

The UV-visible (Figure S6) and rR (Figure 4) spectra for the WT and M292A proteins, as well as the dependence of the rR spectrum on pH (Figure S7), were almost identical. In the MCD, as well, the M292A spectra were remarkably similar to the WT protein, with only slight differences in absorbance maxima (Figure S6). The overall similarity between the spectral signatures of the WT and M292A proteins suggest that Met is not an axial ligand.

Figure 4.

Comparison of the Soret-excited rR spectra of WT CdHmuT, M292A, H136A, and Y235A. Protein concentrations were 80, 70, 25 and 36 μM, respectively. All samples were prepared in 50 mM Tris-Cl at pH 7.0. The spectra were recorded with 406.7-nm excitation. A) Low frequency and B) high frequency spectra of WT CdHmuT (blue), M292A (black), H136A (green), and Y235A (red).

Spectroscopy of H136A CdHmuT

H136A CdHmuT gave a broad Soret band that was blue shifted to 400 nm and three defined bands in the α,β region, including one at 620 nm (Figure 2). The H136A mutant was compared with three tyrosine models: ShuT (26), BLC (72–74), and Mb-H93Y (75) (Figure S8). In the UV-visible spectra, the Soret peaks for all four proteins were similar, with maxima located from 400 to 407 nm. In the visible region, the characteristic high-spin peak located past 600 nm was evident for H136A HmuT, BLC, and ShuT and blue-shifted for H93Y Mb. The peaks and troughs in the visible region of the MCD spectrum are evidence for tyrosinate ligation. Together, the data indicate a ferric five-coordinate tyrosinate-bound heme.

The Soret-excited rR spectrum of H136A HmuT is significantly different from that of WT; it comprises ν₃ bands at 1479 and 1487 cm⁻¹, indicating the presence of 6cHS and five-coordinate high-spin (5cHS) hemes, respectively (Figure 4). The presence of only HS species reflects the weakened axial ligand field in the absence of His136 (77;78). The bands at 721 and 693 cm⁻¹ are assigned to γ₅ (symmetric pyrrole ring fold, A_2u in D_4h) and γ₁₅ (symmetric pyrrole ring fold, B_2u in D_4h), respectively, by analogy to Mb (79–81). Intensification of the bands arising from these modes, which are normally Raman forbidden, reveals lowering of the porphyrin symmetry. Pentacoordination of the heme iron center, as anticipated for H136A mutation and as indicated by the core-size marker band frequencies in Figure 4B, is expected to drive the heme iron out of the mean porphyrin plane toward the Tyr235 side chain. This type of coordination typically increases the extent of porphyrin doming. The A_2u and B_2u symmetries of γ₅ and γ₁₅ correlate with A₁ and B₂ in the approximate C_4v symmetry of a domed conformation. Since A₁ and B₂ modes of a C_4v metalloporphyrin are Raman allowed, the intensification of scattering by both γ₅ and γ₁₅ is consistent with the heme in H136A being more domed than the WT protein.

The presence of 5cHS heme in H136A, which is absent in the WT protein, strongly suggests that His136 is a heme ligand in the WT protein. The rR spectrum of H136A does not exhibit a pH dependence over the range of 5.0 to 10.0, as seen in Figure S9, suggesting that neither the 5cHS or the 6cHS H136A species binds hydroxide under alkaline conditions. The inability to form a hydroxide complex has also been reported for HasA(H32A), wherein tyrosinate is the lone amino acid ligand to the heme (82). In fact, there are very few examples of Fe(III) porphyrinates having two anionic axial RO⁻ ligands, including OH⁻, because large overall positive charges are necessary to stabilize such axial ligand sets (83;84). Thus, the rR spectra are consistent with an equilibrium mixture of 5cHS Fe–Tyr⁻ and 6cHS H₂O–Fe–Tyr⁻ axial ligation.

Spectroscopy of Y235A CdHmuT

Y235A CdHmuT had a Soret band that was red-shifted by 7 nm compared to the native protein and a significant shoulder near 350 nm, which was absent from the native and other mutants (Figure 2). The α,β region had bands at 540 and 575 nm and no charge transfer band, consistent with a predominantly LS heme.

Figure 5 compares the UV-visible and MCD spectra of this mutant in the ferric state at pH 10 with two His/OH⁻ complexes, alkaline Hb at pH 10 (85) and horseradish peroxidase (HRP) at pH 12.5 (86). The peaks and troughs in both the Soret and visible regions are located at comparable wavelengths and exhibit similar relative intensities. These similarities indicate that Y235A adopts a His/OH⁻ ligation set at high pH.

Figure 5.

The UV-visible and MCD spectra for Fe(III) Y235A CdHmuT at pH 10 with Fe(III) alkaline Hb (pH 10) and Fe(III) HRP (pH 12.5). The samples were prepared in 50 mM phosphate buffer. The spectra of alkaline Hb and HRP were replotted from (85) and (86), respectively.

The heme speciation of Y235A was found to be sensitive to pH in the Raman spectra. Y235A was shown to shift from a mixture of 6cHS (1473 cm⁻¹) and 6cLS (1501 cm⁻¹) at pH 10.0 to all 6cHS (1477 cm⁻¹) at pH 5.0 (Figure 6). Based on changes in the relative rR intensities as a function of pH, the pK_a of the acidic form is near 6.

Figure 6.

The pH dependence of ferric Y235A monitored by 406.7 nm-excited rR spectra (11 mW power at sample). A) Isotopologs of Y235A at pH 10 prepared in H₂O, D₂O, and H₂¹⁸O. Difference spectra of D₂O-H₂¹⁸O and H₂O-H₂¹⁸O shown at the top of the figure were generated by subtraction of the respective parent spectra at the bottom of the figure. B) Low frequency and C) high frequency spectra of ferric Y235A as a function of pH. Samples were between 25 and 60 μM.

To determine whether the alkaline species comprising the HS/LS equilibrium are hydroxide complexes, the ²H and ¹⁸O isotopologs were generated in D₂O and H₂¹⁸O, and their Soret-excited rR spectra recorded (Figure 6A). Two isotope-sensitive bands were observed. The band at 521 cm⁻¹ in H₂O shifted to 504 and 489 cm⁻¹ in D₂O and H₂¹⁸O, respectively, and is assigned to the ν_Fe─OH mode of LS Y235A–OH. The second isotope-sensitive band appeared at 433 cm⁻¹ and shifted to 412 cm ⁻¹ in H₂¹⁸O and is tentatively assigned to the ν_Fe─OH mode of HS hemin–OH component of the mixture. The difference feature for the HS ν_Fe─OH observed in the D₂O – H₂¹⁸O difference spectrum has considerably less amplitude than that of the LS ν_Fe─OH making the shift due to deuterium in the HS complex difficult to measure. Y235A is similar to Mb, Hb, and various other heme protein hydroxides (87–91) that exist as HS/LS mixtures. Since the alkaline form of Y235A HmuT is a hydroxide complex, it is likely that the 6cHS species that dominates below pH 6 is an aqua complex.

Heme environment in CdHmuT

The carbon monoxide (CO) complex of ferrous heme is a particularly sensitive probe of the coordination and electrostatic character of its protein environment. The C–O and Fe–C stretching frequencies reflect the extent of FeCO π-backbonding, which is sensitive to FeCO geometry, the trans ligand, and nonbonded interactions between the CO and the distal heme pocket. Useful insight into the distal electrostatic landscape and the σ-donor strength of the trans ligand are derived from the position of a heme carbonyl complex on the ν_Fe–CO/ν_C–O correlation plot (92–94). The Fe–¹³CO isotopologs of WT CdHmuT and heme pocket mutants were used to identify rR bands corresponding to the Fe–CO stretching, Fe–CO bending and C–O stretching modes. The Soret-excited rR spectra are shown in Figure 7. Three ¹³C-sensitive bands were observed for the WT and H136A CdHmuT proteins while four were observed for the Y235A mutant. The spectrum of WT reveals isotope-sensitive bands at 535, 585, and 1920 cm⁻¹ which shift to 531, 558, and 1878 cm⁻¹, respectively, in the HmuT–¹³CO spectrum. They are assigned to the ν_Fe–C, δ_FeCO, and ν_C–O modes, respectively. For the H136A mutant, the isotope–sensitive bands shifted from 530, 574, and 1929 cm⁻¹ to 525, 556, and 1884 cm⁻¹, respectively. Thus, its ν_Fe-C frequency was 5 cm⁻¹ lower than those of WT, and its ν_C–O frequency was 9 cm ⁻¹ greater, consistent with a weakened π-backbonding characteristic of the trans-Tyr ligand in this mutant. The largest differences were observed for the Y235A mutant with two ν_Fe–C bands occurring at 491 and 509 cm ⁻¹, a δ_FeCO frequency of 574 cm⁻¹, and ν_C-O band at 1943 cm⁻¹ which shift to 488, 505, 559, and 1898 cm⁻¹, respectively, with ¹³C. The two ν_Fe-C frequencies were assigned by peak fitting the iron-carbon stretching region of the ¹²CO and ¹³CO spectra and generating a simulated difference spectrum; both are shown in Figure S10.

Figure 7.

Resonance Raman spectra of the ferrous carbonyls of WT CdHmuT, H136A, and Y235A recorded using 413.1-nm excitation. Natural abundance HmuT–CO (black), HmuT–¹³CO (red) and difference (blue) spectra are shown for each protein. Spectra of WT and H136A were recorded at pH 8.8 and that of Y235A at pH 8.2. The asterisks in the carbonyl stretching region of the Y235A spectrum mark plasma emission lines from the Kr⁺ laser.

The inverse correlation between ν_Fe–C and ν_C–O frequencies is plotted for CdHmuT and its heme pocket mutants along with a number of heme–CO proteins and model complexes for comparison in Figure 8. The WT HmuT–CO point falls on the ν_Fe–C/ν_C–O correlation plot in a position consistent with its proximal ligand being a weaker donor than His, suggesting that WT HmuT–CO contains a weak proximal Fe(II)–O bond, such as Fe(II)–Tyr or Fe(II)–OH₂ (95).

Figure 8.

Backbonding correlation plot of ν_Fe-CO versus ν_C-O for ferrous carbonyls of heme proteins showing the dependences of their positions on axial ligation and distal pocket properties. WT (blue), H136A (green), Y235A (red) are shown as stars on the plot. Catalase, hexagon; HasA(Y75A), ◇; HasA(WT), ○; HasA(H83A), ○; HasA(H32A), ○ (105;120). The dashed line is the least squares line for six-coordinate Fe–CO adducts in which the proximal ligand is thiolate or imidazolate; the dotted line is the least squares line for Fe–CO adducts with proximal histidine (neutral imidazole) (91;96;120) (and references therein); and the solid line represents a compilation of “five-coordinate” model complexes (93) (and references therein) and heme proteins which the ligand trans to CO is coordinated through an oxygen atom (98).

Distal H-bond donors to the bound CO ligand and positive charge enhance π-backbonding. Both of these interactions weaken the C–O bond while strengthening the Fe–C bond, placing points high and to the left on the imidazole correlation line (e.g., ν_Fe–C 520 cm⁻¹ and ν_C–O 1935 cm⁻¹) (96). Off-axis Fe–C–O distortion and negative charge weaken backbonding which, absent other factors such as hydrogen bonding, is characterized by positions to the low and to the right on the correlation line. Thus, the positions of WT HmuT–CO and H136A–CO would be interpreted as due to a positive charge near and/or H-bond donation to the bound CO.

The bottom set of spectra in Figure 7 reveal that Y235A HmuT–CO has two conformers whose positions on the ν_Fe–C/ν_C–O correlation plot are distinct from each other and from WT and H136A HmuT–CO. One conformer is located on the imidazole line, indicating neutral histidine axial ligation; its position relative to other proteins on the imidazole correlation line is consistent with a modest distal H-bonding interaction. In contrast, the second conformer is slightly above the imidazolate line suggesting that the proximal histidine (His136) has some imidazolate character, perhaps due to its interaction with an H-bond acceptor in the proximal pocket.

Ferric Y235A CdHmuT–fluoride

Fluoride complexes of heme proteins are sensitive probes of the distal H-bonding environment (97;98). The energy of the charge transfer band at 600 – 620 nm (CT1) together with the Fe–F stretching frequency constitutes a sensitive probe of H-bond strength between a distal H-bond donor and the bound F⁻ ligand. Low ν_Fe─F frequencies correlate with red-shifted CT1 bands in complexes having strong hydrogen bonds. The ν_Fe─F mode of Y235A HmuT–F was identified by exciting into its CT2 band (450 – 460 nm) with 441.6-nm light. Relative enhancement of scattering by the ν_Fe─F mode with 441.6-nm excitation is considerably greater than with Soret excitation; peak fitting of the 441.6-nm excited rR spectrum shown in Figure 9 revealed that the ν_Fe─F band occurs at 392 cm⁻¹. The Y235A HmuT–F CT1 band was observed at 613 nm (16,313 cm⁻¹) (Figure 9A inset). Correlation of its ν_Fe─F and the CT1 energy places Y235A CdHmuT low on the correlation plot in Figure 9B which is consistent with strong hydrogen bonding between the bound F⁻ ligand and the distal pocket.

Figure 9.

Characterization of Y235A–F by correlation of the Fe^III–F stretching frequency and CT1 energy. A) Low frequency window of the rR spectra of Y235A–F using Raman excitation into the CT2 (441.6 nm) and Soret (406.7 nm) bands. Protein was 80 μM in 100 mM sodium phosphate buffer in 330 mM sodium fluoride, pH 5.8. Laser power at the sample was 4.6 mW with 441.6-nm excitation and 9.7 mW with 406.7-nm excitation. Peak fitting analyses of both spectra are overlaid on the original spectra with the calculated Fe^III–F stretching band shown in red; calculated ν₈ and propionate and vinyl bending bands are shown in black; the overall fit is shown in magenta. Inset: Visible spectrum of Y235A–F rR sample. B) Correlation plot of ν_Fe-F frequency and the CT1 energy. Y235A is shown in red. Other points are from Nicoletti and coworkers (98). Open circles are for mutants of truncated Hb from Thermobifida fusca (Tf-trHb) with varying number of hydrogen bonds between the distal pocket and the fluoride (97;98).

Ferrous Y235A CdHmuT

Unlike WT and H136A CdHmuT, which are slow to reduce with aqueous buffered S₂O₄²⁻, Y235A CdHmuT was readily converted to a 5cHS ferrous heme (ν₄, 1354 cm⁻¹; ν₃, 1467 cm⁻¹) upon reaction with S₂O₄²⁻ (spectra not shown). The ν_Fe─His frequency for 5cHS ferrous hemes has been shown to be significantly enhanced with 441.6 nm laser excitation (99) and two ν_Fe─His modes for ferrous HmuT-Y235A are tentatively assigned to bands at 221 and 249 cm⁻¹ based on comparison of their relative enhancements in the 441.6 and 413.1-nm excited spectra in Figure 10. Observation of two ν_Fe─His modes is consistent with two proximal pocket conformers in Y235A CdHmuT, which are distinguished by the extent of imidazolate character of the proximal His ligand. These are likely the same conformers responsible for the two ν_Fe–C frequencies (491 and 509 cm ⁻¹) in the corresponding carbonyl spectrum shown in Figure 7 and Figure S10.

Figure 10.

Comparison of the low frequency RR window of ferrous Y235A spectra obtained with 413.1-nm (violet) and 441.6-nm (blue) excitation. Laser powers at the sample were 4.0 mW and 4.6 mW, respectively. The solutions were 38 μM in protein and 100 mM in Tris-Cl, pH 8.8.

Discussion

His/Tyr ligand set identified for HmuT:heme complex

Ferric species

Sequence alignment, homology modeling, and UV-visible, MCD, and Raman spectroscopies all lead to the conclusion that WT CdHmuT contains a six-coordinate active site with a tyrosine bound to one axial position of the heme and a histidine bound to the other. Mutation studies validated these findings, with M292A showing spectra very similar to the wild-type protein, indicating that this methionine is not an axial ligand.

H136A HmuT exhibited both UV-visible and MCD spectral features of other five-coordinate tyrosine-ligated proteins such as BLC(74), Mycobacterium avium ssp. paratuberculosis (MAP) (74), Plexaura homomalla coral allene oxide synthase (cAOS) (73), S. dysenteriae ShuT (26), and the S. aureus Isd system: IsdA-N1 (29;36;37), IsdB-N2 (38), IsdC-N2 (33;37), and IsdH-N3 (34;38). The ~620 nm charge transfer band appears to be characteristic of a tyrosine bound to the heme.

The Y235A mutant showed loss of the charge transfer band in the UV-visible spectrum, consistent with removal of the axial tyrosine. Both the UV-visible and MCD spectra of Y235A were not comparable with other known 5c His-bound heme proteins, but rather with 6c His/OH⁻ species, such as HRP(86) or alkaline hemoglobin (85). It was concluded that the ferric iron was still ligated to H136, but with the axial position vacated by the mutated Y235 occupied by a hydroxide ligand. This His/OH⁻ motif was seen not only at pH 10, but also at pH 6.5. pK_a values of water trans to histidine in ferric heme proteins vary from > 10 down to at least 6.8 (see examples in Supplementary Table 1). The homology model indicates that the Arg237 side chain is found in the heme pocket on the side opposite the axial histidine. Thus, the low pK_a for Y235A may be attributable to the interaction of water with this cationic side chain, which could serve as the H-bond donor to the axial Tyr235 ligand. Consistent with this proposal, in almost all tyrosine-heme proteins studied to date, the axial tyrosine is hydrogen-bonded to a second residue, common examples include Tyr, Arg and His (see examples in Supplementary Table 2). For heme uptake proteins, the number of residues between the tyrosine and its hydrogen bonding partner (located on the C-terminal side in known examples) can be as few as one or as many as seven.

For heme proteins having abound water ligand, a decrease in the Fe–OH stretching frequency relative to Mb and Hb has been attributed to strong hydrogen bond donation to the hydroxide. For example, in hemoglobin from M. tuberculosis (HbN) a 35 cm ⁻¹ decrease in HS Fe–OH stretching frequency has been attributed to a strong interaction between the bound hydroxide and a distal tyrosine side chain (90). Very strong hydrogen bonding in alkaline HRP similarly gives rise to the LS ν_Fe─OH frequency of 503 cm⁻¹, 47 cm⁻¹ lower than the LS ν_Fe─OH Mb frequency of 550 cm⁻¹(87). For Y235A CdHmuT, the LS and HS Fe–OH stretching frequencies are 29 and 58 cm⁻¹ lower, respectively, than those reported for Mb. This strongly suggests Tyr235 has a hydrogen bonding partner that upon removal of Tyr235 its normal H-bond partner serves as a H-bond donor to the hydroxide ligand. The presence of a strong hydrogen bonding partner on the Tyr235 side of the heme is further supported by the position of the Y235A HmuT–F on the ν_Fe─F frequency/CT1 energy empirical correlation plot (Figure 9B). On the correlation plot, it lies very close to the Thermobifida fusca (Tf-trHb) fluoride adduct which has two strong hydrogen bonds between the fluoride and the distal heme pocket (98).

Ferrous Species

The ferrous spectra of the wild-type and the H136A mutant could not be reduced under standard reductive conditions, e.g., sodium dithionite, unless in the presence of CO. This contrasts with reduction of Y235A, which is readily effected. These observations are consistent with His/Tyr heme ligation. Related observations have been made for HasA(100), which has a very low reduction potential (−550 mV versus SHE) (101). Upon mutation of its axial tyrosine, it is also readily reduced by dithionite (100).

Proteins and engineered mutants that can bind CO with either a trans His or Tyr have a histidine in some instances and an oxygen ligand (tyrosine or water) in others. For example, hemoglobin Saskatoon, with histidine and tyrosine as axial ligands, forms the His–Fe–CO species (102). Sperm whale HisE7Tyr (with a Tyr–Fe–His motif in the ferric form) also forms a His–Fe–CO complex (103). Other hemoglobins with axial tyrosines (e.g., Boston, Hyde Park and Saskatoon) form His–Fe–CO species, presumably utilizing the distal histidine (102). In contrast, HasA, with a His/Tyr ligand set, gives an O–Fe–CO species upon reduction in the presence of CO(100). The CO complex of human heme oxygenase H25Y, with a tyrosine axial ligand in the ferric form, gives an H₂O–Fe–CO species (104). Catalase, with an axial tyrosine, is thought to form the Tyr–Fe–CO species (105).

The ν_Fe–CO band in the Soret-excited rR spectrum of catalase–CO has been reported to be more intense than the totally symmetric ν₇ band (105). This signature was correlated with the anionic character of the proximal Tyr whose coordination to heme iron is stabilized by H-bonding to a His residue. Although the ν_Fe–CO bands for WT and H136A HmuT CO complexes are not more intense than their ν₇ bands, they are quite intense relative to the ν _Fe–CO band of Y235A HmuT–CO. This is taken as further evidence for a charge neutral Tyr–Fe(II)–CO species in H136A HmuT and WT HmuT.

Unlike the wild-type and H136A, Y235A was reduced by aqueous S₂O₄²⁻ in the absence of CO. Based on the two Fe–His stretching frequencies of 221 and 249 cm⁻¹, ferrous Y235A HmuT has one conformer with Fe–His proximal bond of strength comparable to that of Mb and Hb (218 – 224 cm⁻¹) (106;107) and a second conformer with significant imidazolate character in the proximal histidine similar to that observed for peroxidases (i.e., ν_Fe–Im⁻ occurs at 244 cm⁻¹ for HRP)(108).

Why tyrosine?

Although not common in heme proteins (109), the His/Tyr ligand set has also been observed in S. marcescens HasA(110), P. aeruginosa HasA(111;112) and PhuR (47), E. coli CcmE (113;114), Paracoccus denitrificans MauG (115), and Y. pestis HmuT (27). Of these, HasA, PhuR, CcmE and HmuT are all involved in heme transfer. MauG, in contrast, appears to use the tyrosinate axial ligand to stabilize a high oxidation center in the mechanistic pathway of this protein (116). The His/Tyr ligand set is also known in hemoglobin variants such as Hb M Saskatoon (102) and has been created by site-directed mutagenesis of sperm whale Mb (HisE7Tyr) (103). S. aureus IsdA binds heme through His83 and hemin through Tyr166 (36;37;70).

Tyrosine alone is also an axial ligand in a number of proteins in heme uptake pathways characterized to date, including S. dysenteriae ShuT (25), P. aeruginosa PhuT (25;26), Y. pestis HasA (117), N. meningitidis HmbR (46) and the heme uptake proteins from the S. aureus Isd system [IsdA (29), IsdB (32), IsdC (33), and IsdH (34)] as well as IsdX1 and IsdX2-N5 of B. anthracis (41;42). Sequence alignment of the Isd proteins revealed these tyrosine residues are conserved among species, and their role in heme binding is significant.

It is possible that modulation of H-bond donation to the axial tyrosine plays a role in triggering heme release and transfer. In some instances, the hydrogen bonding residue participates in an extended H-bonding network that also involves the heme propionates (17). Disruption of the hydrogen bonding network in these cases could have cooperative effects on the structure of the protein, its affinity for hemin, and potentially on the kinetics and mechanism of hemin transfer (71;82;100).

Tyrosine may also be employed as an axial ligand to ensure the hemin remains in its ferric form (100). Tyrosinate-bound heme proteins are characterized by low reduction potentials, consistent with stabilization of the Fe(III) center by the negative charge of the tyrosinate. For example, the midpoint reduction potential of SmHasA was reported to be −550 mV (101), nearly 0.5 V more negative than the potential of −60 mV for the hemin|heme couple (118). The redox potential indicates that tyrosine binds more strongly with the Fe(III) of hemin than with the Fe(II) of heme. Assuming that the His/Tyr axial ligation in HmuT imposes a similarly negative potential as in HasA, estimation of this difference in binding free energy is facilitated by the thermodynamic cycle shown in Scheme 1. Based on this cycle, (ΔG_III–ΔG_II) = nF(E_b°–E_f°) = −4.7×10⁴ J·mol⁻¹ where ΔG_III and ΔG_II are the free energies of apoHmuT complexation with hemin (Fe^IIIPPIX) to give HmuT^III and heme (Fe^IIPPIX) to yield HmuT^II, respectively. E_b° and E_f° are the reduction potentials of bound (HmuT^III) and free hemin, respectively. Thus, formation of HmuT^III is favored by an estimated 47 kJ·mol⁻¹ over HmuT ^II. This would strongly favor binding of hemin over heme, thereby favoring uptake of Fe(III) by any bacterium having a Tyr⁻-based HBP that delivers hemin to the ABC permease. This discrimination could help guard against the damaging effects of Fenton-type chemistry from buildup of free heme in the reducing environment of the cell.

Scheme 1.

HmuT joins a growing number of extracellular and cell-surface HBPs that use the H-bond assisted axial Tyr ligand motif to bind and stabilize hemin. These proteins share high affinities for hemin and cognate hemin-accepting proteins that, ostensibly, use the free energy of protein-protein complexation to destabilize their hemin-bound states, thereby facilitating hemin transfer to the acceptor protein (10).

Conclusions

Multiple lines of spectroscopic evidence have revealed the hemin axial ligand set in HmuT from C. diphtheriae to be His136/Tyr235 from the N - and C-terminal domains of the protein, respectively. The same axial ligand set is found in YpHmuT, but with the His and Tyr ligands arising from the C-and N-terminal domains of the proteins, respectively. This highlights the variety of binding motifs used by heme binding proteins in bacteria. Solution speciation of the ferric form of CdHmuT is dominated by the 1:1 complex, which, like HasA, exists as a thermal spin state equilibrium between 6cHS and 6cLS complexes. Ferric CdHmuT is slow to reduce with S₂O₄²⁻ in the presence of CO with His136 being replaced by CO upon reduction. The position of the trans-Tyr carbonyl complex on the neutral O- bound ligand line of the π-backbonding correlation plot suggests that, as in HasA–CO, the Tyr-based phenol ligand is a charge neutral heme carbonyl. Thus, HmuT reinforces the emerging theme of extracellular and cell surface heme-binding proteins that use H-bond assisted axial Tyr ligands to stabilize hemin-bound states that require a heme-accepting partner for release and transfer of the hemin substrate. Moreover, the axial Tyr ligand (Tyr235 in CdHmuT) is required for full hemin uptake function in C. diphtheriae.

ABBREVIATIONS

ABC transporter

ATP-binding cassette transporter

BLC

bovine liver catalase

CdHmuT

Corynebacterium diphtheriae HmuT

HBP

heme binding protein

Mb

myoglobin

Hb

hemoglobin

HRP

horseradish peroxidase

MCD

magnetic circular dichroism

rR

resonance Raman spectroscopy

LS

low spin

HS

high spin

WT

wild-type