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Cytoplasmic Heme-binding Protein (HutX) from Vibrio cholerae is an Intracellular Heme Transport Protein for the Heme-degrading Enzyme, HutZ Yukari Sekine, Takehito Tanzawa, Yoshikazu Tanaka, Koichiro Ishimori, and Takeshi Uchida Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01273 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Biochemistry

Cytoplasmic Heme-binding Protein (HutX) from Vibrio cholerae is an Intracellular Heme Transport Protein for the Heme-degrading Enzyme, HutZ

Yukari Sekine1, Takehito Tanzawa2, Yoshikazu Tanaka3,4, Koichiro Ishimori1,5, and Takeshi Uchida1,5 1

Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan

2

Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan

3

Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan

4

Japan Science and Technology Agency, PRESTO, Sapporo 060-0810, Japan

5

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan

To whom correspondence should be addressed: Takeshi Uchida, Phone/Fax: 81-11-706-3501. E-mail: [email protected] This study was supported in part by Grants-in-Aid for Scientific Research (13J04076 to Y.S., 24550182 to T.U., 24000011 to Y.T., and 25109501 to K.I.) and Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Culture, Education, Sports, Science, and Technology (MEXT) of Japan, and JST, PRESTO (Y.T.). KEYWORDS: heme, heme degradation, heme transport ABBREVIATIONS: HO, heme oxygenase; r.m.s.d., root mean square deviation; SPR, surface plasmon resonance; CPR, cytochrome P450 reductase. FOOTNOTE: Accession Code PDB 5EXV (crystal structure of heme binding protein)

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ABSTRACT HutZ is a cytoplasmic heme-binding protein from Vibrio cholerae. Although we have previously identified HutZ as a heme-degrading enzyme (Uchida, T. et al. (2012) Chem. Commun., 48, 6741−6743), the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, hutWXZ. To this end, we constructed an expression system for HutX in Escherichia coli, and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from E. coli O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 × 10–2 and 1.5 × 10–2 s–1, values distinctly larger than those from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of ~400 µM. These results suggest that heme is transferred from HutX to HutZ via a specific protein-protein interaction. Therefore, it can be concluded that HutX is a cytoplasmic heme transport protein for HutZ.


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All living organisms require iron for their survival. However, ferrous iron in the external medium is easily oxidized to the insoluble ferric iron. Therefore, the amount of bioavailable iron for bacteria is limited. Thus, almost all bacteria have developed sophisticated mechanisms for solubilizing, sequestering, and releasing iron within cells1. Free inorganic iron is acquired by small organic molecules called siderophores. Once a siderophore is complexed with iron, it is recognized by outer-membrane siderophore receptor proteins and transported into the cytoplasm across cell membranes in an energy-dependent manner2,3,4. However, the availability of free iron is strongly limited in vertebrate hosts, resulting in insufficient iron acquisition by siderophores, especially for pathogenic bacteria. Therefore, pathogens have evolved another system in which host heme-containing proteins are utilized as an iron source. Because 95% of the total iron in the human body is present as heme iron5,6, the utilization of heme is a common mechanism employed by pathogens6,7. A recent report on the initial stage of infection by Gram-positive pathogens, such as Staphylococcus aureus, highlighted the distinct preference for heme as an iron source over ferrous iron8. For Gram-negative pathogens, proteins encoded within the heme transport operon have been predicted based on genetic studies9,10,11,12. According to these predictions, the heme transport system in Gram-negative bacteria consists of TonB-dependent outer-membrane receptor proteins, which are critical for active transport of heme into the periplasm, and periplasmic heme-binding proteins that transport heme to the cytoplasmic membrane, where cytoplasmic permeases and ATPases facilitate transport of heme into the cytoplasm7. In the cytoplasm, heme is degraded by heme-degrading enzymes, which remove iron from the heme moiety. The mechanism of heme transfer from outside the cell to the cytoplasm of bacteria has been extensively studied5. For example, the crystal structure of the outer-membrane heme receptor protein HasR complexed with the hemophore protein HasA has been reported13,14, as has the crystal structure of the inner-membrane heme receptor protein HmuUV from Yersinia pestis15. However, little is known about the fate of the heme after it enters the cytoplasm. 3 ACS Paragon Plus Environment

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Vibrio cholerae, the intestinal pathogen that causes cholera, also has an absolute requirement for iron, because iron plays a crucial role in the survival and pathogenicity of this bacterium16,17. In V. cholerae, a heme uptake operon called hut (heme utilization) has been identified9. The hut operon encodes genes for outer-membrane receptor protein (HutA), periplasmic

heme-binding

protein

(HutB),

and

ATP-dependent

permease

proteins

(HutC/HutD) (Fig. 1A). We have recently purified and characterized HutZ as a heme-degrading enzyme18, which releases iron into the cytoplasm. Once heme is internalized, it is degraded to release iron, or reused directly in hemoproteins as a chromophore17. Although heme is a fundamental element for bacterial survival and host colonization as described above19, it is also a potentially toxic molecule that can generate free radicals20. Therefore, it is necessary for the balance between heme uptake and degradation to be tightly regulated. The V. cholerae gene for the heme-degrading enzyme HutZ is encoded with two other genes, hutW and hutX, in an operon divergently transcribed from the tonB1 operon9,21 (Fig. 1A). These genes are under the same transcriptional regulation by the ferric uptake regulator (Fur). Henderson and co-workers showed that hugWXZ from Plesiomonas shigelloides is needed for survival when heme is used as an iron source22. HugWXZ from P. shigelloides shares homology with HutWXZ from V. cholerae (25–51% identity and 41–68% similarity)22. Therefore, hutW, hutX, or hutZ are considered necessary to obtain iron from heme. A BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search suggested that HutW belongs to the S-adenosylmethionine (SAM) radical superfamily23. This family of proteins uses a radical-based mechanism to catalyze diverse chemical reactions, such as biosynthesis and repair of DNA, and biosynthesis of antibiotics, other medicinally relevant natural products, and enzyme cofactors. All enzymes in this superfamily contain at least one [4Fe-4S] cluster in which three of its four iron ions are coordinated by cysteine residues, typically found in a CxxxCxxC motif. Therefore, we predicted that HutW serves as an electron carrier for HutZ. HutX, on the other hand, belongs to the ChuX_HutX superfamily. Previous research has 4 ACS Paragon Plus Environment

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shown that ChuX from the pathogenic Escherichia coli O157:H7 binds heme with a stoichiometry of 1:1 and an intermediate dissociation constant (Kd,heme) of 2.0 µM24, and is thought to act as a heme carrier protein. HutX shares 37% sequence identity with ChuX (Fig. 1B). These findings led us to hypothesize that the HutX protein is an intracellular heme transport protein specific for HutZ. Purification and a preliminary crystal structure of HutX was recently reported25. However, the function of HutX remains unknown. Herein, we report the purification and characterization of HutX. Purified HutX binds to heme with a stoichiometry of 1:1 and a Kd,heme of 7.4 nM. HutX transports ferric heme to HutZ through direct interactions. UV-Vis and resonance Raman spectra showed that the heme-HutX complex contains a 5-coordinate high-spin heme that is linked to the protein through a proximal Fe-O bond. The crystal structure of apo-HutX shows that Tyr90 is the only residue present in the predicted heme-binding cleft. These results suggest that the side chain of Tyr90 coordinates the heme iron.

EXPERIMENTAL PROCEDURES Materials. The chemicals used in this study were purchased from Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), or Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. Protein Expression and Purification. The HutX gene was purchased from the PlasmID Repository

(https://plasmid.med.harvard.edu/PLASMID/Home.xhtml;

cloneID:

VcCD00036416) and amplified by PCR. The amplified fragment was cloned into the pET-28b vector (Merck Millipore, Darmstadt, Germany) using the NdeI and EcoRI restriction sites. The thrombin recognition site (Leu-Val-Pro-Arg-Gly-Ser) in the pET-28b construct was mutated to the HRV 3C protease recognition site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) as described previously26. The correct gene sequence was confirmed by DNA sequencing 5 ACS Paragon Plus Environment

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(Eurofins Genomics). E. coli carrying a HutX expression plasmid was grown at 37 °C in LB broth supplemented with 50 µg/mL kanamycin. After cultures reached an optical density at 600 nm (OD600) of 0.6–1.0, expression of the His-tagged fusion protein in E. coli BL21(DE3) was induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside. The cells were further grown at 28 °C overnight and harvested by centrifugation, after which the cell pellet was stored at -80 °C until use. The pellet was thawed on ice, suspended in lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Nonidet P-40 (pH 8.0). Cells (~4.0 g) obtained from a 1-L culture were incubated for 30 min on ice with 1 mg/mL lysozyme and DNase. The sample was centrifuged at 40000 × g for 30 min, and the supernatant was loaded onto a HisTrap HP column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and 20 mM imidazole. The resin was extensively washed with 50 mM Tris-HCl, 500 mM NaCl, and 50 mM imidazole (pH 8.0), and then bound protein was eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and 200 mM imidazole. Eluted HutX was incubated for ~36 h at 4 °C with Turbo3C protease (Accelagen, San Diego, USA) to remove the His6-tag. After cleavage, the reaction mixture was again applied to a HisTrap column. The column flow-through was applied to a gel-filtration column (HiLoad 16/60 Superdex 200 pg; GE Healthcare) equilibrated with 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). The yield of purified HutX was 10–20 mg from 1 L of LB culture. HutX contains 167 amino acid residues and a calculated molecular mass of 18,775 Da. Protein purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels. The Y90F/Y91F mutation was introduced using a PrimeSTAR mutagenesis basal kit from Takara Bio (Otsu, Japan). The primers employed for mutation were 5'-GGG GTT TCT TTA ATT TAA TGG GTC GTG ATG-3' (forward) and 5'-AAA TTA AAG AAA CCC CGT GCA ACT TTC CC-3' (reverse). The introduced mutation was verified by sequencing.


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Absorption Spectroscopy. All absorption spectra were obtained using a V-660 UV-Vis spectrophotometer (Jasco, Japan). Hemin binding studies were carried out by difference absorption spectroscopy. Hemin was dissolved in 0.1 M NaOH, and its concentration was determined on the basis of absorbance at 385 nm using an extinction coefficient (ε385) of 58.44 mM-1cm-1. Aliquots of hemin solution (0.5 mM) were added to both the sample cuvette containing 5 µM apo-HutX and reference cuvette at 25 °C. Spectra were recorded 3 min after the addition of hemin. The absorbance difference at 399 nm was plotted as a function of heme concentration, and the dissociation constant (Kd,heme) was calculated using quadratic binding equation 1: ∆Absorbance =

εbinding − εfree 2 [HutX]total +[heme]total + K d − ([HutX]total +[heme]total + K d ) − 4[HutX]total [heme]total 2

{

}

(1)

The millimolar extinction coefficient was determined using the pyridine hemochrome method27. Heme Transfer from HutX to HutZ. The heme transfer reaction for HutX was carried out in a 2-mL reaction volume containing 2.5 µM heme-HutX and 12.5 µM apo-HutZ or apomyoglobin in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0) at 25 °C. Heme was extracted from myoglobin using the acid/butanone method as previously described28. The reaction was measured as an increase in absorbance at 411 and 408 nm upon addition of apo-HutZ and apomyoglobin, respectively. Measurements were carried out with a Jasco V-660 UV-Vis absorption spectrophotometer. Rate constants of heme transfer were calculated by fitting the data to a double exponential equation using Igor Pro (WaveMetrics, Portland, USA). Protein-Protein Interaction of HutX and HutZ. The interaction between HutX and HutZ was examined by surface plasmon resonance technology using a Biacore-J system (GE Healthcare). His-tagged HutX was immobilized on Sensor Chip NTA. The sensor chip was activated by injection of 500 µM NiCl2 (pH 7.4) for 3 min, after which HutX (200 nM) in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% (v/v) Surfactant P20, pH 7.4) was 7 ACS Paragon Plus Environment

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passed over the chip at flow rate of 30 µL/min for 3 min. A HutZ solution containing 50-750 µM protein, prepared in HBS-P buffer, was passed over the sensor chip for 5 min (association step); this was followed by a 5-min dissociation at 25 °C. The chip was regenerated by washing first with an HBS-P buffer containing 350 mM EDTA for 3 min and then with 500 µM NiCl2 for 3 min. Data were fit to a 1:1 Langmuir model using the BIAevaluation software provided by Biacore. Heme Degradation Reaction of HutZ. The reaction of HutZ was monitored by UV-Vis spectroscopy. Following addition of 2.5 µM heme-HutX to 5.0 µM apo-HutZ in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0), the spectrum was recorded at 1-min intervals for 10 min. H2O2 in the same buffer was added to the mixture of heme-HutX and apo-HutZ to a final concentration of 100 µM. In the case of reactions with ascorbic acid (final concentration, 1 mM), 1 mg/mL of bovine liver catalase was added to suppress H2O2. Absorption spectra between 250 nm and 800 nm were recorded at 2-min intervals for 30 min. Crystallization of HutX. Purified His6-tagged HutX was concentrated to 7 mg/mL. Crystals of His6-tagged apo-HutX were grown from 0.1 M HEPES, 2% PEG400, 2.0 M ammonium sulfate (pH 7.2) using the sitting-drop vapor diffusion method. X-ray Diffraction. X-ray diffraction of His6-tagged HutX was performed on a beamline BL17Aa at the Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). The diffraction data were indexed, integrated, scaled, and merged using X-ray diffraction. The collected data and processing statistics are summarized in Table 2. Structure Solution and Refinement. His6-tagged HutX belonged to the P21 space group with the cell dimensions, a = 61.21 Å, b = 80.88 Å and c =111.1 Å, and diffracted to a resolution of 2.9 Å. The structure was determined by the molecular replacement method with PHENIX.AUTOMR29 using the structure of ChuX from E. coli O157:H7 (PDB code: 2OVI) as a search model. The refinement converged after several cycles of manual model corrections 8 ACS Paragon Plus Environment

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with COOT30,31 and refinement using phenix.refine32. Individual atomic coordinate refinement and individual ADP refinement were performed for four of six molecules in an asymmetric unit, whereas rigid body refinement and group ADP refinement were applied for the remaining two molecules because of their ambiguous electron density. Ramachandran plot analysis was performed using MOLPROBITY33. Superposition of structures and calculation of root mean square deviation (r.m.s.d) were performed using the cealign algorithm of the program PyMOL. Coordinates and structural features have been deposited in the Protein Data Bank (PDB) under the code, 5EXV. Data processing and refinement statistics are given in Table 2. Resonance Raman Spectroscopy. Resonance Raman spectra were obtained using a single monochromator (SPEX500M; Jobin Yvon) equipped with a liquid nitrogen-cooled CCD detector (Spec-10:400B/LN; Roper Scientific). Samples were excited at wavelengths of 413.1 and 441.6 nm using a krypton ion (BeamLok 2060; Spectra Physics) and helium-cadmium (IK5651R; Kimmon Koha, Tokyo, Japan) laser, respectively. The laser power at the sample point was adjusted to ~5 mW for the ferric and ferrous forms, and to 0.1 mW for the CO-bound form to prevent photodissociation. Raman shifts were calibrated with indene, CCl4, acetone, and an aqueous solution of ferrocyanide. The accuracy of the peak positions of well-defined Raman bands was ±1 cm-1. Sample concentrations for Raman experiments were approximately 10 µM in 50 mM sodium phosphate, 150 mM NaCl (pH 8.0).

RESULTS Expression and Purification of HutX. HutX was expressed in E. coli BL21(DE3), and purified primarily as an apoprotein, based on the near absence of heme absorption features in the UV-Vis spectrum (Fig. S1A). The HutX protein was estimated to be ~95% pure by SDS-PAGE with an apparent molecular mass of 18 kDa (Fig. S1B). An analysis of the 9 ACS Paragon Plus Environment

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purified HutX by size-exclusion chromatography indicated that the protein exists as a dimer (45 kDa; Fig. S1C). Heme-Binding Properties of HutX. To test whether HutX is capable of binding to heme, we used UV-Vis absorption spectroscopy. The difference absorption spectra of HutX, obtained by subtracting the spectra of free heme at various concentrations, are shown in Figure 2A. The plot of absorbance difference at 399 nm suggests that HutX binds to heme in a 1:1 stoichiometry (Fig. 2A, inset). This plot was also used to calculate the Kd,heme of HutX for heme using equation 1, which yielded a Kd,heme of 7.4 ± 7.5 nM, a value significantly lower than that for ChuX (1.99 ± 0.02 µM)24. Because the plot of absorbance difference at 412 nm monotonously increased, an intense band at 412 nm would represent nonspecific heme binding. The millimolar extinction coefficient at 390 nm of heme-HutX was determined to be 70 mM-1cm-1 by the pyridine hemochrome method. The gel filtration column elution profile revealed that the heme-reconstituted HutX was a dimer, as was the apoprotein (data not shown), suggesting that the dimeric nature of HutX is not affected by binding of heme. Absorption Spectra of the Heme-HutX Complex. HutX was reconstituted with a 1.2-fold excess of heme, and then unbound heme was removed using a gel-filtration column. Absorption spectra of the heme-reconstituted HutX are depicted in Figure 2B. The Soret absorption maximum of the ferric HutX was 390 nm and the visible maximum was 615 nm, which is typical of a 5-coordinate high-spin heme34,35. In the absorption spectra of the ferrous heme-HutX, the Soret band appeared at 422 nm with Q-bands at 532 and 559 nm, suggesting the presence of a 6-coordinate low-spin heme. Additionally, a shoulder on the Soret band at 388 nm was observed, which is not seen in most hemoproteins, but is found in the heme oxygenase (HO), H25Y mutant, in which the proximal His is replaced with Tyr35. The Soret peak of the ferrous-CO complex of HutX located at 408 nm is indicative of a 5-coordinate CO-bound heme35.


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Heme Transfer Reaction from HutX to HutZ. To investigate the possibility that HutX acts as a heme transport protein for HutZ, we next monitored absorption spectral changes of the ferric heme-HutX upon mixing with apo-HutZ. Addition of a 5-fold excess of apo-HutZ to the heme-HutX complex resulted in a red shift of the Soret band from 390 to 411 nm, suggesting that heme is transferred from HutX to apo-HutZ (Fig. 3A). The heme transfer from ferric heme-HutX was completed within 10 min (Fig. 3C). The time course was fit to a double exponential function, which yielded a koff1 of 8.3 × 10–2 s–1 (51%) and a koff2 of 1.5 × 10–2 s–1 (49%). To determine whether heme is transferred directly from HutX to apo-HutZ, we conducted the same reaction in the presence of apomyoglobin. When ferric heme-HutX was mixed with 5 equivalents of apomyoglobin, the Soret band caused a red shift from 390 to 408 nm (Fig. 3B), as observed in Figure 3A. Using a biphasic model, koff1 and koff2 were determined to be 1.8 × 10–2 s–1 (45%) and 4.5 ×10–3 s–1 (55%), respectively. Both koff1 and koff2 for heme-HutX to apomyoglobin are about 4-fold smaller than those for heme-HutX to apo-HutZ. Considering the considerably higher affinity of apomyoglobin for heme, this result strongly suggests that the heme that apo-HutZ binds is not free heme released by HutX, but instead is transferred from HutX to apo-HutZ via protein-protein interactions. Protein-Protein Interactions between HutX and HutZ. To investigate the interaction of HutX with HutZ, we performed surface plasmon resonance (SPR) analysis. Apo-HutX was immobilized on a Ni-NTA surface through the His-tag. Figure 4 shows association and dissociation curves with five different concentrations of apo-HutZ. The observed association and dissociation phases clearly indicate specific interactions of apo-HutZ with apo-HutX, with rapid dissociation. Using a 1:1 binding model, we calculated association (ka) and dissociation (kd) rate constants as well as the dissociation equilibrium constant (KD), as summarized in Table 1. Unfortunately, it was not possible to obtain an accurate KD value owing to the extremely rapid dissociation of apo-HutZ, although the value for the binding of HutX to HutZ was estimated to be greater than 400 µM. In the presence of heme in HutX and 11 ACS Paragon Plus Environment

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HutZ, the KD value was over 500 µM (data not shown), which was in the same range as that for apo-HutX and apo-HutZ. The quite large KD value for HutX and HutZ binding might raise questions regarding whether HutX interacts with HutZ. However, the sensorgram clearly showed that HutX can bind to HutZ with a ka of 1.8 × 10–2 M-1s-1. Effect of the Interaction of HutX with HutZ on the Heme Degradation Reaction. Next, we examined whether the interaction between HutX and HutZ affects the heme degradation reaction of HutZ. After addition of H2O2 to the solution containing heme-HutX and HutZ, the Soret band at 409 nm was diminished and a new band appeared at 647 nm (Fig. 5A). This spectral change suggests the formation of verdoheme18. The time course of the absorbance change at the Soret maximum is shown in Figure 5A (inset). The apparent kinetic constant obtained by fitting the data to a single exponential curve was 0.62 min-1, which is almost identical to that in the absence of HutX (0.52 min-1; Fig. 5B, inset). Furthermore, HutX could not degrade heme in a reaction containing H2O2 (Fig. S2). These results indicate that the rate of heme-degradation is not affected by HutX. Overall Structure of HutX. The crystal structure of apo-HutX was determined by molecular replacement with Phenix using the structure of ChuX from E. coli O157:H7 as a search probe. Crystallographic Rwork and Rfree converged to 24.6% and 27.8%, respectively, and the resolution achieved was 2.9 Å. Collected data and refinement statistics are listed in Table 2. Figure 6A shows the determined X-ray structure of HutX (PDB entry, 5EXV). Each asymmetric unit contains three HutX dimers, which is in good agreement with the results estimated by gel filtration, and has an approximate molecular mass of ~45 kDa (Fig. S1C). Except for one or two residues in the N- and C-terminus of each subunit, all residues exhibited good electron density. The overall structure of apo-HutX is quite similar to that of ChuX from E. coli O157:H7 (r.m.s.d., 2.44 Å for 312 residues)24. The apo-HutX structure comprises antiparallel 12 ACS Paragon Plus Environment

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eight-strand β-sheets and peripheral α-helices. At the intermolecular interface between each dimer are equivalent sets of antiparallel β-sheets, which bow outward to form an overall saddle motif (Fig. 6A). These central sheets are composed of six β-sheets contributed by one of the HutX monomers and three others contributed through domain swapping with the partnering HutX molecule. Each β-sheet is flanked at its N terminus by the set of three α-helices in an α-loop-α-loop-α configuration and at its C terminus by one pair of antiparallel α-helices. Two large clefts are on opposite sides of the central core of β-pleated sheets. In a previous study, heme was modeled in the heme-binding clefts in ChuX, which led to the proposal that the heme iron is axially coordinated between His98 and Met11524. Both residues are conserved in HutX and correspond to His103 and Met120 (Figs. 1B, 6B), respectively. Because these two residues are oriented in the putative heme-binding cleft, they were presumed to coordinate heme iron. In addition to His103 and Met120, the conserved residues Glu25, Gln26, Ile74, Glu76, Tyr90, Phe119, and Lys139 are also in close proximity to the putative heme-binding cleft (Fig. 6B). Resonance Raman Spectra of Heme-HutX. To further characterize heme coordination in HutX, we measured resonance Raman spectra. In the ferric heme-HutX complex, the spinand coordination-state marker band, ν3, was observed at 1491 cm-1 (Fig. 7A), indicating that the ferric heme is in the 5-coordinate high-spin state36, in good agreement with absorption spectrum results (Fig. 2B). In addition, the small intensity ratio of ν4 to ν3 (Iν4/Iν3 = 0.6), suggests the presence of a weak axial ligand such as an anionic oxygen or sulfur atom37,38,39,40. Because HutX has no Cys (Fig. 1B), but Tyr90 is located in the putative heme-binding cleft (Fig. 6B), we propose that Tyr90 coordinates the heme in heme-bound HutX. To confirm this, we constructed the Y90F/Y91F mutant of HutX. The cell pellet for wild-type HutX-expressing cells was brownish, despite the fact that HutX was purified as an apoprotein. However, the pellet for cells expressing the Y90F/Y91F mutant was whitish, indicating that


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the heme-binding ability was decreased by the mutation. The heme titration plot of the Y90F/Y91F mutant showed a significant reduction in the absorbance difference at 399 nm (Fig. S4), supporting the idea that the axial ligand of HutX is Tyr90. Upon reduction, the ν3 band of the ferrous heme-HutX appeared at 1472 and 1502 cm-1, which represent 5-coordinate high-spin and a 4-coordinate intermediate-spin hemes, respectively (Fig. 7A)36,41. This Raman spectrum also resembles that of the HO H25Y mutant35. Resonance Raman spectra of the ferrous-CO heme complex of HutX are illustrated in Figure 7B. It is evident from these spectra that the 496, 526 and 1960 cm-1 bands were CO-isotope dependent, and downshifted to 486, 515 and 1875 cm-1, respectively, upon 13C18O binding. Accordingly, we assigned the 496 and 526 cm-1 bands to the Fe-CO stretching mode (νFe-CO) and the 1964 cm-1 band to the CO stretching mode (νC-O). In Figure 7C, placement of the νFe-CO and νC-O frequencies onto the correlation plot shows that one plot of νFe-CO versus

νC-O for HutX falls on the line with proteins possessing the histidine-coordinated heme 42,43,44, whereas the other falls on the line of 5-coordinate CO-heme proteins35,45. These results indicate that the heme iron in HutX is a mixture of a 6-coordinate His-Fe-CO heme and 5-coordinate CO heme.

DISCUSSION Heme Transfer from HutX to HutZ via Protein-Protein Interaction. Previously, we found that HutZ from V. cholerae is a cytosolic heme-degrading enzyme18. However, there is no information on how HutZ obtains heme in the cytoplasm. A closer look at the genome sequence of V. cholerae revealed that HutZ is encoded by the hutWXZ operon (Fig. 1A). HutW belongs to the radical SAM superfamily, whose members contain a [4Fe-4S] cluster binding site23; thus, we assumed that HutW is a reductase for HutZ. In contrast, HutX belongs 14 ACS Paragon Plus Environment

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to the ChuX_HutX superfamily, whose members bind and utilize heme. Therefore, we predicted that HutX is the protein that transfers heme to HutZ. Although the cell pellet containing HutX was brownish, HutX was purified as an apoprotein. However, the spectral titration of heme to HutX clearly showed that HutX binds to heme at 1:1 ratio with a moderate heme-binding affinity (Kd,heme = 7.4 ± 7.5 nM) comparable to that of HutZ (52 nM)18. An examination of the heme transfer reaction from HutX to HutZ showed that the Soret band of heme-HutX disappeared immediately after mixing of apo-HutZ, while a new band at 411 nm from heme-HutZ appeared, indicating that heme was transferred from HutX to HutZ. However, the possibility that the free heme released by HutX was captured by excess HutZ could not be ruled out. Thus, the rate of heme transfer from heme-HutX to apomyoglobin was also determined. The rate of heme transfer from heme-HutX to apo-HutZ was found to be 4-fold higher than that from heme-HutX to apomyoglobin (Fig. 3C). The fact that the Kd,heme of myoglobin is 105-fold smaller than that of HutX46 suggests that heme transfer from HutX to HutZ is driven by a transient protein-protein interaction. To observe specific protein-protein interactions between HutX and HutZ, we employed SPR. The resulting sensorgrams showed that HutX binds to HutZ with a KD > 400 µM (Table 1). Compared with the KDs of human HO for cytochrome P450 reductase (CPR)47 and of Pseudomonas aeruginosa HO for PhuS, the KD of HutX for HutZ is about 20- and 6000-fold larger, respectively. These differences are derived from the extremely large kd of HutX, suggesting that dissociation of HutX from the HutX-HutZ complex is so fast that HutX cannot form a stable complex with HutZ. The facts that the heme-degrading reaction of HutZ with H2O2 is not affected in the presence of HutX (Fig. 5), and the resonance Raman spectra of heme-HutX were not altered by addition of apo-HutZ support the weak interaction between HutX and HutZ (Fig. S3). Although this interaction is very weak, the fast dissociation of HutX from the HutX-HutZ complex would appear to favor formation of a complex of 15 ACS Paragon Plus Environment

Biochemistry

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reductase with HutZ for electron transfer. Furthermore, the ka of HutX with HutZ (1.0 × 102 M-1 s-1) is only about 10-fold smaller than that of HO with CPR (1.0 × 103 M-1 s-1)47, which would be acceptable for heme transfer from HutX to HutZ. Heme-Coordination Structure of HutX. To determine the mechanism of heme transfer from HutX to HutZ, we attempted to characterize the heme coordination structure of HutX. Although the crystal structure of heme-HutX was not obtained, we successfully determined the crystal structure of apo-HutX (Fig. 6). Structural superimposition revealed that HutX shares structural similarity with ChuX. Docking simulations of ChuX with heme suggested that the heme iron is coordinated to His98 and Met11524. Sequence alignment of HutX with ChuX revealed that His98 and Met115 of ChuX correspond to His103 and Met120 of HutX, respectively (Fig. 1B), suggesting that these are possible heme ligands of HutX. However, the UV-Vis absorption spectrum displayed a Soret band at 390 nm with a visible maximum at 615 nm, indicating a 5-coordinate ferric heme. This spectral feature of HutX is similar to that of 5-coordinate Tyr-ligated ShuT37 and Rv020348, in which Tyr serves as an axial ligand of heme, but is different from that of ChuX. Furthermore, the resonance Raman spectra of HutX showed an unusually high relative intensity of ν3 with respect to ν4, which indicates an oxygen-based proximal ligand, such as a hydroxide or tyrosinate37,39,40,49. The presence of 4-coordinate heme for the ferrous form and 5-coordinate CO-heme supports the Tyr-coordination. These results imply that the proximal ligand of the heme iron is Tyr. The obtained crystal structures of HutX show that Tyr90 and Tyr91 adjoining His103 are the predicted axial ligands of the heme iron (Fig. 6B). Because only Tyr90 is oriented toward the predicted heme-binding cleft and is conserved in ChuX, we assume that Tyr90, and not His103, is an axial ligand of heme in HutX (Fig. 6B). The possibility of coordination to His103 cannot be completely ruled out, considering that the Y90F/Y91F double mutant still bound heme (Fig. S4). However, spectroscopic data strongly suggest that Tyr coordinates the heme. An ongoing crystallization study of heme-HutX will clarify the coordination structure. 16 ACS Paragon Plus Environment

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Despite the presence of His103 in the heme-binding site, Tyr90 is a heme ligand. Axial ligation of the heme iron by a tyrosine side chain was also encountered in the hemophore HasA14,50,51. A structural characterization of HasA from Serratia marcescens revealed a novel heme-binding motif in which the heme is held between two loops containing His32 and Tyr75 that is hydrogen-bonded to His83. Although His83 is not directly coordinated to the iron, replacement of His83 by Ala resulted in a 260-fold decrease in the heme binding affinity52. The crucial role of His83 in the binding of heme to HasA is to enhance the nucleophilic nature of Tyr75 through hydrogen bonding. Thus, while His103 is shown not to be directly involved as a heme axial ligand, it may stabilize the coordination of Tyr90 through a hydrogen bond interaction with the phenolate group. Mechanism of Heme Transport from HutX to HutZ. The X-ray crystal structure and spectroscopic analysis of HutX predict that the heme-binding sites in dimeric HutX are in two large clefts, which are on opposite sides of the central β-pleated sheet core (Fig. 6). Furthermore, HutZ, which accepts heme from HutX, exists as dimer18, and the heme-binding pockets are also predicted to be on sites opposite the dimer interface53. These configurations clearly show that the two subunits of HutZ could not accept heme at the same time. Therefore, the fact that HutX dissociates from HutZ rapidly would appear to be favorable for transporting two hemes to HutZ, although it is not clear whether HutZ needs either one or two hemes for the heme-degrading reaction. HutX gives biphasic dissociation kinetics, with koff1 and koff2 values of 1.8 × 10–2 s–1 and 4.5 × 10–3 s–1, respectively. One interpretation of this biphasic kinetics feature is that the rate of the first heme dissociation from HutX dimer might be different from that of the second. Furthermore, these rates are comparable to those of proteins with Tyr as a heme ligand, but 10–100 fold larger37,48 than that of HutZ (8.5 × 10–4 s-1). Indeed, similar koff rates for heme were reported for PhuS (3.6 × 10–2 s–1)54, which is a cytoplasmic heme transport protein. If the dissociation rate constant (kd) of HutX from the HutX-HutZ complex is much larger than that (koff) of heme from heme-HutX, heme transfer 17 ACS Paragon Plus Environment

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does not occur via direct protein-protein interaction. However, kd is comparable to koff1 and koff2 (Table 1). Furthermore, it is possible that the interaction of HutZ with heme-HutX affects the coordination of heme in HutX, which would destabilize the heme binding of HutX. In the case of HasA, docking simulations suggest breakage of the bond between the heme and an axial ligand of HasA upon binding to the receptor protein, HasR13. A similar conformational change would be induced to facilitate heme transfer from HutX to HutZ. These results indicate that it is plausible for HutX to act as a transient heme transport protein for HutZ. Titration studies provided Kd,heme values of HutX and HutZ for heme of 7.6 and 52 nM18, respectively (Fig. 2A). The heme binding affinity of HutX is comparable to that of HutZ. Furthermore, Tyr-coordination of heme-HutX seems to disfavor ferrous heme binding owing to the anionic character of Tyr-O–, which raises the question of whether HutX can function as a heme-transfer protein. In the process of heme transport in V. cholerae, heme is transported into the periplasm across the outer membrane heme receptors, HutA, HutR, or HasR. In the periplasm, HutB binds heme and transports it to HutC/HutD complexes, which in turn transfer heme into the cytoplasm across the inner membrane17. Among these proteins, HutB is known to coordinate heme through Tyr (Uchida et al., in preparation), suggesting that heme is transported from the outside to the inside of a bacterial cell as the ferric form, and not the ferrous form. Furthermore, the affinity of heme for HutX and/or HutZ can be modulated by the protein-protein interaction between HutX and HutZ. Therefore, Tyr-coordination of HutX would be appropriate for heme-transport to HutZ in V. cholerae. In summary, we present here the first characterization of an intracellular heme transport protein, HutX, to which the heme is bound in a 5-coordinate high-spin state with Tyr90. Additionally, we conclude that HutX interacts with HutZ, and transports the ferric heme, but does not inhibit heme degradation by HutZ. Therefore, HutX and HutZ act as heme transporter and heme-degrading enzyme, respectively, in V. cholera, and HutW may play the


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role of reductase for the heme-degrading reaction by HutZ. Supporting Information: The Supporting Information is available free of charge on the ACS Publications web site at http://pubs.acs.org.


Biochemistry

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[16] Wyckoff, E. E., Mey, A. R., and Payne, S. M. (2007) Iron acquisition in Vibrio cholerae, Biometals 20, 405-416. [17] Wyckoff, E. E., Schmitt, M., Wilks, A., and Payne, S. M. (2004) HutZ is required for efficient heme utilization in Vibrio cholerae, J Bacteriol 186, 4142-4151. [18] Uchida, T., Sekine, Y., Matsui, T., Ikeda-Saito, M., and Ishimori, K. (2012) A heme degradation enzyme, HutZ, from Vibrio cholerae, Chem Commun 48, 6741-6743. [19] Stoebner, J. A., and Payne, S. M. (1988) Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae, Infect Immun 56, 2891-2895. [20] Everse, J., and Hsia, N. (1997) The toxicities of native and modified hemoglobins, Free Radic Biol Med 22, 1075-1099. [21] Heidelberg, J. F., Eisen, J. A., Nelson, W. C., Clayton, R. A., Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K., Peterson, J. D., Umayam, L., Gill, S. R., Nelson, K. E., Read, T. D., Tettelin, H., Richardson, D., Ermolaeva, M. D., Vamathevan, J., Bass, S., Qin, H., Dragoi, I., Sellers, P., McDonald, L., Utterback, T., Fleishmann, R. D., Nierman, W. C., White, O., Salzberg, S. L., Smith, H. O., Colwell, R. R., Mekalanos, J. J., Venter, J. C., and Fraser, C. M. (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae, Nature 406, 477-483. [22] Henderson, D. P., Wyckoff, E. E., Rashidi, C. E., Verlei, H., and Oldham, A. L. (2001) Characterization of the Plesiomonas shigelloides genes encoding the heme iron utilization system, J Bacteriol 183, 2715-2723. [23] Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods, Nucleic Acids Res 29, 1097-1106. [24] Suits, M. D., Lang, J., Pal, G. P., Couture, M., and Jia, Z. (2009) Structure and heme binding properties of Escherichia coli O157:H7 ChuX, Protein Sci 18, 825-838. [25] Su, T., Chi, K., Wang, K., Guo, L., and Huang, Y. (2015) Expression, purification and

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preliminary crystallographic analysis of a haem-utilizing protein, HutX, from Vibrio cholerae, Acta Cryst F71, 141-144. Uchida, T., Sasaki, M., Tanaka, Y., and Ishimori, K. (2015) A Dye-Decolorizing Peroxidase from Vibrio cholerae, Biochemistry 54, 6610-6621 Berry, E. A., and Trumpower, B. L. (1987) Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra, Anal Biochem 161, 1-15. Teale, F. W. (1959) Cleavage of the haem-protein link by acid methylethylketone, Biochim Biophys Acta 35, 543. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J Appl Crystallogr 40, 658-674. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr D Biol Crystallogr 60, 2126-2132. 21 ACS Paragon Plus Environment

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[31] Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr D Biol Crystallogr 66, 486-501. [32] Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr D Biol Crystallogr 66, 213-221. [33] Chen, V. B., Arendall, W. B., III, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography, Acta Crystallogr D Biol Crystallogr 66, 12-21. [34] Egeberg, K. D., Springer, B. A., Martinis, S. A., Sligar, S. G., Morikis, D., and Champion, P. M. (1990) Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis, Biochemistry 29, 9783-9791. [35] Liu, Y., Moenne-Loccoz, P., Hildebrand, D. P., Wilks, A., Loehr, T. M., Mauk, A. G., and Ortiz de Montellano, P. R. (1999) Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase, Biochemistry 38, 3733-3743. [36] Rice, R. H., Lee, Y. M., and Brown, W. D. (1983) Interactions of heme proteins with hydrogen peroxide: protein crosslinking and covalent binding of benzo[α]pyrene and 17 beta-estradiol, Arch Biochem Biophys 221, 417-427. [37] Eakanunkul, S., Lukat-Rodgers, G. S., Sumithran, S., Ghosh, A., Rodgers, K. R., Dawson, J. H., and Wilks, A. (2005) Characterization of the periplasmic heme-binding protein ShuT from the heme uptake system of Shigella dysenteriae, Biochemistry 44, 13179-13191. [38] Stevens, J. M., Uchida, T., Daltrop, O., Kitagawa, T., and Ferguson, S. J. (2006) Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme, J Biol Chem 281, 6144-6151. [39] Caillet-Saguy, C., Piccioli, M., Turano, P., Lukat-Rodgers, G., Wolff, N., Rodgers, K. R., Izadi-Pruneyre, N., Delepierre, M., and Lecroisey, A. (2012) Role of the iron axial ligands of heme carrier HasA in heme uptake and release, J Biol Chem 287, 26932-26943. [40] Mokry, D. Z., Nadia-Albete, A., Johnson, M. K., Lukat-Rodgers, G. S., Rodgers, K. R., and Lanzilotta, W. N. (2014) Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor, Biochim Biophys Acta 1840, 3058-3066. [41] Uchida, T., Sato, E., Sato, A., Sagami, I., Shimizu, T., and Kitagawa, T. (2005) CO-dependent activity-controlling mechanism of heme-containing CO-sensor protein, neuronal PAS domain protein 2, J Biol Chem 280, 21358-21368. 22 ACS Paragon Plus Environment

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[42] Ray, G. B., Li, X. Y., Ibers, J. A., Sessler, J. L., and Spiro, T. G. (1994) How far can proteins bend the FeCO unit? Distal polar and steric effects in heme proteins and models, J Am Chem Soc 116, 162-176. [43] Lou, B. S., Snyder, J. K., Marshall, P., Wang, J. S., Wu, G., Kulmacz, R. J., Tsai, A. L., and Wang, J. (2000) Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase, Biochemistry 39, 12424-12434. [44] Spiro, T. G., and Wasbotten, I. H. (2005) CO as a vibrational probe of heme protein active sites, J Inorg Biochem 99, 34-44. [45] Lukat-Rodgers, G. S., Rodgers, K. R., Caillet-Saguy, C., Izadi-Pruneyre, N., and Lecroisey, A. (2008) Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release, Biochemistry 47, 2087-2098. [46] Hargrove, M. S., Barrick, D., and Olson, J. S. (1996) The association rate constant for heme binding to globin is independent of protein structure, Biochemistry 35, 11293-11299. [47] Higashimoto, Y., Sakamoto, H., Hayashi, S., Sugishima, M., Fukuyama, K., Palmer, G., and Noguchi, M. (2005) Involvement of NADPH in the interaction between heme oxygenase-1 and cytochrome P450 reductase, J Biol Chem 280, 729-737. [48] Owens, C. P., Du, J., Dawson, J. H., and Goulding, C. W. (2012) Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake, Biochemistry 51, 1518-1531. [49] Sun, J., Loehr, T. M., Wilks, A., and Ortiz de Montellano, P. R. (1994) Identification of histidine 25 as the heme ligand in human liver heme oxygenase, Biochemistry 33, 13734-13740. [50] Arnoux, P., Haser, R., Izadi, N., Lecroisey, A., Delepierre, M., Wandersman, C., and Czjzek, M. (1999) The crystal structure of HasA, a hemophore secreted by Serratia marcescens, Nat Struct Biol 6, 516-520. [51] Kumar, R., Matsumura, H., Lovell, S., Yao, H., Rodriguez, J. C., Battaile, K. P., Moenne-Loccoz, P., and Rivera, M. (2014) Replacing the axial ligand tyrosine 75 or its hydrogen bond partner histidine 83 minimally affects hemin acquisition by the hemophore HasAp from Pseudomonas aeruginosa, Biochemistry 53, 2112-2125. [52] Deniau, C., Gilli, R., Izadi-Pruneyre, N., Letoffe, S., Delepierre, M., Wandersman, C., Briand, C., and Lecroisey, A. (2003) Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake, Biochemistry 42, 10627-10633. [53] Liu, X., Gong, J., Wei, T., Wang, Z., Du, Q., Zhu, D., Huang, Y., Xu, S., and Gu, L. (2012) Crystal structure of HutZ, a heme storage protein from Vibrio cholerae: A structural mismatch observed in the region of high sequence conservation, BMC Struct Biol 12, 23. 23 ACS Paragon Plus Environment

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[54] Bhakta, M. N., and Wilks, A. (2006) The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the δ-regioselective heme oxygenase of Pseudomonas aeruginosa, Biochemistry 45, 11642-11649. [55] Spencer, A. L., Bagai, I., Becker, D. F., Zuiderweg, E. R., and Ragsdale, S. W. (2014) Protein/protein interactions in the mammalian heme degradation pathway: heme oxygenase-2, cytochrome P450 reductase, and biliverdin reductase, J Biol Chem 289, 29836-29858. [56] Lansky, I. B., Lukat-Rodgers, G. S., Block, D., Rodgers, K. R., Ratliff, M., and Wilks, A. (2006) The cytoplasmic heme-binding protein (PhuS) from the heme uptake system of Pseudomonas aeruginosa is an intracellular heme-trafficking protein to the δ-regioselective heme oxygenase, J Biol Chem 281, 13652-13662.


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Biochemistry

TABLES Table 1: Comparison of the dissociation (KD) constants with other hemoproteins Proteins

ka –1 –1

M s HutX

HutZ

1.0 × 10

2a 2a

kd s–1 0.04

KD a a

Ref.

µM 400a a

heme-HutX heme-HO-1

heme-HutZ CPR

1.0 × 10 1.0 × 103

0.05 2.0 × 10-3

500 20

heme-PhuS

pa-HOb

1.68 × 104

1.07 × 10-3

0.064

a) The accurate value was not determined due to the rapid dissociation rate. b) Pseudomonas aeruginosa HO


This study This study 55 56

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Table 2: Data collection and refinement statistics for HutX Data collection Beamline

Photon Factory BL17A

Wavelength (Å)

0.98

Resolution range (Å)

48.66 - 2.90 (3.08 - 2.90)

Space group

P21

Cell dimensions a, b, c (Å)

61.21, 80.88, 111.1

α, β, γ (°)

90, 95.6, 90

Total reflections

80821 (13143)

Unique reflections

23689 (3750)

Multiplicity

3.41 (3.50)

Completeness (%)

98.33 (96.67)

10.3 (2.34) 2

Wilson B-factor (Å )

58.57

Rsym (%)

10.8 (53.5) Refinement

Rwork

0.2464

Rfree

0.2779

Number of atoms

7856

Protein residues

994

RMS (bonds) (Å)

0.009

RMS (angles) (°)

1.32

Ramachandran favored (%)

95.11

Ramachandran allowed (%)

4.89

Average B-factor

95


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Biochemistry

FIGURE LEGENDS Figure 1: (A) Gene map of the V. cholerae hutWXZ operon. The direction of transcription is indicated by the arrows, and the smaller boxes indicate the potential Fur boxes. (B) Amino acid sequence alignment of HutX with E. coli O157:H7 ChuX. The alignment was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The candidates of heme axial ligands are shown in bold with black background. Figure 2: Heme titration and electronic absorbance spectrum of the heme-HutX complex. (A) Absorption difference spectra of heme-binding to HutX. The difference at 399 nm following incremental addition of heme (1–12.5 µM) to HutX (5 µM) in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 against a blank containing buffer alone, as shown in the insets. (B) Optical absorption spectra of heme-HutX. Ferric (solid line), ferrous (dotted line), and ferrous-CO (dashed-dashed line) complexes in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0. Figure 3: Heme displacement from the heme-HutX complex. (A) Heme displacement from HutX (2.5 µM) by apo-HutZ (12.5 µM) in 50 mM Tris-HCl, 150 mM NaCl, pH8.0 spectra measured at 1-min intervals over a period of 60 min. (B) Heme displacement from HutX by apomyoglobin as described above. (C) Time courses for hemin displacement from the heme-HutX to apo-HutZ (a) and to apomyoglobin (b), measured by the change of maximum absorptions over time relative to their respective maximum absorptions at 0 min. Figure 4: Kinetic analysis of HutX interactions with HutZ examined by surface plasmon resonance. Apo-HutZ samples injected over Ni-NTA immobilized apo-HutX at five concentrations (50, 100, 250, 500, and 750 µM). Figure 5: Enzymatic activity assays of HutZ. Reaction of HutZ (5.0 µM) with H2O2 (100 µM) in the presence (A) and absence (B) of HutX (2.5 µM). The spectra were recorded at an


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

interval of 1 min for 10 min over. Enzymatic activities of HutZ, measured by the change of maximum absorptions in inset. All of reactions in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0. Figure 6: Crystal structure of HutX. (A) Dimeric structure of HutX. The two monomers are displayed in green and cyan. (B) The putative heme-binding site with conserved residues Glu25, Gln26, Ile74, Glu76, Tyr90, His103, Phe119, Met120, and Lys139 in stick representation, where nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. Figure 7: (A) Resonance Raman spectra of the high-frequency regions of HutX excited at 413.1 nm in 50 mM Sodium phosphate, 150 mM NaCl, pH 8.0. (B) Resonance Raman spectra of the ferrous-CO complex of HutX in the low-frequency (left) and high-frequency (right) regions with excitation at 413.1 nm. Spectra of the ferrous heme-HutX complexes of 12C16O (a),

13 18

C O (b), and the difference (12C16O –

13 18

C O) (c). (C) Correlation plot between

frequencies of the νFe-CO and νC-O stretching modes. The two solid lines correspond to correlations for proximal imidazoles (solid circles), proximal imidazolates (solid triangles), thiolate-ligated hemoproteins (solid diamonds) and 5c hemoproteins (solid inverted triangle). The data point for HutX is presented as two open circles.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A Fur boxes hutD

hutC

hutB exbDI

exbBI

tonBI

hutW

hutX

hutZ

B HutX ChuX

1 MESLQQQVAQ LLEQQPTLLP AAMAEQLNVT EFDIVHALPE EMVAVVDGSH 50 1 --MSHVSLQE FLKTEPDGTL EVVAEQYNTT LLEVVRNLPS S--TVVPGDK 46 : .: : :*: :* .:*** *.* :::*: **. . :** *.:

HutX ChuX

51 AQTILESLPE WGPVTTIMTI AGSIFEVKAP FPKGKVARGY YNLMGRDGEL 100 47 FDTVWDTVCE WGNVTTLVHT ADVILEFSGE LPSGFHRHGY FNLRGKHG-M 95 :*: ::: * ** ***:: *. *:*... :*.* :** :** *:.* :

HutX 101 HGHLKLENIS HVALVSKPFM GRESHYFGFF TAQGENAFKI YLGRDEKREL 150 ChuX 96 SGHIKAENCT HIALIERKFM GMDTASILFF NKEGSAMLKI FLGRDDHRQL 145 **:* ** : *:**:.: ** * :: : ** . :*. :** :****::*:* HutX 151 IPEQVARFKA MQQQHKQ-- 167 ChuX 146 LSEQVSAFHT LAASLKEHA 164 :.***: *:: : . *:

Figure 1

ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

0.4

Absorbance

412 0.3

399

Abs (399 nm)

Biochemistry

Page 30 of 36

0.2 0.1 0.0 0

0.2

1

2

[heme] / [HutX]

0.1

0.0 300

400

500

600

700

800

Wavelength /nm

B 0.24

408

Absorbance

0.20

390

0.16 422

532 559

0.12 615 0.08

551

0.04

5

388

0.00 300

Figure 2

400

500 600 700 500 600 700 Wavelength /nm


800

Page 31 of 36

Biochemistry

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

411

Absorbance

0.3

390 0.2

0.1

0.0 300

400

500

600

700

800

700

800

Wavelength /nm

B

0.5

408

Absorbance

0.4 0.3

390

0.2 0.1 0.0 300

400

500

600

Wavelength /nm

Relative Abs (Peak)

C

1.2

(a) apo-HutZ

1.0 0.8

(b) apomyoglobin

0.6 0.4 0.2 0.0 0

Figure 3

250

500

Time /sec ACS Paragon Plus Environment

750

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

1200

750 M Response/ RU

1000

500 M

800 600

250 M

400

100 M 200

50 M

0 0

100

200

300

Time /sec

Figure 4


400

Page 33 of 36

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Biochemistry

Absorbance

0.25

409

0.20

Abs (409 nm)

A 0.24 0.20 0.16 0.12

0.15

0

2

4

6

8

10

Time /min.

0.10

647

0.05 0.00 300

400

500

600

700

800

Wavelength /nm

Absorbance

0.25

412

0.20

Abs (412 nm)

B

0.15

0.25 0.20 0.15 0.10 0.05 0

2

4

6

8

10

Time /min.

0.10

645

0.05 0.00 300

400

500

600

Wavelength /nm

Figure 5


700

800

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 34 of 36

B

M120

F119

Q26

K139 E25 E76 I74

Y90 H103

Figure 6


Page 35 of 36

4

1556 1585

1472 1502

Intensity

ferrous

10

2

3

4

1620

1357

A

10

1568

1372

2

1491

3

1590 1629

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ferric 1600

1700

1960

528

b

b 496

Intensity

-1

c 500

c

1500

479

B

1400

Raman shift /cm

1869

1300

526

a

a

1900 2000 600 1800 -1 -1 Raman shift /cm shift /cm Raman shift Raman /cm

400

500

-1

C

Fe-CO

/cm-1

540 520 HutX 500 480 460 1900

Figure 7

1920

1940

1960 -1 /cm C-O


1980

2000

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

Graphic for the Table of Contents

heme Tyr90

HutZ HutX


Cytoplasmic Heme-Binding Protein (HutX) - ACS Publications

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