Heme Proximal Hydrogen Bonding between His170 and Asp132

Article pubs.acs.org/biochemistry

Heme Proximal Hydrogen Bonding between His170 and Asp132 Plays an Essential Role in the Heme Degradation Reaction of HutZ from Vibrio cholerae Takeshi Uchida,*,†,‡ Nobuhiko Dojun,‡ Yukari Sekine,‡ and Koichiro Ishimori†,‡ †

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan

‡

S Supporting Information *

ABSTRACT: HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from Helicobacter pylori, HugZ, predicts that Asp132 in HutZ is located within hydrogen-bonding distance of the heme axial ligand His170. Hydrogen bonding between His170 and Asp132 appears to be disfavored in heme-degrading enzymes, because it can contribute to the imidazolate character of the axial histidine, as observed in most heme-containing peroxidases. Thus, we investigated the role of this potential hydrogen bond in the heme degradation reaction by mutating Asp132 to Leu, Asn, or Glu and by mutating His170 to Ala. Heme degradation activity was almost completely lost in D132L and D132N mutants, whereas verdoheme formation through reaction with H2O2 was comparable in the D132E mutant and wild-type enzyme. However, even at pH 6.0, when the heme is in a high-spin state, the D132E mutant was inactive toward ascorbic acid because of a significant reduction in its affinity (Kd) for heme (4.1 μM) compared with that at pH 8.0 (0.027 μM). The heme degradation activity of the H170A mutant was also substantially reduced, although this mutant bound heme with a Kd of 0.067 μM, despite the absence of an axial ligand. Thus, this study showed that proximal hydrogen bonding between Asp132 and His170 plays a role in retaining the heme in an appropriate position for oxygen-dependent heme degradation.

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proteins, HutZ was predicted to be a heme-storage protein. Although HutZ was reported to lack heme oxygenase activity,10 a recent study showed that HutZ degrades heme in a pHdependent manner; HutZ is inactive at pH 8.0, whereas it is active at pH 6.0.11 We found that the activity is correlated with the ratio of high-spin heme (Fe3+−H2O) to low-spin heme (Fe3+−OH−) (T. Uchida et al., unpublished observations). This pH dependence of the heme degradation activity of HutZ reflects a structural feature; the imidazolate character of the proximal His of the heme−HutZ complex represses the activity at pH 8.0. A plot of frequencies of the Fe−CO (νFe−CO) and C−O (νC−O) stretching modes of the CO-bound heme−HutZ complex falls between lines of proteins having a neutral His as an axial ligand and proteins having imidazolate as an axial ligand.11 Because of the imidazolate character of the heme− HutZ complex, the rate of reduction of heme by ascorbic acid is quite low (T. Uchida et al., unpublished observations). Thus, the heme degradation reaction is inhibited at the step of heme reduction when ascorbic acid is used as an electron source. Although the heme complex of HutZ has not been crystallized,

ron is an essential element for bacteria, because it acts as a cofactor in playing key roles in metabolic processes such as oxygen transport, electron transfer, and energy transduction. To obtain iron, bacteria have evolved sophisticated ironacquisition systems that reflect the limited amount of available iron under growth conditions of most bacteria. For example, a number of bacteria synthesize and secrete low-molecular weight compounds called siderophores,1 which bind ferric iron from many iron sources, such as soil, water, and transferrin, with high affinity.2−5 Iron-bound siderophores are taken up through receptor proteins in the outer membrane. Most pathogens also utilize a siderophore-independent iron-uptake system, in which heme (iron-protoporphyrin IX) is a common iron source.6−8 Heme receptor proteins in the outer membrane accept heme directly, or from hemoglobin, and transfer it into the cell periplasm. There, periplasmic heme-binding proteins carry heme to inner-membrane receptors, which transfer heme across the inner membrane to the cytoplasm. In the cytoplasm, the porphyrin ring of heme is oxidized by a heme-degrading enzyme and broken to release iron. The Gram-negative bacterium Vibrio cholerae is the causative agent of the disease cholera. Analyses of the genome sequence of V. cholerae and bioinformatics-based predictions have identified putative genes encoding proteins for heme acquisition, termed Hut (heme utilization).9 Among Hut © XXXX American Chemical Society

Received: February 20, 2017 Revised: April 18, 2017 Published: May 8, 2017 A

DOI: 10.1021/acs.biochem.7b00152 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

fast autoxidation rate of the oxyferrous heme−HutZ complex supports this conclusion.11 The strong electron donation from the imidazolate group of the axial His is known to promote heterolytic cleavage of the O−O bond to form a ferryl species (called a “push” effect).29 Because the ferryl species is not susceptible to heme degradation,26 heme-degrading enzymes have evolved to avoid this pathway. Therefore, the presence of a strong hydrogen bond between the proximal His and nearby Asp in HutZ is not expected to be favored in a heme-degrading enzyme. In this study, we employed two approaches to assess the role of hydrogen bonding between Asp132 and His170. In the first, we replaced Asp132 with Leu, Asn, or Glu; in the second, we substituted Ala for His170. Mutations at both sites led to a significant loss of heme degrading activity, except for the substitution of Glu for Asp132. When the proximal hydrogen bond was intact, the heme−HutZ complex was inactive, because heme could not be reduced to ferrous heme because of strong electron donation from the proximal His. However, disruption of the hydrogen bond did not lead to an increase in enzymatic activity, likely because the heme was not held in an appropriate position; thus, the proximal hydrogen bond is necessary for HutZ to bind heme. Collectively, only a slight modulation of the strength of the hydrogen bond between His170 and Asp132 is key to activation of the degradation reaction of the heme−HutZ complex.

a crystal structure for the homologous protein, HugZ, from Helicobacter pylori is available.12 This structure suggests that Asp132 in HutZ is located within hydrogen-bonding distance of the heme axial ligand His170 (Figure 1). A hydrogen bond between His170 and Asp132 could contribute to the imidazolate character of the axial His, as observed in oxygenactivating heme enzymes (i.e., peroxidases).13−15

Figure 1. Crystal structure of HugZ from H. pylori (Protein Data Bank entry 3GAS). Residues are numbered according to the amino acid sequence of HutZ from V. cholerae. α and β indicate the subunits.

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MATERIALS AND METHODS 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) and were used without further purification. Expression and Purification of HutZ. Mutant HutZ proteins were expressed in Escherichia coli and purified as described previously.11 The hutZ gene was subcloned into pET28b (Merck Millipore, Darmstadt, Germany) via NdeI and EcoRI sites, and the thrombin recognition site (Leu-Val-ProArg-Gly-Ser) in the pET-28b construct was mutated to the HRV 3C protease recognition site (Leu-Glu-Val-Leu-Phe-GlnGly-Pro).30 Mutagenesis was conducted utilizing a PrimeSTAR mutagenesis basal kit from Takara Bio (Otsu, Japan). DNA oligonucleotides were purchased from Eurofins Genomics Inc. (Tokyo, Japan). The mutated genes were sequenced (Eurofins Genomics Inc.) to ensure that only the desired mutations were introduced. Measurement of Binding of Heme to HutZ. Heme binding was tracked by difference spectroscopy in the Soret region of the UV−visible spectrum. Successive aliquots of 0.5 mM hemin in 0.1 M NaOH were added to both the sample cuvette, which contained 10 μM apo-HutZ, and the reference cuvette. Spectra were recorded 3 min after the addition of each heme aliquot. The absorbance difference at 411−413 nm was plotted as a function of heme concentration, and the dissociation constant (Kd,heme) was calculated using the quadratic binding equation

In mammalian heme oxygenase-1 (HO-1), the proximal His is hydrogen bonded with Glu29.16,17 However, this hydrogen bond is not as strong as those observed in peroxidases, because the redox potential is −65 mV,18 which is far from that of most peroxidases with imidazolate as an axial ligand.19 Additionally, the Fe−His stretching mode, νFe−His, was observed at 218 cm−1,20 which is almost identical to those observed for myoglobin (220 cm−1)21,22 and hemoglobin (215 and 221 cm−1),23 which contain a typical neutral His as an axial ligand, and is far from that of most peroxidases.24,25 The weaker hydrogen bond between the proximal His and Glu29 in HO-1 seems to be favorable for the heme degradation reaction, because it prevents the O−O bond of the hydroperoxy species from being cleaved. Considering that the high-valent iron−oxo species has no heme degradation activity,26 O−O bond cleavage prior to hydroxylation of a meso carbon of the porphyrin ring would not be preferable for heme degradation. Therefore, the role of this weak hydrogen bonding imposed on HO-1 by Glu29 is considered to define the orientation of the proximal His.27 Despite the prediction of the presence of a hydrogen bond between His170 and Asp132, the νFe−His of the ferrous heme− HutZ complex is identical to that of heme-HO,11,28 indicating a moderate hydrogen bond. In contrast, on the basis of a correlation plot of νFe−CO versus νC−O, the hydrogen bond between His170 and Asp132 in the ferrous CO-bound heme− HutZ complex is predicted to be stronger than that in other heme-degrading enzymes.11 This discrepancy seems to be attributed to the presence of the distal heme ligand in the heme−HutZ complex. Formation of the distal hydrogen bond between Arg92 and the heme ligand (O2, CO, or OH−) would be coupled with formation of the proximal hydrogen bond. The

ΔA = ΔA max ×

[P] + [H] + Kd,heme −

([P] + [H] + Kd,heme)2 − 4[P][H] 2[P]

(1) B

DOI: 10.1021/acs.biochem.7b00152 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry where [P] and [H] represent the total protein and hemin concentrations, respectively. The pH of the protein solution was not changed by the addition of the alkaline hemin solution. Spectroscopy. Optical spectra of purified proteins were recorded with a UV−visible spectrophotometer (V-660, Jasco, Tokyo, Japan) at room temperature. Resonance Raman spectra were obtained with a single monochromator (SPEX500M, Jobin Yvon, Edison, NJ) equipped with a liquid nitrogen-cooled CCD detector (Spec-10:400B/LN, Roper Scientific, Princeton, NJ). Excitation wavelengths of 413.1 nm, delivered by a krypton ion laser (BeamLok 2060, Spectra Physics, Mountain View, CA), and 441.6 nm, delivered by a helium−cadmium laser (IK5651R, Kimmon Koha, Tokyo, Japan), were employed. The laser power at the sample point was adjusted to ∼5 mW for the ferric and ferrous forms; a lower power (0.1 mW) was used 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 peak positions of well-defined Raman bands was ±1 cm−1. Samples for resonance Raman experiments were prepared at a concentration of approximately 10 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). Heme Degradation Activity. The heme degradation reaction of HutZ was monitored by spectrophotometry. Briefly, 1.9 mL of a hemin-reconstituted protein solution (final concentration, 10 μM) in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0) was placed in a cuvette, and the reaction was started by adding 100 μL of 4 mM H2O2 or 20 mM ascorbic acid in the same buffer at 25 °C. Spectra were recorded at 1 min intervals for 10 min for H2O2 and at 2 min intervals for 30 min for ascorbic acid. In the case of reactions with ascorbic acid (final concentration, 1 mM), 1 mg/mL bovine liver catalase was added to suppress H2O2. After the reaction, ferrozine (Dojindo, Kumamoto, Japan) was added to a final concentration of 1 mM. The amount of released iron was calculated by measuring the absorbance at 562 nm using an extinction coefficient (ε562) of 29 mM−1 cm−1. Cyanide Binding Rate Constants. Cyanide (CN) binding was measured using a stopped-flow apparatus (Unisoku, Osaka, Japan) by following the decrease in absorbance at 412 nm. In a typical CN binding experiment, one syringe contained 3 μM HutZ [50 mM NaPi and 150 mM NaCl (pH 8.0)], and the second syringe contained an at least 100-fold excess of CN. Three determinations were performed for each ligand concentration. The mean of the pseudo-first-order rate constants, kobs, was used to calculate second-order rate constants, obtained from the slope of a plot of kobs versus ligand concentration (kobs = kon[CN] + koff).

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RESULTS Purification and Heme Reconstitution of Asp132 Mutants. Three Asp132 mutant proteins (D132L, D132N, and D132E) were purified using the same procedure that was used for wild-type (WT) HutZ.11 Elution profiles of gelfiltration columns showed that all three mutants, like the WT, exist as a dimer (data not shown). Titration of the mutants with heme was monitored using difference absorption spectra, which provided dissociation constant (Kd,heme) values of 0.47 ± 0.07, 0.15 ± 0.02, and 0.027 ± 0.004 μM for D132L, D132N, and D132E mutants, respectively (Figure 2, insets; Table 1). A comparison of these values with that for WT HutZ (0.052 ± 0.004 μM)11 showed that replacement of Asp132 with Leu or

Figure 2. Absorption spectra of the heme−HutZ complex. The protein concentration was 5 μM (on a per heme basis) in 50 mM TrisHCl and 150 mM NaCl (pH 8.0). Spectra are for the ferric (), ferrous (···), and CO-bound (---) forms. The inset shows the heme binding curve generated from difference absorbance spectra by plotting ΔA vs the heme:protein molar ratio: (A) heme−D132L, (B) heme−D132N, and (C) heme−D132E.

Asn resulted in an approximately 3−10-fold decrease in heme affinity, whereas replacement with Glu had little effect. Absorption Spectra of Heme−Asp132 Mutants. Absorption spectra of heme−Asp132 mutants are shown in C

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Table 1. Heme Dissociation Constants (Kd) and Kinetic Constants of H2O2 and CN (kSoret and kCN) for Heme-Bound WT HutZ and Its Asp132 Mutants Kd,heme (μM) WT D132L D132N D132E a

pH 8.0

pH 6.0

0.052 ± 0.004 0.47 ± 0.07 0.15 ± 0.02 0.027 ± 0.004

0.13 ± 0.06 NDa 1.9 ± 0.3 4.1 ± 6.5

kSoret (min−1) 0.52 0.24 0.35 0.42

± ± ± ±

0.06 0.01 0.02 0.02

kCN (mM−1 s−1)

ref

± ± ± ±

11 this study this study this study

0.88 1.26 0.77 0.99

0.01 0.02 0.01 0.01

Not determined because of the extremely weak affinity.

evaluate the accessibility of ligands to the active site, and the rate of CN binding (kon,CN) was determined using a stoppedflow apparatus. A typical time trace for the reaction of the heme−D132L mutant with CN ion, monitored at 407 nm, is shown in Figure 4A. Under conditions with an excess of CN, the pseudo-first-order rate constants (kobs) obtained from single-exponential fits increased linearly with increasing concentrations of CN (Figure 4B). The second-order rate constant (kon,CN) for the heme−D132L mutant obtained from the slope was 1.26 mM−1 s−1, which is somewhat faster than that of heme−WT HutZ (0.88 mM−1 s−1) (Table 1). Therefore, the decreased accessibility of the ligand to the heme does not account for the small amount of verdoheme formation in the D132L and D132N mutants. Reaction of Heme−Asp132 Mutants with Ascorbic Acid. The heme degradation reaction of heme−Asp132 mutants was also examined using ascorbic acid as an electron donor. All reactions were conducted in the presence of catalase to scavenge H2O2. Spectral changes following addition of ascorbic acid at pH 8.0 are shown in Figure 5. In reactions of all three Asp132 mutants, the intensity of the Soret band diminished very little, and no clear band was observed in the 500−800 nm region derived from the products of heme degradation.31 These spectral changes suggest that no heme degradation occurred for the heme−Asp132 mutants under these reaction conditions, similar to previously reported results for heme−WT HutZ.11 As reported previously,11 the heme degradation reaction of heme−WT HutZ with ascorbic acid is significantly accelerated at pH 6.0. This activation is related to a change in the heme spin state: when the heme is in a low-spin state, the enzyme is inactive, whereas it is active when the heme is in a high-spin state. When the pH is decreased to 6.0, the Soret band of all three Asp132 mutants shifted to 403 nm (Figure 6A), indicating that some low-spin heme was converted to highspin heme. Additionally, the absorbance at ∼370 nm was significantly enhanced, suggesting that heme was partially released from the protein. In the reaction of the heme−D132E mutant, which showed a similar amount of verdoheme in the reaction with H2O2, the intensity of the Soret band at 401 nm was significantly diminished with ascorbic acid at pH 6.0 (Figure 6B). To quantify the amount of Fe2+ released by the reaction, we added ferrozine to the solution after the reaction. No increase in absorbance at 562 nm, which is derived from the Fe2+−ferrozine complex, was observed. The yields of Fe2+ for the all three Asp132 mutants were 100-fold larger than that at pH 8.0. In the case of WT HutZ, Kd,heme at pH 6.0 was only 2-fold larger than that at pH 8.0 (data not shown). The occurrence of a fivecoordinate heme in the heme−D132E mutant indicates that the proximal His is prone to dissociate from the heme. The fact that the other two Asp132 mutants also exhibit a fivecoordinate heme and higher Kd,heme values suggests that Asp132 is essential for heme to coordinate to the protein, especially in the high-spin form. Because of the weak bond between His170 and heme in the heme−Asp132 mutants, the proximal His can be released from the heme when O2 binds to the ferrous heme. Such breakage of the Fe−His bond would account for the lower activity of the heme−Asp132 mutants even in the high-spin state. Role of His170 in Heme Degradation by HutZ. To investigate the role of proximal hydrogen bonding in HutZ, we also replaced His170 with Ala. A heme titration plot showed that heme bound to the H170A mutant at a 1:1 ratio and yielded a Kd,heme comparable to that of WT HutZ (Figure 8). Absorption and resonance Raman spectra showed that the heme in the hemin-reconstituted H170A mutant is composed of the six-coordinate species (Figure 10A). The appearance of the νFe−His mode in the heme−H170A mutant (Figure 10B) suggests that His is coordinated to the heme, despite replacement of His170 with Ala. A closer look at the structure of HugZ suggests that His63, which is within hydrogenbonding distance of the heme propionate, is the sole His in the putative heme-binding site (Figure 1).12 Additional replacement of His63 with Leu in the H170G mutant resulted in a significant loss of heme binding ability (Figure S2), implying that His63 in the H170A mutant is capable of coordinating heme. Although the Kd,heme of the H170A mutant is similar to that of WT HutZ (Figure 8), exogenous imidazole replaced His63, considering that addition of imidazole to the ferrous heme−H170A mutant shifted the frequency of νFe−His by 3 cm−1 and significantly enhanced it (Figure 10B). Therefore, J

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structure, and coupling of the tryptophan free radical to the heme. Biochemistry 32, 3313−3324. (14) Poulos, T. L. (1993) Peroxidases. Curr. Opin. Biotechnol. 4, 484− 489. (15) Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T., and Poulos, T. L. (1997) Crystal structure of horseradish peroxidase C at 2.15 Å resolution. Nat. Struct. Biol. 4, 1032−8. (16) Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Crystal structure of human heme oxygenase-1. Nat. Struct. Biol. 6, 860−867. (17) Sugishima, M., Omata, Y., Kakuta, Y., Sakamoto, H., Noguchi, M., and Fukuyama, K. (2000) Crystal structure of rat heme oxygenase1 in complex with heme. FEBS Lett. 471, 61−66. (18) Liu, Y., Moënne-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. (19) Harbury, H. A. (1957) Oxidation-reduction potentials of horseradish peroxidase. J. Biol. Chem. 225, 1009−1024. (20) Takahashi, S., Wang, J., Rousseau, D. L., Ishikawa, K., Yoshida, T., Host, J. R., and Ikeda-Saito, M. (1994) Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation. J. Biol. Chem. 269, 1010−1014. (21) Kitagawa, T., Kyogoku, Y., Iizuka, T., and Saito, M. I. (1976) Nature of the iron-ligand bond in ferrous low spin hemoproteins studied by resonance Raman scattering. J. Am. Chem. Soc. 98, 5169− 5173. (22) Kitagawa, T., Nagai, K., and Tsubaki, M. (1979) Assignment of the Fe-Nε (His F8) stretching band in the resonance Raman spectra of deoxy myoglobin. FEBS Lett. 104, 376−378. (23) Nagai, K., Kitagawa, T., and Morimoto, H. (1980) Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering. J. Mol. Biol. 136, 271−289. (24) Hashimoto, S., Teraoka, J., Inubushi, T., Yonetani, T., and Kitagawa, T. (1986) Resonance Raman study on cytochrome c peroxidase and its intermediate. J. Biol. Chem. 261, 11110−11118. (25) Smulevich, G., Mauro, J. M., Fishel, L. A., English, A. M., Kraut, J., and Spiro, T. G. (1988) Heme pocket interactions in cytochrome. Biochemistry 27, 5477−5485. (26) Wilks, A., and Ortiz de Montellano, P. R. (1993) Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species. J. Biol. Chem. 268, 22357− 22362. (27) Unno, M., Matsui, T., and Ikeda-Saito, M. (2007) Structure and catalytic mechanism of heme oxygenase. Nat. Prod. Rep. 24, 553−570. (28) Takahashi, S., Wang, J. L., Rousseau, D. L., Ishikawa, K., Yoshida, T., Takeuchi, N., and Ikedasaito, M. (1994) Heme-heme oxygenase complex: Structure and properties of the catalytic site from resonance Raman scattering. Biochemistry 33, 5531−5538. (29) Dawson, J. H. (1988) Probing structure-function relations in heme-containing oxygenases and peroxidases. Science 240, 433−439. (30) Uchida, T., Sasaki, M., Tanaka, Y., and Ishimori, K. (2015) A dye-decolorizing peroxidase from Vibrio cholerae. Biochemistry 54, 6610−6621. (31) Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-saito, M. (1996) Oxygen and one reducing equivalent are both required for the conversion of α-hydroxyhemin to verdoheme in heme oxygenase. J. Biol. Chem. 271, 6618−6624. (32) Kitagawa, T., Hashimoto, S., Teraoka, J., Nakamura, S., Yajima, H., and Hosoya, T. (1983) Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases. Biochemistry 22, 2788−2792. (33) Spiro, T. G., Soldatova, A. V., and Balakrishnan, G. (2013) CO, NO and O2 as vibrational probes of heme protein interactions. Coord. Chem. Rev. 257, 511−527.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00152. Resonance Raman spectra of the heme−Asp132 mutants and the heme−WT HutZ complex in the CO-bound form (Figure S1), heme titration plot of the H63L/ H170G double mutant (Figure S2), resonance Raman spectra of the heme−D132E mutant in the CO-bound form (Figure S3), and resonance Raman spectra of the heme−HutZ complex in the presence of 10 mM imidazole (Figure S4) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. E-mail: uchida@sci. hokudai.ac.jp. ORCID

Takeshi Uchida: 0000-0001-9270-8329 Funding

This study was supported in part by Grants-in-Aid for Scientific Research (16K05835 to T.U. and 15H00909 to K.I.) from the Ministry of Culture, Education, Sports, Science, and Technology (MEXT) of Japan. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Braun, V., and Killmann, H. (1999) Bacterial solutions to the iron-supply problem. Trends Biochem. Sci. 24, 104−109. (2) Andrews, S. C., Robinson, A. K., and Rodríguez-Quiñones, F. (2003) Bacterial iron homeostasis. FEMS Microbiol. Rev. 27, 215−237. (3) Miethke, M., and Marahiel, M. A. (2007) Siderophore-based iron acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71, 413− 451. (4) Hider, R. C., and Kong, X. (2010) Chemistry and biology of siderophores. Nat. Prod. Rep. 27, 637−657. (5) Soares, M. P., and Weiss, G. (2015) The iron age of host-microbe interactions. EMBO Rep. 16, 1482−1500. (6) Wandersman, C., and Stojiljkovic, I. (2000) Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. Curr. Opin. Microbiol. 3, 215−220. (7) Braun, V., and Hantke, K. (2011) Recent insights into iron import by bacteria. Curr. Opin. Chem. Biol. 15, 328−334. (8) Contreras, H., Chim, N., Credali, A., and Goulding, C. W. (2014) Heme uptake in bacterial pathogens. Curr. Opin. Chem. Biol. 19, 34− 41. (9) Wyckoff, E. E., Mey, A. R., and Payne, S. M. (2007) Iron acquisition in Vibrio cholerae. BioMetals 20, 405−416. (10) 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. (11) 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. (12) Hu, Y., Jiang, F., Guo, Y., Shen, X., Zhang, Y., Zhang, R., Guo, G., Mao, X., Zou, Q., and Wang, D.-C. (2011) Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori. J. Biol. Chem. 286, 1537−1544. (13) Goodin, D. B., and McRee, D. E. (1993) The Asp-His-Fe triad of cytochrome c peroxidase controls the reduction potential, electronic K

DOI: 10.1021/acs.biochem.7b00152 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry (34) Wilks, A., Sun, J., Loehr, T. M., and Ortiz de Montellano, P. R. (1995) Heme oxygenase His25Ala mutant - Replacement of the proximal histidine iron ligand by exogenous bases restores catalytic activity. J. Am. Chem. Soc. 117, 2925−2926. (35) Wilks, A., and Moenne-Loccoz, P. (2000) Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine. J. Biol. Chem. 275, 11686−11692. (36) Chu, G. C., Couture, M., Yoshida, T., Rousseau, D. L., and Ikeda-Saito, M. (2000) Axial ligation states of five-coordinate heme oxygenase proximal histidine mutants, as revealed by EPR and resonance Raman spectroscopy. J. Am. Chem. Soc. 122, 12612−12613. (37) Ishikawa, K., Takeuchi, N., Takahashi, S., Matera, K. M., Sato, M., Shibahara, S., Rousseau, D. L., Ikeda-Saito, M., and Yoshida, T. (1995) Heme oxygenase-2. Properties of the heme complex of the purified tryptic fragment of recombinant human heme oxygenase-2. J. Biol. Chem. 270, 6345−6350. (38) Ito-Maki, M., Ishikawa, K., Matera, K. M., Sato, M., Ikeda-Saito, M., and Yoshida, T. (1995) Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1. Arch. Biochem. Biophys. 317, 253−258. (39) 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. (40) Sugishima, M., Sakamoto, H., Kakuta, Y., Omata, Y., Hayashi, S., Noguchi, M., and Fukuyama, K. (2002) Crystal structure of rat apoheme oxygenase-1 (HO-1): Mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms. Biochemistry 41, 7293−7300. (41) Lad, L., Schuller, D. J., Shimizu, H., Friedman, J., Li, H., Ortiz de Montellano, P. R., and Poulos, T. L. (2003) Comparison of the hemefree and -bound crystal structures of human heme oxygenase-1. J. Biol. Chem. 278, 7834−7843. (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.

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DOI: 10.1021/acs.biochem.7b00152 Biochemistry XXXX, XXX, XXX−XXX