A Dye-Decolorizing Peroxidase from Vibrio cholerae - Biochemistry

Oct 2, 2015 - (18, 19) The structure was refined with phenix.refine.(20) To monitor the refinement, a random 5% subset was set aside for calculation o...
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A Dye-Decolorizing Peroxidase from Vibrio cholerae Takeshi Uchida,*,†,‡ Miho Sasaki,‡ Yoshikazu Tanaka,§ 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 060-0810, Japan § Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0808, Japan ‡

S Supporting Information *

ABSTRACT: The dye-decolorizing peroxidase (DyP) protein from Vibrio cholerae (VcDyP) was expressed in Escherichia coli, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis−Menten equation, giving kcat and Km values of 1.3 ± 0.3 s−1 and 50 ± 20 μM, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of VcDyP was highest at pH 4. A mutational study showed that two distal residues, Asp144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of VcDyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H2O2. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. VcDyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that VcDyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid.

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as well as anthraquinone dyes.12,13 Most fungal DyPs are classified into class D. The genomic sequence of Vibrio cholerae and bioinformaticsbased analysis predicted a protein at gene locus VC2145 as a DyP-type peroxidase. The sequence analysis by PeroxiBase suggests that VC2145 belongs to class B, which is supported by the high degree of similarity of the sequences of VC2145 and that of DyP from Escherichia coli (45% identical). Two residues (Asp and Arg) in the heme distal pocket are conserved in all DyPs.7,14,15 However, the roles of these resides are different in distinct families. The distal Asp is considered to be an acid− base catalyst in the class D DyPs, while it would not be essential for class A and B DyPs.7,16 Instead, a distal Arg is crucial for the catalytic activity. To clarify the reaction of the DyP from V. cholerae, we constructed a system to express VC2145 in E. coli and elucidated the mechanism of the catalytic reaction on the basis of tertiary structure analysis.

eroxidases (EC 1.11.1.x) make up a large group of oxidoreductases that catalyze the oxidation of substrates using hydrogen peroxide (H2O2) as an electron acceptor. Peroxidase activity has been observed in plants, microorganisms, and animals, where these enzymes play key roles in important biological processes, including cell wall synthesis and degradation, stress responses, signaling during oxidative stress, and removal of xenobiotics.1,2 Most peroxidases are hemoproteins, which contain Fe(III)-protoporphyrin IX (PPIX) as a prosthetic group. A novel family of hemecontaining peroxidases, the dye-decolorizing peroxidases (DyP) (EC 1.11.1.19), was recently identified.3−5 DyP-type peroxidases were first discovered in fungi and were shown to be capable of degrading a wide range of dyes.3 These enzymes, however, were unrelated to other superfamilies of peroxidases, with negligible sequence homology and no structural similarity. The DyP peroxidase family is subdivided into four subfamilies (classes A−D) according to PeroxiBase.6−9 Class A involves bacterial DyPs containing a Tat signal sequence, indicating that they function outside the cytoplasm.10 Many bacterial DyPs, which are predicted to be expressed in cytoplasm, belong to classes B and C, which suggests that these proteins play a role in an intracellular metabolic pathway. Although DyPs were initially characterized for their ability to degrade dyes,5,7 they are known to react with different substrates.11 In particular, DyPs in class B have a wide range of substrate specificities; they can oxidize manganese and lignin © XXXX American Chemical Society

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EXPERIMENTAL PROCEDURES Materials. All chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Sigma-Aldrich (St. Louis, MO) and used without further purification. Received: August 26, 2015 Revised: September 25, 2015

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DOI: 10.1021/acs.biochem.5b00952 Biochemistry XXXX, XXX, XXX−XXX

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Crystallization. Crystallization of VC2145 was conducted using the sitting drop vapor diffusion method at 4 °C, in which drops of 4 μL were prepared by adding 2 μL of 10 mg/mL protein and 2 μL of crystallization solution. The best diffraction quality crystals grew from 0.2 M potassium thiocyanate and 20% (w/v) PEG 3000. Crystals were soaked into a crystallization solution containing 20% PEG400 as a cryoprotectant and then frozen in liquid nitrogen for cryogenic data collection. Structure Determination. X-ray diffraction data were collected at 100 K on the BL17 beamline of the Photon Factory (Tsukuba, Japan) using ADSC Q270 CCD detector. The wavelength of the incident X-rays was 1.0 Å. Diffraction data were processed and scaled with XDS (Kabsch, 2010). The crystal belonged to space group P32 with the following unit cell dimensions: a = b = 77.7 Å, and c = 156.9 Å. The structure was determined using the molecular replacement method with PHASER17 and the previously reported structure of DyP from Rhodococcus jostii RHA1 [Protein Data Bank (PDB) entry 3QNR] as a search model. COOT was used to manually replace the appropriate residues.18,19 The structure was refined with phenix.refine.20 To monitor the refinement, a random 5% subset was set aside for calculation of Rfree. The resultant FO − FC electron density map showed significant electron density for heme. Then, heme was added to the model and refined. Finally, water molecules were added to the model and refined. Diffraction and refinement statistics are summarized in Table 1. The coordinates and structure factors of VcDyP have been deposited in the PDB as entry 5DE0. Spectroscopy. Optical spectra of purified VC2145 were recorded with a Hitachi U-3310 or Jasco V-660 UV−visible spectrophotometer. Resonance Raman spectra were recorded with a single monochromator (SPEX500M, Jobin Yvon), equipped with a liquid nitrogen-cooled CCD detector (Spec10:400B/LN, Roper Scientific). The excitation wavelengths were 413.1 nm from a krypton ion laser (BeamLok 2060, Spectra Physics) and 441.6 nm from a helium−cadmium laser (IK5651R, Kimmon Koha, Tokyo, Japan). 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 resonance Raman experiments were ∼10 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). Dye Decolorizing Assay. Dye-decolorizing activity was determined by spectrophotometrically measuring the rate of H2O2-mediated decomposition of reactive blue 19 (RB19). Briefly, 900 μL of a hemin-reconstituted protein solution (final concentration of 0.1 μM) in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0) was placed in a 1 mL cuvette, along with 10−50 μL of the substrate solution to be tested. The reaction was started by the addition of 100 μL of 2 mM H2O2 (final concentration of 0.2 mM) in the same buffer, with incubations normally for 3 min at room temperature. The decomposition of the substrate was measured at 595 nm (ε595 = 26.6 mM−1 cm −1 ) using a Jasco V-660 spectrophotometer. H 2 O 2 concentrations were determined spectrophotometrically using an absorption coefficient at 240 nm of 43.6 M−1 cm−1.21 Peroxidase Assay. The peroxidase assay was measured spectrophotometrically. The standard assay was performed in 2 mL of the reaction mixture with the enzyme, 0.2 mM H2O2,

Protein Overexpression and Purification. The VC2145 gene was purchased from the PlasmID Repository (http:// plasmid.med.harvard.edu/PLASMID/Home.xhtml) (clone VcCD00018619) and amplified by polymerase chain reaction (PCR) using primers 5′-CGCCATATGTTTAAGTCACAGACCGC-3′ and 5′-GGGAATTCATTGATTCTTGAGTGTCAGTTC-3′. The amplified fragment was cloned into the pET28b vector (Merck Chemicals) using the NdeI and EcoRI restriction sites. The thrombin recognition site composed of Leu-Val-Pro-Arg-Gly-Ser (LVPRGS) of vector pET-28b was mutated to the HRV 3C protease recognition site composed of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (LEVLFQGP). The correct gene sequence was confirmed by DNA sequencing (Eurofins Genomics). E. coli strains carrying plasmids for VC2145 were grown at 37 °C in LB broth or M9 minimal medium supplemented with 50 μg/mL kanamycin. Expression of the His6-tagged fusion protein in E. coli BL21(DE3) was induced with 1 mM isopropyl β-D-thiogalactopyranoside when the optical density at 600 nm reached 0.6−1.0. The cells were further grown at 28 °C overnight and harvested by centrifugation, and the cell pellet was stored at −80 °C until it was used. The pellet was thawed on ice, suspended in lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl (pH 8.0), 0.1% Nonidet P-40, 1 mg/mL lysozyme, and DNase, and incubated on ice for 60 min. The sample was centrifuged at 40000g for 30 min, and the supernatant was loaded onto a HisTrap HP column (GE Healthcare) pre-equilibrated with 50 mM Tris-HCl buffer containing 500 mM NaCl and 20 mM imidazole (pH 8.0). The resin was extensively washed [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 containing 500 mM NaCl and 200 mM imidazole (pH 8.0). Eluted VC2145 was incubated with HRV 3C protease (Accelagen, San Diego, CA) for ∼16 h at 4 °C to remove the His6 tag. After the cleavage, the reaction mixture was again applied to the 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 protein purity was assessed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) on 12.5% polyacrylamide gels to be ∼95%. Heme Titration Assay. The binding of hemin to VC2145 was assayed by tryptophan fluorescence quenching. Aliquots of 0.5 mM hemin in 0.1 M NaOH were added successively to the sample cuvette, which contained 10 μM protein as purified. Fluorescence spectra at 310−500 nm were recorded 3 min after the addition of each heme aliquot on a FP-8500 spectrofluorimeter (Jasco), with excitation at 295 nm. The fluorescence intensity at 329 nm was plotted against the concentration of hemin. The dissociation constant, Kd, was calculated using the following equation: IF = IF0 + ΔIF

KD + Pt + L t −

(KD + Pt + L t)2 − 4L tPt 2Pt (1)

where IF is the fluorescence intensity at a given hemin concentration, IF0 is the fluorescence intensity in the absence of hemin, ΔIF is the change in fluorescence intensity upon addition of hemin, and Pt and Lt are the total protein and hemin concentrations, respectively. B

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oligomeric,25 which is consistent with the result observed for VcDyP. During purification, we observed that the soluble fraction had a wine-red color, which became more pronounced after purification, suggesting that this protein contains some pigment. Figure 1 shows the absorption (panel A) and fluorescence (panel B) spectra of VcDyP as purified. The absorption spectrum shows the Soret band at 406 nm and Qbands at 510, 548, 569, and 623 nm. This absorption spectrum is not typical of those of heme-containing proteins, but rather similar to those of apomyoglobin-containing protoporphyrin IX (PPIX) with bands at 409, 508, 543, 574, and 626 nm,28 or IsdC containing PPIX with bands at 406, 509, 546, 570, and 625 nm.29 Furthermore, the peaks of the Soret and Q bands of VcDyP as purified were almost identical to those of VcDyP reconstituted with 1 equiv of PPIX (405, 506, 544, 577, and 630 nm). The fluorescence spectrum of VcDyP as purified with excitation at 407 nm exhibited an intense band at 624 nm (Figure 1B), similar to that of the PPIX complexed with apomyoglobin.28 These results indicate that some percentage of VcDyP was purified as a complex with PPIX, but not with heme. The ratio of absorbance at 406 nm to that at 280 nm suggested that the PPIX-bound protein was estimated to be at most 10% of the total protein when the protein was expressed in LB medium, but it was reduced to ∼5% when M9 minimal medium was used. Binding of Heme to VcDyP. Binding of heme to VcDyP as purified was monitored by fluorescence quenching of endogenous Trp residues. The relative fluorescence intensities were plotted as a function of hemin concentration (Figure 1C), and the dissociation constant, Kd, was determined to be 72 ± 5 nM using eq 1. Figure 1D shows the absorption spectra of the ferric, ferrous, and carbon monoxide (CO)-bound forms of hemin-reconstituted VcDyP. The Soret maximum of ferric VcDyP appeared at 405 nm with visible bands at 509 and 635 nm, indicating that the heme moiety of VcDyP is a five-coordinate high-spin moiety as observed in most heme peroxidases.30 Upon reduction of heme with sodium dithionite, the Soret band underwent a red shift to 431 nm, together with a marked increase in intensity around 559 nm with a small shoulder at 539 nm. This spectrum is characteristic of those of proteins with five-coordinate ferrous heme such as deoxymyoglobin.31 When CO was added to the ferrous form, the Soret maximum shifted to 422 nm with Q-bands at 539 and 571 nm, similar to those of CO-bound myoglobin. This property is typical of sixcoordinate CO-bound heme with His coordination31 and indicated that heme of CO-bound VcDyP possesses histidine as a ligand. Overall Structure of VcDyP. The crystal structure of VcDyP was determined by molecular replacement using phenix20 at a resolution of 2.2 Å. The structure of DyP from R. jostii RHA1 (PDB entry 3QNR)13 was used as a search probe. Crystallographic Rwork and Rfree converged to 23.6 and 24.8%, respectively. The data collection and refinement statistics are listed in Table 1. Figure 2A shows the determined X-ray structure of a subunit of VcDyP (PDB entry 5DE0). VcDyP assembles as a homodimer, which is in good agreement with the results estimated by gel filtration with an approximate molecular mass of ∼70 kDa for the wild-type enzyme. The dimensions of the monomer unit are roughly 55 Å × 35 Å × 30 Å. The overall structure, including the arrangement of catalytically important residues surrounding heme (panel B),

Table 1. X-ray Diffraction Data Collection and Refinement Statistics of VcDyP Data Collection beamline wavelength (Å) temperature (K) space group cell dimensions a, b, c (Å) α, β, γ (deg) resolution range (Å) total no. of reflections no. of unique reflections completeness (%) I/σ(I) Rsym redundancy

Photon Factory BL17A 0.9800 100 P32 77.7, 77.7, 156.9 90, 90, 90 41.3−2.24 (2.38−2.24) 296477 (46763)a 50860 (8179)a 99.8 (99.0)a 12.5 (2.4)a 12.0 (76.7)a 5.8 (5.7)a Refinement

Rwork Rfree root-mean-square deviation bond lengths (Å) bond angles (deg) no. of atoms protein water ligand Ramachandran plot (%) most favored additionally allowed

0.236 0.248 0.003 0.745 9260 252 172 97.46 2.54

a

Numbers in parentheses correspond to values in the highestresolution shell.

and an appropriate amount of guaiacol (5−150 μM) or 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) (10−100 μM) at 20 °C. The concentrations of the enzyme were 0.2 μM and 1.0 nM for guaiacol and ABTS, respectively. Reactions were initiated with the addition of H2O2, and initial rates for guaiacol and ABTS oxidation were monitored at 470 nm (ε470 = 22.6 mM−1 cm−1)22 and 414 nm (ε414 = 36.6 mM−1 cm−1),23 respectively. Data were fitted to the Michaelis−Menten equation.

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RESULTS Cloning, Expression, and Purification of VC2145. The VC2145 gene from V. cholerae, hereafter called VcDyP, was amplified by PCR and cloned into vector pET-28b, yielding a plasmid with VcDyP fused to an N-terminal His6 tag sequence. Recombinant VcDyP was expressed in E. coli strain BL21(DE3) and purified using nickel affinity chromatography. The His6 tag was removed by cleavage with protease followed by a nickel affinity column. Following removal of the His6 tag, hemin reconstitution, and size exclusion chromatography, only one band was observed on an SDS−PAGE gel, with an apparent molecular mass of approximately 35 kDa, very close to the mass predicted for VcDyP (33 kDa). Analysis of the purified VcDyP by size exclusion chromatography showed that the experimentally determined molecular mass of this protein was ∼70 kDa (Figure S1), indicating that the protein exists as a dimer in vitro. Although the DyP family contains both monomeric24,25 and oligomeric26,27 members, most bacterial DyPs are C

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Figure 1. (A) Absorption and (B) fluorescence spectra of VcDyP as purified. The protein concentration is ∼5 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). (C) Heme binding curve of VcDyP. (D) Absorption spectra of the heme−VcDyP complex. The protein concentration is ∼10 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). Spectra shown are for the ferric (), ferrous (···), and CO-bound (---) forms.

Figure 2. Crystal structure of VcDyP (PDB entry 5DE0). (A) α-Helices and β-strands of a VcDyP subunit are colored red and yellow, respectively. The heme is colored pink. (B) Close-up around the heme-binding site, including the catalytically relevant residues. Putative pH-dependent radical transfer pathway at lower pH (a) and higher pH (b).

folds, comprising antiparallel four-strand β-sheet and peripheral α-helices.7,32,33 The heme is almost completely buried in the

is the same as that of DyP from Bacteroides thetaiotaomicron.27,32 The VcDyP structure consists of two ferredoxin-like D

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Figure 3. Dye-decolorizing activity of VcDyP. (A) pH dependence of absorbance changes during the reaction of VcDyP (0.1 μM) with RB19 (40 μM) in the presence of H2O2 (0.2 mM) at 25 °C and pH 8.0, 7.0, 6.0, 5.0, and 4.0. (B) Kinetic analysis of the H2O2-dependent reaction of VcDyP. Initial velocity of degradation of RB19 at 25 °C as a function of the substrate. The data were fit to the Michaelis−Menten equation. Errors were determined from at least three independent trials.

suggests that VcDyP is less effective as a dye-decolorizing peroxidase.

protein and adopted a planar conformation with little displacement of the iron from the mean porphyrin plane (∼0.05 Å toward the proximal His). The side chain of His213 contributes to a proximal heme ligand, while no molecule serves as a distal ligand (Figure 2B). This coordination structure of heme without coordination of a water molecule corresponds to the spectroscopic characterization (Figure 1D). The distance between heme iron and Nε of His213 is 2.50 Å. The carbonyl oxygen of Asp272 is located at a hydrogen bonding distance of 2.63 Å to Nδ of His213. This means that the evolutionarily conserved hydrogen bonding between proximal His and Asp in most heme peroxidases34−36 is also conserved in VcDyP. The structure of the heme distal site showed that Asp144 is a sole charged residue whose side chain is oriented to the heme iron. This finding suggests that Asp144 plays an important role in the catalytic reaction. Arg230 is another polar residue in the heme pocket. Although the distal Arg is a highly conserved residue in most peroxidases,36 the guanidyl group of Arg230 points to a heme peripheral propionate group to make a hydrogen bond. Dye-Decolorized Activity of VcDyP. The H2O2-dependent dye-decolorizing activity of VcDyP was monitored by UV− visible absorption spectroscopy. In this study, the enzyme was rapidly mixed with the desired amount of a substrate dye, reactive blue 19 (RB19), in the presence of 200 μM H2O2, and the degradation of the dye was monitored over time by measuring the absorbance at 595 nm. Figure 3A shows a typical time course of this reaction. Because it has been reported that the catalytic activity of most DyP enzymes was dependent on the pH,24,37 the pH profile of the DyP activity for VcDyP using RB19 as a substrate was examined. As shown in Figure 3A, degradation of RB19 by VcDyP was accelerated at acidic pH, with the maximal decrease in absorbance at pH 4.0. This pH dependence of the DyP activity for VcDyP is similar to those observed for other DyP enzymes.6,15,24 The initial rates using various substrate concentrations at pH 4.0 are plotted in Figure 3B, and the kinetic parameters of the enzyme were calculated using the Michaelis−Menten equation. The kcat and Km were 1.3 ± 0.3 s−1 and 50 ± 20 μM, respectively (Table 2). The kcat for VcDyP is slightly smaller but the Km slightly larger than those reported previously,15,38 which

Table 2. Kinetic Constants (Km, kcat, and kcat/Km) for DyP on Reactive Blue 19 (RB19)a RB19 VcDyP TfuDyP AjPI

Km (μM)

kcat (s−1)

kcat/Km (s−1 μM−1)

ref

50 ± 20 29 6.5 ± 0.9

1.3 ± 0.3 10 35 ± 2

0.026 ± 0.016 0.35 4.8 ± 0.9

this study 15 38

a

Kinetic constants were obtained at the optimal pH. Abbreviations: Tf uDyP, DyP from Thermobif ida f usca; AjPI, DyP from Auricularia auricula-judae.

To examine the amino acid residues involved in the DyP activity, we focused on two residues (Asp144 and Arg230) in the heme distal pocket, because these two residues are conserved in almost all DyPs.14 Asp144 and Arg230 were substituted with Val and Leu, respectively. The time courses of degradation of RB19 for the D144V and R230L mutants are shown in Figure S2A. Both mutant enzymes almost completely lost the dye-decolorizing activity, indicating that both Asp144 and Arg230 are necessary for the DyP activity of VcDyP. We also focused on the histidine residue to examine its possible involvement in the pH dependence of the DyP activity. Because two His residues (His178 and His242) are located near heme (Figure 2B), we replaced each with Leu. While the H242L mutant retained the DyP activity, substitution of His178 resulted in a significant loss of activity, indicating that His178 is involved in the pH dependence of the DyP activity of VcDyP (Figure S2B). Peroxidase Activity of VcDyP. The peroxidase activity of VcDyP was monitored using guaiacol as a reducing substrate under the steady state condition. In the reaction of peroxidases with H2O2, guaiacol is reduced to guaiacol oligomer providing absorption at 470 nm.39 The obtained rate constants were plotted against substrate concentration and fit to the Michaelis−Menten equation (Figure 4A), where kcat = 0.027 ± 0.001 s−1 and Km = 10 ± 2 μM (Table 3). Although VcDyP is E

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The oxidation activity for ABTS was also measured to investigate the substrate specificity. Plots of the steady state activity as a function of ABTS concentration are shown in Figure 4B. Because the plot exhibited nonsaturation kinetics, precise kinetic parameters were not obtained. However, the kcat and Km values were estimated by fitting the data to the Michaelis−Menten equation and are listed in Table 3. kcat and Km were 500 s−1 and 180 μM, respectively. The kcat for ABTS is much faster than that for guaiacol in VcDyP and comparable to those of other peroxidases. Resonance Raman Spectra of VcDyP. Resonance Raman spectra of the ferric, ferrous, and CO-bound forms of VcDyP excited at 413.1 nm are shown in Figure 5. A heme skeletal marker band, ν3, at 1480−1510 cm−1 has been known to be sensitive to the spin state of the heme iron.40 The ν3 band of the ferric form of VcDyP was observed at 1491 cm−1 (Figure 5, spectrum d), which is typical of five-coordinate high-spin heme. The ν3 band of the ferrous form was observed at 1470 cm−1 (Figure 5, spectrum e), again suggesting that the heme is a fivecoordinate high-spin form in good agreement with the result of the absorption spectra (Figure 1D). Because resonance Raman spectra of ferrous five-coordinate high-spin heme provide the Fe−His stretching mode (νFe−His), which generally appears at 200−250 cm−1,41 we attempted to observe νFe−His of VcDyP. Spectra b and e in Figure 5 are the resonance Raman spectra of ferrous heme-reconstituted VcDyP with excitation at 441.6 nm. The intense band observed at 229 cm−1 would be the Fe−His stretching mode. The frequency of νFe−His reflects the hydrogen bonding status of the proximal imidazole with the surrounding amino acid residue.41 The frequency of 229 cm−1 is slightly higher than that of myoglobin (220 cm−1),42−44 but not as high as those observed for heme peroxidases (∼245 cm−1),45,46 suggesting that the proximal histidine forms a weaker hydrogen bond with an adjacent residue in comparison with those of other peroxidases. Although this is the first report about νFe−His of the DyP family, it has been reported that chlorite dismutase, which is a member of the DyP family, also has a weak hydrogen bond between the axial histidine and proximal glutamate (νFe−His = 224 cm−1).47 The resonance Raman spectra of the CO-bound form are also shown in Figure 5 (spectra c and f). The low-frequency spectrum shows an isotope-sensitive band at 504 cm−1, which was regarded as the νFe−CO stretching mode due to the 25 cm−1 isotope shift for the 13C18O-bound form (Figure 5, inset). To understand the preference of lower pH for the DyP activity of VcDyP, the νFe−CO band at pH 4.0 was compared with that at pH 8.0, because the frequency of νFe−CO is known to reflect the electrostatic interaction between iron-bound CO and surrounding amino acid residue(s) in the heme distal site.48 At pH 8.0, the νFe−CO band was broad with a peak around 504 cm−1 (Figure 6, spectrum a), which was comprised of a band at 504 cm−1 with a shoulder at 518 cm−1. At pH 4.0, the band at 518 cm−1 became dominant (spectrum c), indicating that the positive electrostatic interaction between iron-bound CO and the surrounding amino acid residue(s) was enhanced when the pH was lowered.48,49 To account for the difference in the reactivity of VcDyP to RB19 and guaiacol, substrate-dependent structural changes around heme were investigated using resonance Raman spectroscopy. The νFe−CO mode of VcDyP, which was observed at 518 cm−1 and pH 4.0 (Figure 6, spectrum c), was not affected by the addition of 1 mM RB19 (spectrum d). This result indicates that RB19 binds to the protein far from the

Figure 4. Peroxidase activity of VcDyP. (A) Absorbance changes during the reaction of VcDyP (0.2 μM) with guaiacol (5−150 μM) in the presence of H2O2 (0.2 mM) at 25 °C. (B) Absorbance changes during the reaction of VcDyP (1.0 nM) with ABTS (10−100 μM) in the presence of H2O2 (0.2 mM) at 25 °C. Initial velocity of degradation of RB19 at 25 °C as a function of the substrate. The data were fit to the Michaelis−Menten equation. Errors were determined from at least three independent trials.

Table 3. Kinetic Constants (Km and kcat) for Peroxidase Activities guaiacol

VcDyP LiP HRP CcP APX IDO Mb

ABTS

Km (μM)

kcat (s−1)

Km (μM)

kcat (s−1)

ref

10 ± 2.0 160 107 ± 6 53 × 103 12.2 NDb 3.2 × 104

0.027 ± 0.001 7.7 400 ± 25 4.3 ± 0.3 16.9 NDb 0.36 ± 0.01

180a 868 11.5 6.2 NDb 61 77

500a 210 52.5 15.5 NDb 61 0.43

this study 65, 66 67, 68 69 70 71 72, 73

a Km and kcat values were not determined because of nonsaturation of the enzyme, but catalytic efficiencies were estimated from the slope of the observed activity vs substrate concentration. bNot determined.

a member of the peroxidase family, the activity observed with guaiacol is much lower than those of plant-type peroxidases and comparative to those of myoglobin. F

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Figure 5. Resonance Raman spectra of VcDyP in the low-frequency (left) and high-frequency (right) regions. The protein concentration was 10 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). Spectra shown are those for the ferric (a and d), ferrous (b and e), and CO-bound (c and f) forms. The excitation wavelength for ferric and CO-bound forms is 413.1 nm, and that for the ferrous form is 441.6 nm. The inset shows a difference spectrum between CO-bound and 13C18O-bound forms.

suggests that guaiacol binds near the heme distal site, which affects the population of the two conformers of iron-bound CO. Cross-Linking of VcDyP by Reaction with H2O2. Both the crystal structure and resonance Raman spectra of VcDyP suggested that RB19 is not present near heme; rather, it is far from heme (Figures 2 and 6). To react with the substrate, the reactive species would be formed on the surface of the protein. Indeed, in the reaction of hemoproteins, such as myoglobin and hemoglobin, with H2O2, radical generated at the heme is transferred to the protein surface, where an intermolecular covalent bond is formed.50,51 Therefore, to confirm if a radical transfer from the heme to the protein surface is occurring, the formation of the covalent bond during the reaction of VcDyP with H2O2 was verified by SDS−PAGE analysis. Figure 7A shows the gel image of VcDyP after incubation with H2O2 for 20 min under various pH conditions. At pH 3−5, no bands except for the monomeric form (∼35 kDa) were observed, while at pH 6−10, a new band appeared at ∼70 kDa. This newly appearing band, which corresponds to dimer, suggested the formation of an intermolecular covalent bond. The amount of the band of ∼70 kDa depends on the pH with a maximum at pH 8.0. Tyrosine and tryptophan are possible amino acid residues that can be oxidized to be radical. There are nine Tyr and two Trp residues in the amino acid sequence of VcDyP. Therefore, we mutated each of the nine tyrosine and two tryptophan residues to identify the site of cross-linking. Neither Trp mutant (W64L and W183L) demonstrated any significant difference in the amount of dimer formation in the reaction with H2O2, whereas two Tyr mutants (Y109F and Y133L) almost completely lost the ability to form the dimer (Figure 7B,C). Because the crystal structure of VcDyP shows that both residues

Figure 6. Resonance Raman spectra of CO-bound VcDyP. The protein concentration was 10 μM in 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). The excitation wavelength is 413.1 nm: (A) pH 8.0, (B) pH 8.0 in the presence of 1 mM guaiacol, (C) pH 4.0, and (D) pH 4.0 in the presence of 1 mM RB19.

heme pocket. In contrast to RB19, the addition of guaiacol caused a slight enhancement of the 518 cm−1 band at the expense of the 504 cm−1 band (spectrum b). This change G

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alterations in the ability to decompose the dye or form crosslinks, are located at the dimer interface of VcDyP (Figure 8).

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DISCUSSION Reaction Mechanism of the Dye-Decolorizing Reaction by VcDyP. As predicted from the protein sequence, VC2145 of V. cholerae (VcDyP) has a dye-decolorizing activity (Figure 3). The catalytic activity of VcDyP depends on the pH. The greatest amount of decomposition was observed at pH 4.0, whereas almost no decomposition was observed at neutral pH. The frequency of νFe−CO of the CO-bound form is also pHdependent. The νFe−CO band of VcDyP is comprised of two bands exhibiting frequency maxima at 505 and 518 cm−1 (Figure 6). The intensity of the former band is slightly larger than that of the latter band at pH 8.0, whereas the latter band is dominant at pH 4.0. The frequency of νFe−CO is known to reflect the electrostatic interaction between iron-bound CO and the surrounding amino acid residue(s).48,49,52 The frequency of νFe−CO (504 cm−1) observed for VcDyP at pH 8.0 is roughly the same as that observed for sperm whale myoglobin,53 in which distal histidine interacts with the iron-bound CO.54 These results suggest that electrostatic interaction is imposed on the iron-bound ligand in the heme pocket of VcDyP at pH 8.0. Enhancement of the band with a higher frequency at pH 4.0 suggests the presence of additional positive charge near the heme distal ligand. The polar amino acid residues present in the heme pocket of VcDyP are only Asp144 and Arg230 (Figure 2A). The side chain of Asp144 points toward the heme iron, whereas that of Arg230 points toward the heme propionate, but not the heme iron. Thus, this frequency shift of νFe−CO when the pH is lowered indicates that Asp144 accepts a proton at lower pH, which imposes additional positive interaction on the heme-bound CO leading to the higher-frequency shift of νFe−CO. The crucial step of the binding of H2O2 to heme is deprotonation of H2O2 to form the HOO− anion, because H2O2 with a pKa of 11.6 exists predominantly as the H2O2 form, not as the deprotonated form, at a pH lower than its pKa value.55,56 Therefore, the presence of a proton acceptor inside the heme pocket is preferred during the H2O2-dependent reactions. After the hydroperoxy species (Fe3+−OOH) is formed, heterolytic cleavage of the O−O bond results in the formation of compound I (oxoferryl heme with porphyrin πcation radical) in most peroxidases. In this step, the donor of a proton to the hydroperoxy species is necessary for heterolytic cleavage. Therefore, formation of compound I requires the presence of an acid−base catalyst in the heme distal site. In most peroxidases, His works as a general acid−base catalyst.57,58 However, no His is present in the heme pocket of VcDyP (Figure 2). Instead, Asp144 and Arg230 are the only polar residues present in the heme pocket. The pKa values of the side chains of Asp and Arg in water are 3.9 and 12.5, respectively. The optimal pH of the DyP activity of VcDyP (Figure 3A) suggests that Asp144 rather than Arg230 of VcDyP would act as a general acid−base catalyst in the DyP reaction. Arg230 is another polar residue in the heme distal pocket of VcDyP. The distal Arg residue is highly conserved in most peroxidases, including those of a DyP family.36 The guanidyl nitrogen of Arg230 is located to make a hydrogen bond interaction with the heme propionate-8, and thus, it seems to be unrelated to the catalytic reaction. In cytochrome c peroxidase, an Arg is present in the heme distal pocket. The crystal structure of cytochrome c peroxidase in the resting state

Figure 7. SDS−PAGE analysis of cross-linking formation. The samples were reacted with H2O2. After 10 min, the reaction was stopped by addition of loading buffer: lane M, standard molecular mass marker; lane −, protein without H2O2 at pH 4. (A) pH dependence of VcDyP: lanes 3−10, VcDyP reacted with H2O2 at pH 3−10, respectively. (B) Y109F mutant. (C) Y133L mutant. (D) Y129L mutant. (E) Y235L mutant. The reaction of the mutant enzymes was conducted at pH 8.0. The following concentrations were used: 5 μM proteins, 312 μM H2O2, and 12.5% polyacrylamide.

are present at the dimer interface (Figure 8), we predicted that Tyr109 and Tyr133 are the radical sites for dye decolorizing.

Figure 8. Distribution of the catalytically relevant Tyr at the dimer interface.

To confirm this prediction, the DyP activity of the Y109F and Y133L mutants was investigated. Unexpectedly, both mutant enzymes retained the DyP activity (Figure S2C). In contrast, the Y129L and Y235L mutants, which retain the ability to form the dimer (Figure 7D,E), almost completely lost their DyP activity (Figure S2D). Interestingly, all these amino acid residues (Tyr109, Tyr129, Tyr133, and Tyr235), related to H

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However, even if the substrate is in this pocket, the high-valent iron−oxo species cannot react with it directly, because Asp144 and Arg230 are present between this site and heme. Therefore, it is likely that the heme distal pocket of VcDyP can provide room for small substrates such as guaiacol but cannot for larger substrates such as RB19 and ABTS. Thus, to degrade RB19, radical transfer from the porphyrin ring to amino acid residue(s) on the protein surface would be inevitable. The crystal structure and quantum mechanics/molecular mechanics calculation of DyP from A. auricula-judae predicated that oxidation of substrate occurs at solvent-exposed Trp, not Tyr.60 A combination of mutational study and EPR analysis confirmed that most dye-decolorizing activity DyP from A. auricula-judae is associated with Trp377.38 VcDyP has two tryptophan residues (Trp64 and Trp183). In contrast to DyP from A. auricula-judae, both Trp mutant enzymes (W64L and W183L) retained approximately half of the WT activity (Figure S2E), indicating that the contribution of Trp radical to the dyedecolorizing reaction of VcDyP would not be significant compared with those of other DyP proteins. In some hemoproteins, such as myoglobin and hemoglobin, it has been reported that radicals generated at the heme are transferred to Tyr on the surface.50,51 When sperm whale myoglobin was reacted with H2O2, dimer was formed through a covalent bond between Tyr103 and Tyr151.51 Via the treatment of VcDyP with H2O2, the SDS−PAGE gel showed the formation of cross-linking between proteins (Figure 7). Mutation of Tyr109 or Tyr133 leads to a complete loss of the band derived from dimer, indicating that the cross-link is formed between Tyr109 and Tyr133. The crystal structure clearly showed that Tyr109 faces Tyr133 in another subunit (Figure 8). The distance between heme and Tyr109 is 24.6 Å, while that between heme and Tyr133 is 17.9 Å. These results imply that radical generated at heme is transferred to Tyr133 first, which then oxidizes nearby Tyr109 from another subunit in an intermolecular radical transfer. The second radical generated at heme moves to Tyr133 to form a radical, which finally makes a cross-link with Tyr109 radical. This cross-linking reaction of VcDyP by H2O2 depends on the pH (Figure 7). The cross-linking was observed above pH 6, while no cross-linking was observed at pH 3−5. The DyP activity of VcDyP is also pH-dependent (Figure 3), and it is complementary to the pH dependence of cross-linking. The DyP reaction is active at pH 4−5, whereas cross-linking reaction occurs above pH 6. These results imply that radical is formed at heme at every pH, but the site where the radical is transferred is different in a pH-dependent manner. The Y109F and Y133L mutant enzymes, whose ability of dimer formation was lost, still have approximately half of the dye-decolorizing activity of the WT enzyme (Figure S2C). We concluded that neither Tyr109 nor Tyr133 is the active site of the DyP reaction. In contrast, Y129L and Y235L lost the DyP activity completely even though they retain cross-linking ability. The crystal structure of VcDyP shows that both Tyr129 and Tyr235 are located at the dimer interface but are slightly far from Tyr133 (Figure 8). These results indicate that the large, hydrophobic substrate binds to a space in the dimer interface but does not enter into the heme pocket. According to the ClustalW analysis of the amino acid sequence of DyPs, Tyr129 of VcDyP is not conserved even in class B whereas Tyr235 is highly conserved in the all classes of DyPs (Figures S4−S7). The ability to donate an electron from Tyr to the ferryl iron correlates with solvent exposure of the side chain.63 In sperm

showed that this Arg is in two different positions; one is in an “out” form, in which the side chain of the distal Arg is oriented toward the heme propionate, and the other is in an “in” form, in which the side chain points to the heme iron.59 In contrast, in the crystal structure of compound I, the distal Arg occupies only one conformation of the “in” form, because the positively charged arginine moves to the heme center to stabilize the oxoferryl species by hydrogen bonding.59 The loss of the DyP activity of the R230L mutant (Figure S2A) raises the possibility that the side chain of Arg230 in VcDyP is also flexible to stabilize the intermediate during the catalytic reaction as suggested for fungal DyP from Auricularia auricula-judae.60 As described above, two distal residues (Asp and Arg) are conserved in all DyPs.7,14,15 However, the roles of these residues are different in distinct DyP families. The distal Asp was proposed to act as an acid−base catalyst in the class D DyPs, while it is not essential for DyPs of classes A and B.7,16 Instead, the distal Arg is crucial for the catalytic activity. In the DyP from R. jostii RHA1 (hereafter called RjDyP), mutation of Asp153, which corresponds to Asp144 in VcDyP, had no effects on the catalytic activity while mutation of Arg244, which corresponds to Arg230 in VcDyP, completely eliminated the activity.16 Therefore, Singh et al. proposed that Arg244, not Asp153, is the acid−base catalyst with a pKa of ∼5.0. The structure of the heme distal site of VcDyP is compared with that of RjDyP in Figure S3. Asp153 of RjDyP contacts Asn246, which is replaced with Ser232 in VcDyP. A slightly larger space between Asp144 and Ser232 in VcDyP would relieve the strain of the side chain of Asp144, so that the side chain rotated slightly compared with that of Asp153 of RjDyP (Figure S3). Such a change causes displacement of the side chain of Asp144 closer to the heme iron by ∼0.3 Å (the distance between iron and Oδ of Asp is 4.74 Å for VcDyP but 5.05 Å for RjDyP). This location of the side chain would be adequate for Asp144 in VcDyP to work as an acid−base catalyst. Accordingly, both Asp144 and Arg230 are catalytically relevant in the case of VcDyP. Active Site of the Dye-Decolorizing Reaction. Addition of RB19 to CO-bound VcDyP had no influence on the frequency of νFe−CO (Figure 6), indicating that RB19 is not located inside the heme pocket. We predicted ligand-binding pockets of VcDyP using POCASA.61,62 The four large pockets are shown in Figure 9. Pocket 1 is closest to the heme.

Figure 9. Ligand-binding pocket predicted by POCASA. I

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molecular size as discussed above. If the radical transfer process is rate-limiting, it is not essential whether compound I or II is generated by the reaction with H2O2. Therefore, kcat for ABTS oxidation of VcDyP would be comparable to those of other peroxidases. This inference would be also applied to the DyP reaction for VcDyP. In summary, DyP from V. cholerae was expressed in E. coli, purified, and characterized in this study. The dye-decolorizing activity of VcDyP depends on the pH with maximum activity at pH 4. Asp144 and Arg230, which are the sole polar resides in the heme distal pocket and well conserved in all DyPs, are essential for the activity. The crystal structure and cross-linking reaction suggest that the radical sites formed by the reaction with H2O2 are dependent on the pH. The radical is transferred from heme to Tyr129 and Tyr235 at acidic pH, while to Tyr109 and Tyr133 at alkaline condition. His178 is a probable switch of the radical transfer pathway.

whale myoglobin, the solvent exposure of Tyr151, Tyr103, and Tyr146 was calculated to be 78.15, 35.12, and 2.44 Å2, respectively. Considering that the cross-link is formed between Tyr151 and Tyr103, solvent exposure of the side chain is a key factor for predicting a radical site. The solvent accessibility of each amino acid residue of VcDyP was calculated by GetArea.64 Among nine Tyr residues, only four possess ≥5% solvent accessibility: Tyr109 (40%), Tyr129 (22%), Tyr133 (38%), and Tyr235 (5%). Furthermore, the predicted pocket 3 is located at the subunit−subunit interface and surrounded by the amino acid residues containing Tyr129 and Tyr235 (Figure 9). These results support the idea that electrons are derived from Tyr109, Tyr129, Tyr133, and Tyr235, and protein radicals are formed at these residues. pH Dependence of the Dye-Decolorizing Reaction. The pH dependence of the DyP reaction and cross-linking activity suggests the pH-dependent switch of the radical transfer pathway. In the crystal structure of VcDyP, two histidine residues, His178 and His242, exist between heme and radical sites (Figure 2B). The DyP activity of the H178L mutant was ∼30% of that of WT (Figure S2B), indicating that mutation of His178 inhibits the radical transfer from heme to Tyr129 and/or Tyr235. In contrast, the formation of the crosslinking was not altered by the mutation of His178 (data not shown). The crystal structure shows that His178 makes a hydrogen bond with Thr277. Disruption of this hydrogen bond can serve as a trigger for a conformational change in a pHdependent manner. His178 would therefore act as a switch that possibly connects heme to the active site of the dyedecolorizing reaction (Tyr129 and Tyr235) (radical transfer pathway a in Figure 2B) or to the site of dimer formation (Tyr109 and Tyr133) (radical transfer pathway b in Figure 2B). In contrast to that of His178, mutation of His242 did not affect the DyP activity or cross-link formation (Figure S2B). His242 is slightly far from the lines connecting heme and Tyr129, and heme and Tyr235, suggesting that His242 is not involved in radical transfer from heme to Tyr129 or Tyr235. Peroxidase Activity of VcDyP. As discussed above, radicals that are generated at heme are rapidly transferred to the protein surface in VcDyP. At pH 3−5, radicals move to Tyr129 and Tyr235 and are utilized for the degradation of dyes. In contrast, they move to Tyr109 (and then Tyr133) at pH 6− 10 and are utilized for the formation of a covalent bond, although this reaction would not be biologically important. During the reaction with H2O2, the O−O bond of the hydroperoxy intermediate is cleaved and compound I or compound II is formed.65 In most peroxidases, the axial His makes a hydrogen bond with nearby Asp or Glu, which provides imidazolate character to the axial His.66 The strong electron donation from the axial His promotes heterolytic cleavage to form compound I rather than homolytic cleavage. Because compound I is more reactive to guaiacol, peroxidases prefer homolytic cleavage. The imidazolate character of the axial His is confirmed by the frequency of νFe−His of resonance Raman spectra. In most peroxidases, νFe−His is observed at ∼245 cm−1.45,46 However, the νFe−His of VcDyP is 229 cm−1 (Figure 5), which is far from those of peroxidases, indicating moderate electron donation from the axial His in VcDyP. Therefore, a lower peroxidase activity was predicted for VcDyP. Indeed, the kcat for guaiacol oxidation of VcDyP is much smaller than those of other peroxidases (Table 3). In the case of ABTS, the binding site is not near the heme site, probably the surface of the protein because of its larger

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b00952. Figures S1−S7 and Table S1 (PDF) Accession Codes

PDB entry 5DE0 (dye-decolorizing peroxidase from V. cholerae).

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +81-11-706-3501. E-mail: [email protected]. jp. Funding

This study was supported in part by Grants-in-Aid for Scientific Research (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. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DyP, dye-decolorizing peroxidase; PPIX, protoporphyrin IX; RB19, reactive blue 19. REFERENCES

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