Hemozymes Peroxidase Activity Of Artificial Hemoproteins

Bioconjugate Chem. 2008, 19, 899–910

899

Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces liWidans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins Rémy Ricoux,† Roger Dubuc,‡ Claude Dupont,‡ Jean-Didier Marechal,§ Aurore Martin,† Marion Sellier,† and Jean-Pierre Mahy*,† Institut de Chimie Moléculaire et des Matériaux d’Orsay, UMR 8182 CNRS, Laboratoire de Chimie Bioorganique et Bioinorganique, Bât. 420, Université Paris XI, 91405 Orsay Cedex, France, Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec, H7V 1B7, Canada, and Unitat de Química Física, Departament de Química, Universitat Autònoma de Barcelona, Edifici C.n., 08193 Cerdonyola (Barcelona), Spain. Received November 28, 2007; Revised Manuscript Received January 19, 2008

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-R4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 µM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV–visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.

Hemoproteins not only play a key role in the transport (hemoglobin) and storage (myoglobin) of dioxygen, the most abundant element of our environment, but some of them are also able to catalyze its activation (cytochromes P450) or to control the level of some of its reductive metabolites such as hydrogen peroxide (peroxidases, catalases). Using, respectively, dioxygen and hydrogen peroxide as oxidant, cytochromes P450 and peroxidases are able to catalyze, under biological conditions, the oxidation of numerous endogenous substrates including steroids, fatty acids, prostaglandins, and leukotrienes, as well as exogenous substrates such as drugs, insecticides, solvents, and hydrocarbons, even the most poorly reactive ones such as n-alkanes. Consequently, the design of artificial metalloenzymes that are able to efficiently catalyze oxidation reactions under mild conditions and with selectivities that are comparable to those exhibited by these enzymes is of great interest for the study and prediction of the oxidative metabolism of new biologically active molecules such as drugs. In addition, it represents a promising route to catalytic systems that can be used in organic synthesis for oxidation reactions that are * Corresponding author. Jean-Pierre Mahy, Institut de Chimie Moléculaire et des Matériaux d’Orsay, UMR 8182 CNRS, Laboratoire de Chimie Bioorganique et Bioinorganique, Bât. 420, Université Paris XI, 91405 Orsay Cedex, France, Fax: [33] 1 69 15 72 81, E-mail: [email protected]. † UMR 8182 CNRS. ‡ INRS-Institut Armand-Frappier. § Universitat Autònoma de Barcelona.

important in industrial and fine chemistry, such as the stereoselective oxidation of alkanes and epoxidation of alkenes (1). Considering the structure–activity relationships of hemoproteins, at least three structural elements have to be mimicked to design functional biomimetic systems (Figure 1): (i) the prosthetic group, a heme or iron(III)-protoporphyrin IX, which is responsible for electron transfer (peroxidases) (2) or oxene transfer reactions (cytochromes P450) (1, 3); (ii) the apoprotein which binds and site-isolates heme, preventing its aggregation and oxidative degradation, and its active-site amino acids, which not only provide a hydrophobic environment for the substrate and control its access to the heme (cytochromes P450), but also participate in some cases in catalysis (peroxidases) (2); (iii) the proximal axial ligand of the iron atom (histidine in peroxidases or cysteinate in cytochromes P450) which has a role in controlling the heme redox potential and the reactivity of the high-valent iron-oxo intermediates involved in their catalytic cycle (4). In the past 10 years, several strategies have been reported for the design of artificial metalloproteins, either to increase or modify the reactivity of an already existing metalloprotein or to induce a new reactivity in a nonmetal protein. They include the introduction of metal binding sites in the active site of a protein by site-directed mutagenesis (5–7), the design of substrate binding cavities (8–11), chemical modification of prosthetic groups (12–15), and covalent attachment of metal cofactors (16–20). In particular, the covalent modification of proteins through the covalent binding of a metal cofactor to a cysteine residue is a powerful tool for the generation of new

10.1021/bc700435a CCC: $40.75  2008 American Chemical Society Published on Web 03/07/2008

900 Bioconjugate Chem., Vol. 19, No. 4, 2008

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Figure 1. Schematic view of the structure of hemoproteins and their hybrid metalloporphyrin-Xln10A mimics: hemozymes.

metalloenzymes, although the efficiency of the modification is greatly dependent on the position and reactivity of the cysteinyl thiol functional group. However, on one hand, artificial hemoproteins with interesting peroxidase activities have also been obtained by the noncovalent association of a synthetic Fe(III)-R3β-tetra-o-carboxyphenylporphyrin (21–25) or microperoxidase 8 (MP8) (26–29) with monoclonal antibodies raised against these cofactors. The later MP8-antibody complexes were additionally shown to catalyze the stereoselective oxidation of sulfides by H2O2 (29). Furthermore, new artificial hemoproteins, also displaying interesting peroxidase activities, were obtained by insertion of modified hemes into mutated sperm whale myoglobin (30–33). Heat-resistant hemoproteins that are able to bind and release O2 in an aqueous medium like hemoglobin and myoglobin were also obtained by incorporating Fe(II)-R4-tetra-o-pivalamidophenylporphyrin into a thermoresistant xylanase (34, 35) or into human serum albumin (HSA) (36). In order to generate new hemoproteins, we have thus chosen a simple strategy that involves the noncovalent incorporation of Fe(III)-R4-tetra-o-carboxyphenylporphyrin (Fe(ToCPP)), Fe(III)-tetra-p-carboxyphenylporphyrin (Fe(TpCPP)), and MP8 (Scheme 1) into β-1,4-endoxylanase or xylanase A (Xln10A) from Streptomyces liVidans. Xylanase A hydrolyzes β-1,4 bonds in the main chain of xylan, the major component of hemi cellulose in the plant cell wall. In the pulp bleaching process, the enzymatic hydrolysis of xylan at high temperature has gained importance from an environmental point of view. Consequently, growing interest in xylanases that are able to work at high temperatures under extreme pH and ionic strength conditions has led to the discovery, cloning, and characterization of numerous enzymes, such as xylanase A from Streptomyces liVidans, which shows optimal activity at 60 °C and pH 6. This enzyme is a 47 kDa

protein which contains two domains: a 14 kDa C-terminal domain, named XBD, which belongs to the CBM13 family of carbohydrate binding modules for the binding of the substrate; and a 33 kDa N-terminal catalytic domain belonging to the GH10 family of glycosyl hydrolases. The three-dimensional structure of this domain has been elucidated and refined to a resolution of 1.2 Α (37). It presents a standard (β/R)8 TIMbarrel fold that contains the two amino acids that are essential for catalysis, Glu 128 and Glu 236. The catalytic site globally appears as a crevice that is rich in positively charged amino acids. Thus, the porphyrins Fe(ToCPP), Fe(TpCPP), and MP8 were chosen because of the negative charges of their carboxylate substituents, which should be complementary to the positive character of the active site of Xln10A. Furthermore, these groups render the porphyrins more water-soluble. Our results show that, among the three iron-porphyrins studied, Fe(TpCPP) is the porphyrin which inserts the best in Xln10A with a KD value of 0.5 µM, and the corresponding Xln10A-Fe(TpCPP) complex shows an interesting peroxidaselike activity characterized by a kcat value of 52 min-1 with o-dianisidine as cosubstrate. In the hybrid hemoprotein obtained, the Xln10A protein brings two interesting features that would be useful in its application as catalyst for selective oxidation reactions: first, it protects the porphyrin macrocycle from oxidative degradation, and second, it confers steric hindrance around the iron atom. In addition, pH dependence studies show that an amino acid side chain characterized by a pKA value of about 7.5 could be participating in the catalysis.

EXPERIMENTAL SECTION Preparation of Fe(III)-Porphyrins. Purification of Microperoxidase 8. Microperoxidase 8 (MP8) was prepared by sequential peptic and tryptic digestion of horse-heart cytochrome

Peroxidase-Like Fe-Porphyrin-Xylanase A Complexes

Bioconjugate Chem., Vol. 19, No. 4, 2008 901

Scheme 1. Structure and Nomenclature of the Porphyrins Discussed in This Paper

c (Sigma) as previously described (38). The heme content was determined by the pyridine chromogen method (38). The purity of the sample was over 97% based on MALDI-TOF mass spectroscopy analysis. Preparation of Fe(III)(ToCPP) and Fe(III)(TpCPP). The synthesis of the four atropoisomers of iron(III)-meso-tetrakis(ortho-carboxy-phenyl)porphyrin Fe(ToCPP) as well as that of iron(III)-meso-tetrakis(para-carboxy-phenyl)porphyrin Fe(TpCPP) was carried out in three steps as described in a previous paper (40). The ortho-carboxymethyl- and para-carboxymethylsubstituted tetraaryl porphyrins were synthetized by reaction at room temperature of ortho- and para-carbomethoxybenzaldehyde, respectively, with pyrrole in CH2Cl2 in the presence of BF3-etherate as a catalyst according to an already described procedure (39, 40). The atropoisomers of meso-tetrakis(orthocarboxymethyl-phenyl)porphyrin (ToCMePP) were then separated on a silicagel column and have been identified, as was meso-tetrakis(para-carboxymethyl-phenyl)porphyrin (TpCMePP), by UV–visible absorption, 1H NMR, and mass spectroscopies (40). The iron atom was inserted by reaction of (TpCMePP) and the isolated R4-(ToCMePP) atropoisomer with Fe(CO)5 in the presence of I2 in toluene at room temperature to avoid isomerization (41). Finally, the para- and R4-orthocarboxy-substituted tetraarylporphyrin isomers were subsequently obtained by saponification of the methyl ester substituents in 2 M KOH in 80% EtOH at room temperature (42). Production and Purification of Xylanase A (Xln10A). Seven-day-old cultures of S. liVidans from Bennett-thiostrepton plates were used as the initial inoculum. The spores were scraped from the plates and inoculated into 12.5 mL minimal M14 medium (composition per liter: glucose, 10 g; K2HPO4, 5.0 g; (NH4)2SO4, 1.4 g; KH2PO4, 1.0 g; CaCl2 · 2H2O, 300 mg; MgSO4 · 7H2O, 300 mg; FeSO4 · H2O, 5.0 mg; CoCl2 · 6H2O, 2.0 mg; MnSO4 · H2O, 1.6 mg; ZnSO4 · 7H2O, 1.4 mg; Tween 80, 2.0 mL; pH 7.4) and incubated for 18 h at 34 °C with agitation. Bacteria were recovered by centrifugation, used to inoculate 500 mL of the same medium, and allowed to grow for 72 h under the same conditions. Proteins contained in the supernatant of S. liVidans culture were first concentrated by ultrafiltration with a 3 kDa cutoff membrane (Omega). The concentrated proteins were then loaded at 4 °C on a Sephacryl S100 beaded column (2.6 × 60 cm; Pharmacia) with 100 mM sodium phosphate pH 6.0, as the eluant. Purified Xln10A containing fractions were pooled, dialyzed, and freeze-dried.

Assay of Xylanase Activity. The glycosidic activity of Xln10A and of the Xln10A-Fe(TpCPP) complex enzyme was determined using a diffusion agar assay. 100 µL of a 1 µM enzyme solution in 50 mM PBS pH 7.5 were poured into 5 mm wells coated with RBB (Remazol-Brillant-Blue)-xylan and incubated at 37 °C for 2, 6, and 24 h. At each time, the diameter of the clearing zone around the well was measured and was indicative of the glycosidic activity. Absorption Spectroscopy Measurements. Absorption spectra were recorded at 25 ( 0.1 °C, using an UVIKON 860 XL UV–visible spectrophotometer. For the insertion of Fe(III)-porphyrins into Xln10A, a typical procedure was followed. The Xln10A-Fe-porphyrin complexes were prepared in 50 mM PBS, pH 7.5, by incubation for 2 h at 4 °C of 1.2 µM MP8, Fe(III)(ToCPP), or Fe(III)(TpCPP) with 1.8 µM Xln10A. The UV–visible characteristics of the Xln10AFe-porphyrin complexes obtained were recorded at 25 ( 0.1 °C and compared to those of the MP8, Fe(III)(ToCPP), and Fe(III)(TpCPP) recorded under the same conditions. In the case of Fe(III)(TpCPP), a titration with Xln10A was done as follows: 0.97 µM Fe(III)(TpCPP) in 50 mM PBS, pH 7.5, was incubated for 20 min with Xln10A at concentrations increasing from 0.05 to 4.96 µM and visible spectra were recorded between 350 and 650 nm. The absorbance at 412 nm was plotted against the Xln10A/Fe-porphyrin ratio, which allowed the determination of the Xln10A/Fe-porphyrin stoichiometry and the value of the dissociation constant (KD). For the reactions with imidazole, difference spectroscopy was used. The sample cuvette contained either 1.85 µM Fe-porphyrin or 1.85 µM Fe-porphyrin preincubated for 40 min at 4 °C with 1.85 µM Xln10A in 50 mM PBS, pH 7.5. The reference cuvette contained 50 mM PBS, pH 7.5. Equal increasing amounts of imidazole (2 M in the same buffer) were then added to both cuvettes, so as to obtain a final concentration ranging between 0.33 and 25 mM, and difference spectra were recorded between 350 and 650 nm. In most cases, the spectral evolution observed involved the formation of well-defined isobestic points indicating the presence of two absorbing species. The reaction could then be represented by (1) PFeIII + nL h PFeIII(L)n P ) MP8, ToCPP, TpCPP, Xln10A-MP8, Xln10A-ToCPP, Xln10A-TpCPP


Ricoux et al.

L ) imidazole According to Brault and Rougee (43), it could then be analyzed by means of the standard equation 1 ⁄ ∆A ) 1 ⁄ ∆A∞ + KD ⁄ ∆A∞ × 1 ⁄ [L]n

(2)

where ∆A ) A - A0, ∆A∞ ) A - A∞, and A0, A∞, and A are the absorbances of the initial, final, and mixed species, respectively. The linearity of the graph representing 1/∆A as a function of 1/[L]n was then assayed with n ) 1 and n ) 2, and KD and A∞ could be determined graphically. It is noteworthy that, when n ) 1, C50 ) KD, whereas when n ) 2, C50 ) KD1/2, with C50 representing the concentration of ligand for which half of the starting Fe-porphyrin or Fe-porphyrin-Xln10A complex has been converted into porphyrin-Fe(L)n or Xln10A-porphyrinFe(L)n. Assay of Peroxidase Activity. To assay the peroxidase activity of Fe(TpCPP) and its complex with Xln10A, the oxidation of cosubstrates, namely, guaiacol, o-dianisidine and 2,6-dimethoxyphenol, by H2O2 was performed at 19 ( 0.1 °C in 50 mM PBS, pH 7.5. The absorbance was monitored for 6 min, respectively, at 490, 450, and 469 nm using an UVIKON 860 UV–visible spectrophotometer. The initial rates of oxidation were determined from the slope at the origin of the curve representing the variations of the absorbance at 490, 450, and 469 nm as a function of time, using respective values of 5500, 7500, and 27 500 M-1 cm-1 (26, 44). The influence of the H2O2 concentration on the oxidation of the cosubstrates was assayed. For this, guaiacol, o-dianisidine, and 2,6-dimethoxyphenol (1.2 mM) were oxidized by H2O2 at concentrations varying between 0.080 and 8 mM, in the presence of 1.2 µM Fe(TpCPP) or 1.2 µM Fe(TpCPP) preincubated for 2 h at 4 °C with 1.8 µM Xln10A as catalyst. In a second set of experiments, the influence of the cosubstrate concentration on the rate of oxidation was assayed. The cosubstrates, at concentrations varying between 50 and 1200 µM, were oxidized by H2O2 2.4 mM in the presence of 1.2 µM Fe(TpCPP) or 1.2 µM Fe(TpCPP) preincubated for 2 h at 4 °C with 1.8 µM Xln10A as catalyst. For the pH dependence studies, solutions of 1.2 µM Fe(TpCPP) or 1.2 µM Fe(TpCPP) preincubated for 2 h at 4 °C with 1.8 µM Xln10A were prepared in 0.1 M citrate/phosphate buffer pH 4.1, 4.6, 5.0, 5.5, 6.0, 6.5, and 7.0, and in 0.1 M Tris-HCl buffer pH 7.3, 7.6, 7.9, and 9.1. 890 µM o-dianisidine was first added and the reaction was initiated by the addition of 320 µM H2O2. The absorbance was monitored at 450 nm as a function of time for 5 min, and the initial rate of oxidation was plotted as a function of the pH value. The influence of imidazole on the kinetic parameters of the oxidation of o-dianisidine by H2O2 in the presence of 1.2 µM Fe(TpCPP) or 1.2 µM Xln10A-Fe(TpCPP) prepared as described above was examined as follows. The catalyst was further incubated for 2 h at 4 °C with imidazole at concentrations ranging between 0 and 50 mM. 890 µM o-dianisidine was then added, and the reaction was started by the addition of 320 µM H2O2. The initial rates of oxidation were then measured as described above. Molecular Modeling. The atomic coordinates of Xln 10A were downloaded from the Protein Data Bank (45) (PDB code 1V0L) (46). The exploration of the protein cavity volumes and shapes were performed with CASTp (47) and Q-sitefinder (48) webserver. The structures of the different meso-tetraphenyl-porphyrins have been generated using the Chem3D package (CambridgeSoft Corporation). A short minimization was performed on each compound using the MM2 force field (49) implemented in Chem3D. For the MP8 system, the environment of the heme has been generated from one of the crystal structures available

Figure 2. Incorporation of Fe(TpCPP) into Xln10A: ( - - - ) UV–visible spectrum of Fe(TpCPP), 1.2 µM in 50 mM PBS, pH 7.5; (s) UV–visible spectrum obtained after addition of 1.8 µM Xln10A corresponding to the Xln10A-Fe(TpCPP) complex. Inset: variations of the R,β bands under the same conditions.

of the human cytochrome C (PDB code 1CRI (50)). The minimization was followed by a short molecular dynamic run to better relax the system. All the volumes of the ligands have been calculated using the Connolly solvent excluded volume package included in Chem3D. Docking studies were performed using the GOLD v3 program (51) with the Chemscore (52) fitness function. The accessible docking space was defined as a 20 Å sphere around one of the residues at the core of the cleft. Fifty solutions were generated for each porphyrin macrocycle and ranked according to their Chemscore values. We selected this approach since it had been applied with success in predicting protein–ligand interactions for metal containing systems like cytochrome P450 (53). Since the ligand remains quite exposed to the solvent, a large number of low-energy solutions were analyzed in detail. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (54).

RESULTS Insertion of Fe(III)-Porphyrins into Xylanase A. The insertion of three Fe-porphyrins bearing negatively charged carboxylate substituents was examined by UV–visible spectroscopy as described in the experimental section. For this, 1.5 mol equiv of Xln10A was added to solutions of either microperoxidase 8 (MP8), Fe(TpCPP), or Fe(ToCPP), 1.2 µM in 50 mM PBS pH 7.5, a concentration for which no aggregation of the porphyrin complexes was observed. In the case of microperoxidase 8 (MP8), no significant change in its UV–visible spectrum was observed, except for a decrease in intensity of its Soret band at 397 nm (data not shown). This is characteristic of a simple hydrophobic interaction between MP8 and Xln10A with no amino acid side chain of the protein binding to the iron atom of MP8, leaving a vacant coordination site on the iron available for the binding of exogenous ligands and catalysis. Similarly, the addition of Xln10A to Fe(ToCPP) caused only a decrease of its Soret band at 396 nm (data not shown), showing that no amino acid side chain of Xln 10A was coordinating the iron atom in this complex either, leaving two vacant axial coordination positions on the iron atom. Finally, the addition of 1.5 equiv Xln10A to Fe(TpCPP) 1.2 µM in 50 mM PBS, pH 7.5, was examined by UV–visible spectroscopy. The spectrum for Fe(TpCPP) before the addition of Xln10A (with maxima at 409, 567, and 611 nm) was characteristic of a high-spin pentacoordinate porphyrin-iron(III) species (Figure 2). A clear change in this spectrum was observed on addition of Xln10A, with one clear isosbestic point at 445 nm and two others, less clear, at around 575 and 612 nm, leading to a final spectrum with maxima at 412, 570, and 614 nm for the corresponding Xln10A-Fe-porphyrin complex. In addition,



Figure 3. Titration of Fe(TpCPP) by Xln10A: evolution of the UV–visible spectrum of Fe(TpCPP), 0.97 µM in 50 mM PBS, pH 7.5, after addition of Xln10A at concentrations increasing from 0.05 µM to 4.96 µM. Inset: Double reciprocal plots of the absorbance at 412 nm versus the Xln10A/ Fe-porphyrin ratio.

these changes in the UV–visible spectrum were independent of the pH, as similar variations in the spectra were recorded at pH 4.0, 5.1, 6, 6.6, 7.6, and 10.6 (data not shown). Since the final spectrum is also characteristic of a high-spin pentacoordinate iron(III)-porphyrin complex, this clearly indicates that the environment of the iron atom changes upon insertion of Fe(TpCPP) into Xln10A, which could be due to the binding of an amino acid side chain of Xln10A to the iron atom. A titration of Fe(TpCPP), 0.97 µM in 50 mM PBS, pH 7.5, with Xln10A at concentrations increasing from 0.05 to 4.96 µM was performed (Figure 3). Double reciprocal plots of the absorbance at 412 nm versus the Xln10A/Fe-porphyrin ratio (Figure 3, inset) allowed us to determine a 1/1 Xln10A/Feporphyrin stoichiometry and a dissociation constant value KD ) 0.5 µM. This value was about 5-fold lower than those found for complexes of Fe(II)-2-[8-(2-methylimidazolyl)octanoyloxymethyl]-tetrakis-R4-(o-pivalamidophenyl)porphyrin with xylanases from Thermotoga maritima (KD ) 2.2 µM) and Dictioglomus thermophylum (KD ) 2.9 µM) (34, 35) and was in agreement with a good affinity of Xln10A for Fe(TpCPP). This difference in affinity could be due to the rather flat shape of Fe(TpCPP) that better entered the cleft of Xln10A than the more bulky pivaloyl porphyrin did in the cleft of xylanases from Thermotoga maritima and Dictioglomus thermophylum. Coordination of Imidazole on Fe(III)-Porphyrins and Their Complexes with Xylanase A. In order to evaluate the size of the cavity around the iron atom in the complexes of xylanase A with the three iron(III)-porphyrins, the coordination of imidazole on the iron atom was examined. To this effect, the progressive addition of imidazole (0–95 µM) to solutions of MP8, Fe(ToCPP), Fe(TpCPP), and the corresponding Fe(porphyrin)-Xln 10A complexes, 1.85 µM in 50 mM PBS, pH 7.5, was analyzed by differential UV–visible spectroscopy as described in the Experimental Section. In all cases, the progressive appearance of a new spectrum, characteristic of an iron(III)imidazole species, was observed (Table 1). The particular case of Fe(TpCPP) and Xln10A-Fe(TpCPP) is shown in Figure 4. In the case of Fe(TpCPP), a new spectrum with absorption maxima at 412, 545, and 581 nm and isobestic points at 402

Table 1. Visible Characteristics, KD and C50 Values of the Complexes of Fe(porphyrin) and Xln10A-Fe(porphyrin) with Imidazole in 50 mM PBS, pH 7.5, at 20 °C Fe(porphyrin) or Xln10A+Fe(porphyrin) MP8 Xln10A+MP8a Fe(ToCPP) Xln10A+Fe(ToCPP)a Fe(TpCPP) Xln10A-Fe(TpCPP)b complex

number of imidazole ligands λmax (nm) C50 (mM) 1 1 2 2 2 1

403 402 413 412 415 417

0.05 0.06 0.69 0.67 3.4 3.3

KD 0.05 mM 0.06 mM 0.48 mM2 0.49 mM2 11.6 mM2 3.3 mM

a In these cases no complex coming from the insertion of MP8 or FeToCPP into the cleft of Xln10A was obtained. b Xln10A-Fe(TpCPP) was formed by insertion of FeTpCPP into the cleft of Xln10A.

and 439 nm is formed, whereas in the case of Xln10AFe(TpCPP), a new spectrum with absorption maxima at 415, 538, and 579 nm and isobestic points at 400 and 420 nm appears upon addition of imidazole. The inverse of the absorbance at the absorption maximum of the Fe-imidazole complex, 1/∆Aλmax, was then plotted against the inverse of the concentration of imidazole added, 1/[ImH], and against the reverse of the square of the concentration of imidazole added, 1/[ImH]2, in order to determine whether one or two imidazoles were bound per iron atom and the corresponding KD value, as described in the Experimental Section. With both MP8 and Xln10A-MP8, 1/∆A403 varied linearly as a function of 1/[ImH] and not as a function of 1/[ImH]2, which showed that only one imidazole was bound per iron atom in both cases. In addition, similar KD values of 0.05 ( 0.01 and 0.06 ( 0.01 mM, respectively, were calculated for both complexes, which suggested that the xylanase protein had no effect on the binding of imidazole as a sixth ligand, trans to its histidine on the iron atom in microperoxidase 8. With both Fe(ToCPP) and Xln10A-Fe(ToCPP), 1/∆A413 varied linearly as a function of 1/[ImH]2 and not as a function of 1/[ImH], which showed that two imidazole ligands were bound per iron atom in both complexes. Here also, similar KD values of 0.48 ( 0.01


Ricoux et al.

Figure 4. (A) Addition of imidazole to iron(III)-meso-tetrakis(para-carboxyphenyl)porphyrin (Fe(TpCPP)). Spectral evolution observed for the addition of 0 to 20 mM ImH to 1.85 µM Fe(TpCPP) in 50 mM PBS, pH 7.5, at 20 °C. Inset: corresponding values of 1/∆A412 plotted against 1/[ImH] (1) and 1/[ImH]2 (2). (B) Addition of imidazole to the iron(III)-meso-tetrakis(para-carboxyphenyl)porphyrin Fe(TpCPP)-Xln10A complex. Spectral evolution observed for the addition of 0 to 20 mM ImH to 1.85 µM Fe(TpCPP)-Xln10A in 50 mM PBS, pH 7.5, at 20 °C. Inset: corresponding values of 1/∆A412 plotted against 1/[ImH] (3) and 1/[ImH]2(4).

and 0.49 ( 0.01 mM2, respectively, were calculated for both complexes, again suggesting that the xylanase protein had no effect on the binding of two imidazole ligands on the iron atom to give a hexacoordinate (ToCPP)Fe(ImH)2 complex. In the case of Fe(TpCPP), 1/∆A413 varied linearly as a function of 1/[ImH]2 (Figure 4), whereas in the case of Xln10A-Fe(TpCPP), 1/∆A413 varied linearly as a function of 1/[ImH] (Figure 4). This showed that, whereas free Fe(TpCPP) was able to bind two imidazole ligands as expected, Fe(TpCPP) incorporated into xylanase A was able to bind only one. It is thus likely that the xylanase protein brings steric hindrance around the plane of the porphyrin, leaving only one face accessible to ligands to coordinate to the iron atom. Molecular Modeling. A series of molecular modeling studies have been carried out to address two major issues: (1) the characterization of the most probable binding site of the iron(III)-porphyrin complexes into the xylanase A structure and (2) the nature of the most stable binding modes of the Xln10Airon(III)-porphyrin complexes when MP8, Fe(ToCPP), and Fe(TpCPP) are inserted into Xln10A. The three iron(III)-porphyrin complexes discussed in this work have molecular structures substantially different from the natural substrate of Xln10A. Therefore, reasonable doubt exists as to whether the binding site of our porphyrin derivatives is the same as that of xylanes. We investigated this question by characterizing all possible accessible cavities of the Xln10A structure using a combination of different methods designed for this task. All the results agreed in predicting that the xylanase cleft is by far the largest accessible cavity. For example, the Q-sitefinder algorithm (48) showed that the cleft had a volume of 338 Å3 (summing the volume of its different subpockets), while the three next cavities in size had volumes of 145, 100, and 98 Å3, respectively. Since the porphyrin complexes are quite large (the approximated volume of the Fe(ToCPP) and Fe(TpCPP) entities is around 600 Å3), the Xln10A cleft appears as

Figure 5. Upper view of the Xln10A cleft. Secondary structures of the protein are depicted in ribbon format. The solvent-accessible area of the cleft is shown in gray scale.

the most probable binding site for these compounds, though they are not expected to entirely fit into it. For MP8, not only its volume (approximately 1400 Å3) but also its shape (mostly globular) suggests that its incorporation into this site is extremely poor. The optimized structure of the MP8 complex shows that this complex has a globular form, which suggests a very small complementarity of form with the narrow channel the cleft displays (Figure 5). This is in agreement with the experimental data that show that the activity of MP8 is not altered in solution in the presence of Xln10A. On the contrary, the volumes of Fe(ToCPP) and Fe(TpCPP) (614 and 613 Å3, respectively), as well as their planar shapes,


Figure 6. Best binding mode of the Fe(TpCPP)-Xln10A complex obtained using Gold with the Chemscore scoring function. The main interactions between the carboxylates of the Fe(TpCPP) and the polar patches {His80, Lys248, Trp266} (A) and {Ser212, Tyr172, Asn173} (B) are indicated.

suggest a substantial insertion of both iron(III)-porphyrin complexes into the cleft. To investigate the nature of the molecular complexes between these porphyrin derivatives and Xln10A, a series of molecular docking experiments were performed. From the 50 solutions obtained with the docking program Gold using the ChemScore scoring functions, striking differences in the structural properties of the predicted complexes were observed. In general, all 50 solutions obtained from the docking of Fe(TpCPP) into Xln10A present a similar structural behavior. In low-energy solutions, two aromatic groups and part of the porphyrin ring are substantially anchored into the cleft. A strong hydrogen bonding network is observed between the carboxylates of two consecutive aromatic substituents of Fe(TpCPP) and the protein. One of the carboxylates interacts with a patch of three residues; two positive residues {His80, Lys48} and Trp266 and a second with another polar patch of three residues at the site opposite to the cleft {Ser212, Tyr172, Asn173} (Figure 6). It is noteworthy that a hydrogen bond is also frequently observed between a third carboxylate and Gln94: a residue that lies at the very surface of the protein (Figure 6). Finally, the general form of the Fe(TpCPP)-Xln10A complex shows the porphyrin tilted with respect to the average plane represented by the surface of the protein, and therefore, only one face of Fe(TpCPP) seems accessible to the solvent (Figure 7a). The solutions obtained from the docking of Fe(ToCPP)Xln10A complexes have a large structural variability, which depends mainly on which face of the porphyrin is occupied by the carboxylates. Although it is well-known that in mesotetraphenyl-porphyrins the complete rotation of the phenyl is energetically demanding, our dockings have been performed allowing free rotation around the Cmeso-phenyl bond in order to better explore of the conformational space. As a general feature, Fe(ToCPP)-Xln10A interactions are mainly based on hydrophobic contacts and have few hydrogen bond interations with the Xln10A cleft. In fact, Fe(ToCPP) appears to be unable to penetrate deeply into the cleft, and protein–ligand interactions mainly take place at the external part of the site (Figure 7b). At most, only one aromatic moiety fits into the cleft and most of the porphyrin remains exposed to the solvent. None of the lowenergy Fe(ToCPP)-Xln10A complexes present an interaction of the carboxylates with the {His80, Lys48, Trp266} patch. The comparison of calculated Fe(ToCPP)-Xln10A and Fe(TpCPP)-Xln10A structures suggests that the carboxylates in the ortho position significantly alter the ability of the porphyrin to


penetrate into the cleft, while on the contrary, the linear form of the para-substituted phenyls allows better penetration. This favors a strong polar interaction with the {His80, Lys48, Trp266} positive patch which lies slightly deeper into the cleft. This selectivity of Xln10A for the para- against the orthosubstituted porphyrin is confirmed by the computed binding energies with a significantly stronger predicted binding affinity for Fe(TpCPP) than Fe(ToCPP) (-28.8 and -24.4 kJ.mol-1 for best and average values of Fe(TpCPP) dockings and -23.8 and -18.8 kJ.mol-1 for best and average values of Fe(ToCPP) dockings, respectively). Study of Peroxidase Activity of the Xln10A-Fe(TpCPP) Complex. Oxidation of Various Peroxidase Cosubstrates. Since the binding studies described above suggested that, among the assayed porphyrins, xylanase was able to influence only the accessibility of the iron of Fe(TpCPP), the complex associating Xln10A and Fe(TpCPP) appeared as the best candidate to lead to a selective catalyst for oxidation reactions. Consequently, the oxidation by H2O2 of several peroxidase cosubstrates, specifically two phenolic cosubstrates, guaiacol and 2,6-dimethoxyphenol, and a biaryl cosubstrate, o-dianisidine, was examined in the presence of Xln10A-Fe(TpCPP) as catalyst and compared to that catalyzed by free Fe(TpCPP). The influence of the H2O2 concentration on the oxidation of the cosubstrates was first assayed. The rates of oxidation were measured as described in the Experimental Section, and the kinetic parameters were determined in each case from Lineweaver–Burk plots. The kcat and KM values calculated for the three substrates in the presence of Fe(TpCPP) and Xln10A-Fe(TpCPP) are reported in Table 2. The KMvalue is higher in the case of the Xln10A-Fe(TpCPP) complex than for Fe(TpCPP) alone by factors of about 1.1, 1.6, and 5, respectively for guaiacol, 2,6dimethoxypenol, and o-dianisidine. This showed that the affinity of H2O2 for the porphyrin was lower when it was incorporated into xylanase, which confirmed that access to the iron atom is restricted in the xylanase complex. With Xln10A-Fe(TpCPP) as catalyst, the kcat value was lower for guaiacol and 2,6dimethoxypenol, by factors of 3.1 and 1.6, respectively, whereas it was higher by a factor of 1.8 in the case of o-dianisidine. As a consequence, the catalytic efficiency kcat/KM was lower by a factor of 2.6, in the case of dimethoxyphenol, to 3.6, in the case of o-dianisidine, with Xln10A-Fe(TpCPP) when compared to Fe(TpCPP) alone as catalyst. This can probably be explained by the fact that guaiacol and 2,6-dimethoxyphenol are smaller hydrophobic substrates that can compete with H2O2 for the active site whereas o-dianisidine is a larger bicyclic compound, that hardly enters the porphyrin binding site in Xln10A. However, the catalytic lifetime of Xln10A-Fe(TpCPP) is longer than that of Fe(TpCPP) alone, and when the plateau of catalytic activity is reached, more substrate has been oxidized, with all three substrates examined (data not shown). In addition, with higher concentrations of H2O2, the difference between the catalysts becomes more important. Typically, when o-dianisidine is oxidized under the conditions described above but with concentrations of H2O2 increasing from 80 to 640 µM, the concentration of o-dianisidine oxidized increases with the H2O2 concentration, from 18 to 37 µM with Xln10A-Fe(TpCPP) as catalyst (Figure 8) but decreases from 17 to 12 µM with Fe(TpCPP) as catalyst. This shows that xylanase has an important protecting effect toward oxidative degradations of the porphyrin. The influence of the cosubstrate concentration on the rate of oxidation was then assayed in the cases of guaiacol and o-dianisidine. Table 3 shows the kinetic parameters that were determined in each case. In the case of o-dianisidine, the KM value increases by a factor of 4 when Fe(TpCPP) is incorporated into xylanase, whereas at the same time, the kcat value does not


Ricoux et al.

Figure 7. General view of the best binding mode predicted by Gold 3 with the Chemscore scoring function for Fe(TpCPP) (panel a) and Fe(ToCPP) (panel b). Table 2. Comparison of Kinetic Parameters, Relative to H2O2, for the Oxidation of Various Reducing Cosubstrates by H2O2 Catalyzed by Fe(TpCPP) or Xln10A-Fe(TpCPP) reducing cosubstrate guaiacol

catalyst

Fe(TpCPP) Xln10A-Fe(TpCPP) 2,6-dimethoxyphenol Fe(TpCPP) Xln10A-Fe(TpCPP) o-dianisidine Fe(TpCPP) Xln10A-Fe(TpCPP)

kcat/KM KM kcat (mM) (min-1) (M-1 min-1) 0.83 0.92 0.28 0.45 0.16 0.80

17.8 5.6 9.3 6.1 29.2 52.0

2.15 × 104 6.1 × 103 3.43 × 104 2.16 × 104 1.82 × 104 6.5 × 104

vary. Thus, the affinity for o-dianisidine decreases when Fe(TpCPP) is incorporated into xylanase, whereas the catalytic activity does not vary. In the case of guaiacol, on the contrary, the affinity increases, as shown by the KM value which decreases from 0.44 to 0.24 mM, and the catalytic activity decreases as shown by the 11-fold decrease in the kcat value, when Fe(TpCPP) is incorporated into xylanase. It is likely that o-dianisidine, which is a larger biaryl cosubstrate, does not enter easily inside the active site of xylanase, explaining the increase in KM. However, the nature of the cosubstrate does not prevent H2O2 from interacting with the iron atom, leading to an unchanged catalysis. On the contrary, guaiacol, a smaller hydrophobic cosubstrate, enters the xylanase binding site more readily due to hydrophobic interactions, and KM decreases. It also hinders the interaction of H2O2 with the iron atom causing a reduction in catalysis, and thus, kcat decreases. Influence of Imidazole on the Kinetic Parameters of the Oxidation of o-Dianisidine by H2O2. The influence of imidazole on the kinetic parameters of the oxidation of o-dianisidine by H2O2 in the presence of 1.2 µM Fe(TpCPP) or of 1.2 µM Xln10A-Fe(TpCPP) was examined. The catalyst was first incubated for 2 h at 4 °C with imidazole at concentrations ranging between 0 and 50 mM; then the o-dianisidine cosubstrate (890 µM) was added and the reaction was initiated by the addition of H2O2 (320 µM). The initial rates of oxidation were measured as described above. With both catalysts, the addition of imidazole caused an inhibition of the peroxidase activity. With Fe(TpCPP) as catalyst, the reaction is inhibited with an IC50 of about 11 mM, whereas with Xln10A-Fe(TpCPP), it is inhibited with an IC50 of about 25 mM. pH Dependence Studies. To study the pH dependence of the peroxidase activity of Fe(TpCPP) and Xln10A-Fe(TpCPP), the oxidation of o-dianisidine by H2O2 was performed in the presence of these two catalysts at pHs ranging between 4.1 and 9.1, as described in the Experimental Section. Figure 9 shows that, in the case of Fe(TpCPP) alone, the rate of oxidation increases linearly with pH for pH values above 5. In the case

Figure 8. Variation of the concentration of o-dianisidine oxidized as a function of the concentration of H2O2. Concentration of o-dianisidine oxidized at the plateau, when 890 µM o-dianisidine is oxidized by H2O2 at concentrations increasing from 0.08 to 0.64 mM, with 1.2 µM Fe(TpCPP) or Xln10A-Fe(TpCPP) as catalyst, in 50 mM PBS, pH 7.5, at 20 °C. Table 3. Comparison of Kinetic Parameters, Relative to the Reducing Cosubstrate, for the Oxidation of Guaiacol and o-Dianisidine by H2O2 Catalyzed by Fe(TpCPP) or Xln10AFe(TpCPP) reducing cosubstrate guaiacol

catalyst

Fe(TpCPP) Xln10A-Fe(TpCPP) o-dianisidine Fe(TpCPP) Xln10A-Fe(TpCPP) 2-methoxyphenol wild type Mb (32) 2-methoxyphenol RMb(H64D)-2 (32) p-OH-phenyl wild type Mb (33) propionic acid p-OH-phenyl T67R/S92D Mb-H (33) propionic acid

kcat kcat/KM KM (mM) (min-1) (M-1 min-1) 0.44 0.24 0.23 0.80 54 0.052 72

6.4 0.6 17.5 17.6 168 72 60

20

396

1.45 × 104 2.4 × 103 7.5 × 104 2.2 × 104 3.1 × 103 1.4 × 106 8.4 × 102 2.0 × 104

of Xln10A-Fe(TpCPP), a sigmoid curve is found with an inflection point at about pH 7.5.

DISCUSSION The insertion into Xln10A of three Fe-porphyrins which are water-soluble and bear negatively charged carboxylate substituents, microperoxidase 8 (MP8), Fe(III)-R4-tetra-o-carboxyphenyl porphyrin Fe(ToCPP), and Fe(III)-tetra-p-carboxyphenylporphyrin Fe(TpCPP) (Figure 1), was studied in order to define a new hybrid hemoprotein that would have interesting properties as a catalyst for selective oxidation reactions. Our results clearly show that, among the three Fe-porphyrins studied,


Figure 9. Influence of the pH on the peroxidase activity of Fe(TpCPP) and Fe(TpCPP)-Xln10A. Variation, as a function of the pH, of the initial rate of oxidation of 890 µM o-dianisidine by 320 µM H2O2 in the presence of 1.2 µM Fe(TpCPP) (- - -) or Fe(TpCPP)-Xln10A (s), in 0.1 M citrate/phosphate buffer pHs 4.1, 4.6, 5.0, 5.5, 6.0, 6.5, and 7.0, and in 0.1 M Tris-HCl buffer pHs 7.3, 7.6, 7.9, and 9.1.

only Fe(TpCPP) could lead to a new hemoprotein having such properties. First of all, UV–visible studies showed that most probably neither MP8 nor Fe(ToCPP) were inserted inside the cleft of Xln10A. Indeed, their titration by this protein did not lead to any significant change in their UV–visible spectrum, except a decrease in their Soret band which is characteristic of a nonspecific hydrophobic interaction between the protein and these two porphyrins, without any amino acid side chain coordinating to the iron atom. In agreement with this, similar results were observed when MP8 and Fe(ToCPP) were titrated by imidazole in the presence or absence of Xln10A. Both MP8 and MP8-Xln10A bound one imidazole ligand with a similar KD value of about 0.05 mM, and both Fe(ToCPP) and Fe(ToCPP)-Xln10A bound two imidazole ligands with similar KDs of about 0.48 mM (Table 1). This showed that in both cases the Xln10A protein does not interact closely with the porphyrin macrocycle and does not prevent imidazole from binding on the iron atom. On the contrary, addition of Xln10A to Fe(TpCPP) led to a significant change in its UV–visible spectrum (Figure 2), with a 3 nm shift of its characteristic bands, which could be due to the binding of an amino acid side chain of Xln10A to the iron atom. A titration of Fe(TpCPP) by increasing amounts of Xln10A (Figure 3) clearly indicated a 1:1 Fe(TpCPP)/Xln10A stoichiometry and allowed a KD of 0.5 µM to be measured for the obtained complex, that was close to the KD values reported for complexes of Fe(II)-2-[8-(2-methylimidazolyl)octanoyloxymethyl]-tetrakis-R4-(o-pivalamidophenyl)porphyrin with bacterial xylanases B (34, 35). In addition, different results were observed when Fe(TpCPP) was titrated by imidazole in the presence or absence of Xln10A. Fe(TpCPP) bound two imidazole ligands with a KD of 11.56 mM2, whereas Fe(TpCPP)-Xln10A bound only one with a KD of 3.3 mM (Table 1). This showed that the Xln10A protein interacted closely with the porphyrin macrocycle and brought steric hindrance around one face of the porphyrin, possibly with an amino acid side chain coordinating the iron atom, preventing imidazole from binding on this face. Furthermore, these results were corroborated by molecular modeling studies that allowed a possible explanation of the difference in Xln10A-Fe-porphyrin interactions to be put forward. These analyses suggested that, in agreement with the UV–visible studies, both the large volume (VMP8 ) 1400 Å3 vs Vcav ) 338 Å3) and the globular shape of MP8 lead a very poor incorporation of MP8 into the quite narrow and elongated cleft of Xln10A. On the contrary, the volumes and shapes of Fe(ToCPP) and Fe(TpCPP)


appeared reasonably close to those of the Xln10A cleft and suggested at least a partial insertion of both iron(III)-porphyrin complexes into it. A weak hydrophobic complementarity with the binding site of Xln10A was suggested for Fe(ToCPP) which bound perpendicularly to the surface of the protein with only one aromatic moiety inserted into the cleft and most of the porphyrin exposed to the solvent (Figure 7b). Therefore, both faces of Fe(ToCPP) seemed accessible to the solvent and could accommodate two axial imidazole ligands on its iron atom as showed by the absorption spectroscopy studies. Finally, molecular modeling studies suggested that Fe(TpCPP) could be more deeply inserted into the Xln10A cleft, with two aromatic groups and part of the porphyrin ring anchored into the binding site (Figure 7a). A strong association was then observed, mainly due to a hydrogen-bonding network between the carboxylates of two consecutive aromatic substituents of Fe(TpCPP) and several side chains of protein residues. Indeed, from the docking experiments, it was predicted that the binding energies could be substantially stronger for Fe(TpCPP) than for Fe(ToCPP) by about 5 kJ.mol-1. In the Fe(TpCPP)-Xln10A complex, the porphyrin is tilted with respect to the surface of the protein, and therefore, only one face of Fe(TpCPP) seems accessible to the solvent (Figure 7a) and this can explain why the binding of only one imidazole on the iron atom was observed in this complex. In addition, the glycosidic activity of Xln10A and of the Xln10A-Fe(TpCPP) complex was determined using a diffusion agar assay as described in the Experimental Section. Thus, Xln10A and Xln10A-Fe(TpCPP), 1 µM in 50 mM PBS pH 7.5, were added to wells coated with RBB (Remazol-Brillant-Blue)xylan and incubated at 37 °C for 2, 6, and 24 h. The glycosidic activity was then determined from the diameter of the clearing zone around the well that was due to the hydrolysis of xylan. Under those conditions, no clearing zones could be observed on RBB-xylan, in the presence of Xln10A-Fe(TpCPP) even after 24 h incubation, while Xln10A alone in PBS buffer showed increasing clearing zones with increasing incubation time. This clearly showed that the Xln10A-Fe(TpCPP) complex did not retain any glycosidic activity anymore. This constituted an additional strong argument proving that the Fe(TpCPP) molecule was competitively incorporated into the substrate binding cleft of the (β/R)8 TIM-barrel and affected the original enzymatic activity of the host xylanase. The peroxidase activity of Fe(TpCPP)-Xln10A was then examined. This new hybrid hemoprotein was found able to catalyze the oxidation of several peroxidase cosubstrates, namely, guaiacol, 2,6-dimethoxyphenol, and o-dianisidine, with kcat values between 3.4 and 52 min-1 and kcat/KM values between 6.1 × 103 and 6.5 × 104 M-1 min-1 (Table 2). These values are comparable to those already published for several Feporphyrin-antibody complexes (Table 3) that display kcat values between 63 and 667 min-1 and kcat/KM between 3.7 × 103 and 2.9 × 105 M-1 min-1, except for the 3A3-MP8 complex that displays higher kcat (885 min-1) and kcat/KM (2 × 106 M-1 min-1) values (26). While the kcat values remain comparable to those measured for the enzyme horseradish peroxidase (HRP) itself, the molar efficiency remains low when compared to that observed for this enzyme (kcat/KM ) 6.1 × 108 M-1 min-1) mainly because of the large difference in the affinity of the substrate, H2O2, which is much higher for HRP (KM ) 0.5 µM) (59) than for Fe(TpCPP)Xln10A (KM ) 0.5–0.9 mM) (Table 4). In order to examine the influence of the Xln10A protein on the catalytic activity of Fe(TpCPP), the kinetic parameters obtained for H2O2 in the presence of the three substrates with Fe(TpCPP)-Xln10A as catalyst have been compared with those obtained in the presence of Fe(TpCPP) alone as catalyst (Table 2). It appears that, in all the cases, the KM values are higher with Fe(TpCPP)-Xln10A (KM ) 0.92, 0.45, and 0.80 mM) than


Ricoux et al.

Table 4. Comparison of Kinetic Parameters for Oxidation of Various Cosubstrates by H2O2 Iron-Porphyrin-Protein Complexesf haptena

complex (ref) HRP (59) HRP (60) FeIII(MPIXb)7G12 (55) FeIII(MPIX) (55) FeIII(MPIX)-9A5 (56) FeIII(MPIX)-11D1 (56) FeIII(MPIX) (56) FeIII(TpCPP)c-13–1 L (57) Fe(TMPyP)f (58) Fe(TMPyP)-12E11G (58) FeIII(ToCPP)d-13G10 (24) FeIII(ToCPP)-14H7 (21) FeIII(ToCPP) (21) Fe(MPIX)-2B4 (59) Fe(MPIX) (59) MP8–3A3 (26) MP8 (26) Fe(TpCPP) (this work) Xln10A-Fe(TpCPP) (this work)

N-CH3-MPIX

N-CH3-MPIX N-CH2OH-MPIX TpCPPH2 3MPy1C Fe(ToCPP) Fe(ToCPP) N-CH3-MPIX MP8

substrate

kcat (min-1)

Km(H2O2) (mM)

kcat/Km (M-1 min-1)

leucomalachite green o-dianisidine o-dianisidine ABTSe pyrogallol o-dianisidine pyrogallol pyrogallol pyrogallol pyrogallol pyrogallol pyrogallol ABTS ABTS ABTS o-dianisidine o-dianisidine o-dianisidine o-dianisidine o-dianisidine o-dianisidine

306 700 394 166 132 86 21 667 83 680 560 63 51 330 77 885 590 29.2 52.0

0.0005

6.1 × 108

24 43 35 13 100 2.3

1.6 × 104 1.4 × 104 7.3 × 103 3.8 × 103 3.7 × 103 6.6 × 103 2.4 × 102 2.9 × 105

8.6 16 9 42 43 25 0.45 0.4 0.16 0.80

7.9 × 104 3.7 × 104 7.1 × 103 1.2 × 103 7.7 × 103 3.1 × 103 2 × 106 1.45 × 106 1.82 × 105 6.5 × 104

a in the case of monoclonal antibodies. b MPIX, mesoporphyrin IX. c TpCPP, meso-tetrakis(para-carboxyphenyl)porphyrin. d ToCPP, meso-tetrakis(orthocarboxyphenyl)porphyrin. e ABTS, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid). f TMPyP, meso-tetrakis(4-methylpyridyl)porphyrin.

with Fe(TpCPP) alone (KM ) 0.83, 0.28, and 0.16 mM for guaiacol, 2,6-dimethoxyphenol, and o-dianisidine, respectively). It is likely that this is due both to the steric hindrance and to the increased hydrophobicity brought by the protein around the iron atom of the porphyrin. With Fe(TpCPP)-Xln10A, the kcatvalue was lower than with Fe(TpCPP) alone in the case of guaiacol (5.6 and 17.8 min-1, respectively) and 2,6dimethoxyphenol (6.1 and 9.3 min-1, respectively) but higher with o-dianisidine (52.0 and 29.2 min-1, respectively). This could be explained by the fact that guaiacol and 2,6dimethoxyphenol are smaller hydrophobic substrates that can compete with H2O2 for the active site whereas o-dianisidine is a larger bicyclic compound that hardly enters the porphyrin binding site in Xln10A. The kinetic parameters for o-dianisidine and guaiacol have also been measured. They can be compared with the values reported for phenolic cosubstrates in the presence of myoglobin and new hemoproteins obtained by incorporation of modified hemes into recombinant myoglobin (Table 3). Both myoglobin and new myoglobin-derived hemoproteins lead to higher kcat values (60–396 min-1) (32, 33) than Xln10-Fe(TpCPP) with guaiacol (kcat ) 0.6 min-1) and o-dianisidine (kcat ) 17.6 min-1). Only one of the reported new hemoproteins, rMb(H64D)2, leads to a lower KM value (KM ) 0.052 mM) (32) than Xln10Fe(TpCPP) with guaiacol (KM ) 0.24 mM) and o-dianisidine (KM ) 0.80 mM). As a consequence, it also appears to be the more efficient catalyst, characterized by a kcat/KM value of 1.4 × 106 M-1 min-1 (32), whereas all the other myoglobin-derived hemoproteins display molar efficiencies that are in the same range as those displayed Xln10-Fe(TpCPP) (kcat/KM ) 8.4 × 102 to 2.2 × 104 M-1 min-1) (Table 3). Oxidation of o-dianisidine by increasing amounts of H2O2, in the presence of either Xln10A-Fe(TpCPP) or Fe(TpCPP) alone as catalyst, led to a plateau value that was dependent upon the concentration of H2O2 that was used (Figure 8). This was due in both cases to a bleaching of the metalloporphyrin cofactor as was proven by two experimental observations: (i) UV–visible monitoring of the reaction showed a bleaching of the Soret band at, respectively, 409 nm for Fe(TpCPP) or 412 nm for Xln10AFe(TpCPP), (ii) further addition of the same amount of either H2O2 or o-dianisidine to the reaction mixture, once the plateau was reached, did not lead to any further oxidation of odianisidine. It is noteworthy, however, that another important advantage brought by the protein around the Fe-porphyrin

cofactor could be shown: an important protecting effect toward oxidative degradation of the porphyrin. Indeed, with Xln10AFe(TpCPP) as catalyst, the reaction remains linear as a function of time for a longer time, and when the plateau is reached, more substrate is oxidized. Furthermore, as shown in the particular case of o-dianisidine (Figure 8), the concentration of cosubstrate oxidized increases with the H2O2 concentration over a wide range of concentrations (e.g., 80–640 µM), whereas under the same conditions, with Fe(TpCPP) as catalyst, the concentration of o-dianisidine oxidized significantly decreases. When the effect of the addition of imidazole on the peroxidase activity was assayed, inhibition was observed with both Fe(TpCPP) and Fe(TpCPP)-Xln10A catalysts, with respective IC50 values of 11 and 25 mM. It is likely that this inhibition is due to the binding of imidazole ligands on the iron atom of Fe(TpCPP). With both Fe(TpCPP) and Xln10A-Fe(TpCPP), the IC50 value is larger than the C50 value for the binding of imidazole on the iron atom (C50 ) 3.4 mM and 3.3 mM, respectively; Table 1), which could reflect competition between imidazole and the substrate to enter the active site as the basis of the inhibition. Finally, a study of the catalytic activity as a function of pH showed that, whereas with Fe(TpCPP) alone the rate of oxidation of o-dianisidine by H2O2 increased linearly with pH for pH values above 5, a sigmoid curve with an inflection point at about pH 7.5 was found in the case of Xln10A-Fe(TpCPP). Since the solubility of Fe(TpCPP) decreases at lower pH values, and it cannot be ruled out that this could be involved in the decrease of peroxidase activity of the hemozyme at low pH. However, it is also likely that an amino acid side chain with a pKA of about 7.5 could be participating in the catalysis. The amino acids located inside the binding pocket of Xln10A, that were both characterized by a pKa value between 6 and 8 and close enough to the iron atom of the metalloporphyrin cofactor, were searched using molecular modeling. This allowed us to point out five amino acids that could possess those properties: Lys 48, Asp 50, His 80, Glu 128, and His 207 (Figure 10). Site-directed mutagenesis studies will be undertaken to examine which of them could be participating in catalysis.

CONCLUSION The results described above show that the association of a synthetic iron(III)-tetra-para-carboxy-phenyl substituted porphyrin and a nonrelated protein xylanase A from Streptomyces liVidans,


Figure 10. Amino acids located inside the binding pocket of Xln10A, that could be participating in the catalysis of the peroxidase reaction by the Xln10A-Fe(TpCCP) complex: Lys 48, Asp 50, His 80, Glu 128, and His 207.

leads to a new hybrid hemoprotein that displays interesting peroxidase activity. This new biocatalyst combines the advantages of both partners. First, Fe-tetraaryl-porphyrins are less sensible to oxidative degradations of meso bridges by oxidizing intermediates than natural hemes. The first positive effect of the protein is to increase this protection of the porphyrin ligand toward the highly reactive oxygenated species generated during the catalysis. Second, the protein also brings steric hindrance and an increased hydrophobic environment around the iron, two important characteristics that will be highly useful in the future for the catalysis of selective oxidation reactions. It may also be possible for the protein to bring amino acids that participate in the catalysis and in the binding of the iron. Preliminary molecular modeling experiments have already suggested a set of amino acids that could fill these functions. Further site-directed mutagenesis experiments are underway to confirm these results.

ACKNOWLEDGMENT We thank the Ministère des Relations Internationales du Québec, the Consulat Général de France à Québec and the 61th Commission Permanente de Coopération Franco-Québecoise for a two-years collaboration grant between the team of Prof. J.-P Mahy (France) and that of Prof. C. Dupont (Quebec) that helped to develop this project.

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Hemozymes Peroxidase Activity Of Artificial Hemoproteins

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