Hemoabzymes: towards new biocatalysts for selective oxidations

Journal of Immunological Methods 269 (2002) 39 – 57 www.elsevier.com/locate/jim

Hemoabzymes: towards new biocatalysts for selective oxidations Re´my Ricoux a, He´le`ne Sauriat-Dorizon a, Elodie Girgenti a, Dominique Blanchard b, Jean-Pierre Mahy a,* a

Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, Institut de Chimie Mole´culaire d’Orsay, Baˆtiment 420, Universite´ de Paris-sud XI, 91405 Orsay Cedex, France b Laboratoire de Biotechnologie, Etablissement de Transfusion Sanguine, 34, Boulevard Jean Monnet, 44011 Nantes Cedex 01, France Received 4 January 2002; accepted 14 January 2002

Abstract Catalytic antibodies with a metalloporphyrin cofactor or «hemoabzymes», used as models for hemoproteins like peroxidases and cytochrome P450, represent a promising route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin antibodies, raised against various free-base, N-substituted Sn-, Pd- or Fe-porphyrins. Five of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/Km values of 3.7
103 – 2.9
105 M1 min1. This value remained, however, low when compared to that of peroxidases. This strategy has also led to a few models of cytochrome P450. The best of them, raised against a water-soluble tin(IV) porphyrin containing an axial anaphtoxy ligand, was reported to catalyze the stereoselective oxidation of aromatic sulfides by iodosyl benzene using a Ru(II)porphyrin cofactor. The relatively low efficiency of the porphyrin – antibody complexes is probably due, at least in part, to the fact that no proximal ligand of Fe has been induced in those antibodies. We then proposed to use, as a hapten, microperoxidase 8 (MP8), a heme octapeptide in which the imidazole side chain of histidine 18 acts as a proximal ligand of the iron atom. This led to the production of seven antibodies recognizing MP8, the best of them, 3A3, binding it with an apparent binding constant of 107 M. The corresponding 3A3 – MP8 complex was found to have a good peroxidase activity characterized by a kcat/Km value of 2
106 M1 min1, which constitutes the best one ever reported for an antibody – porphyrin complex. Active site topology studies suggest that the binding of MP8 occurs through interactions of its carboxylate substituents with amino acids of the antibody and that the protein brings a partial steric hindrance of the distal face of the heme of MP8. Consequently, the use of the 3A3 – MP8 complexes for the selective oxidation of substrates, such as sulfides, alkanes and alkenes will be undertaken in the future. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hemoabzymes; Catalytic antibodies; Peroxidases; Cytochrome P450; Microperoxidase 8; Oxidation

Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption ionization – time of flight mass spectrometry; ABTS, 2,2V-azinobis(3ethylbenzothiazoline-6-sulfonic acid); Hb, hemoglobin; Mb, myoglobin; HRP, horseradish peroxidase; NADPH, nicotinamide adenine dinucleotide phosphate; FADH2, flavin adenine dinucleotide; FMNH2, flavin mononucleotide. * Corresponding author. Tel.: +33-1-69-15-74-21; fax: +33-1-69-15-72-81. E-mail address: [email protected] (J.-P. Mahy). 0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 2 2 3 - 5

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R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

1. Introduction Hemoproteins play a fundamental role in many natural processes, such as the transport (hemoglobin), storage (myoglobin) and activation (cytochrome P450) of dioxygen, the most abundant element of our environment, and the control of the level of cellular peroxides (peroxidases). They also catalyze, under biological conditions, the oxidation of numerous endogenous and exogenous substrates (cytochrome P450 and peroxidases). As a consequence, the elaboration of biomimetic systems that exhibit, under mild conditions, efficiencies and selectivities that are comparable to those exhibited by those enzymes is of great interest, not only to better understand their in vivo mechanism, but also to build up catalytic systems. In particular, the design of biomimetic systems that are able to reproduce the reactions catalyzed by peroxidases and cytochrome P450, and which are easier to handle than the enzymes themselves, should provide interesting tools for two main purposes (Mansuy and Battioni, 1994): the development of new catalysts for oxidation reactions which

are important in industrial and fine chemistry, such as the selective oxidation of alkanes and the stereoselective epoxidation of alkenes; and the study and the prediction of the oxidative metabolism of new biologically active molecules, such as drugs. 1.1. Elaboration of models of hemoproteins based on monoclonal antibodies 1.1.1. Requirements for the elaboration of models of hemoproteins The elaboration of model systems of hemoproteins must take into account the structural elements that are involved, at least in part, in their functioning (Fig. 1): (i) the prosthetic group, a heme or iron(III)protoporphyrin IX, which is responsible for the binding of ligands, such as O2 (hemoglobin, myoglobin) (Antonini and Brunori, 1971; Ortiz de Montellano, 1995), electron transfer reactions (peroxidases) (Everse et al., 1991) or oxene transfer reactions (cytochrome P450) (Mansuy and Battioni, 1989, 1994; Ortiz de Montellano, 1995); (ii) the apoprotein which binds and site-isolates heme, preventing its

Fig. 1. Schematic view of the structure of hemoproteins and their anti-metalloporphyrin antibodies mimicks: hemoabzymes.

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

aggregation and its 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 (cytochrome P450), but also participate in some cases in catalysis (peroxidases) (Everse et al., 1991); and (iii) the proximal axial ligand of the Fe atom, histidine in peroxidases or cysteinate in cytochrome P450, which has a role in controlling the heme redox potential and the reactivity of the high-valent Fe-oxo intermediates involved in the catalytic cycle of these enzymes (Poulos, 1996). 1.1.2. Catalytic antibodies as models of hemoproteins The production of monoclonal antibodies raised against transition state analogs has proven to be a powerful strategy to obtain antibodies that are able to catalyze a wide range of reactions (Benkovic, 1992; Thomas, 1994, 1996; Lerner et al., 1991; Schultz and Lerner, 1995; Wentworth and Janda, 1998; Blackburn et al., 1998; Reymond, 1999; Hilvert, 2000; Blackburn and Garcß on, 2000; Stevenson and Thomas, 2000). However, since most of these catalytic antibodies have modest catalytic efficiencies, several other strategies have been envisioned. The first one involves the production of antibodies directed toward the idiotype of anti-enzymes antibodies. This strategy has led to antibodies which display an acetylcholine esterase activity, with the highest efficiency (1.35
105 M1 s1) ever reported for catalytic antibodies (Izadar et al., 1993), or a h-lactamase activity (Avalle et al., 1998). A second strategy is based on the association of antibodies with cofactors, such as inorganic cofactors (Hsieh et al., 1993, 1994), natural cofactors (Shokat, 1988), metal ions (Roberts et al., 1990; Wade et al., 1993a,b; Crowder et al., 1995) or metal cofactors (Schwabacher et al., 1989; Iverson and Lerner, 1990; Cochran and Schultz, 1990a,b; Keinan et al., 1990; Harada et al., 1991; Keinan et al., 1992; Savitsky et al., 1994a,b; Feng et al., 1995; Takagi et al., 1995; Kawamura-Konishi et al., 1996a,b; Quilez et al., 1996; Khoda et al., 1997; Harada et al., 1997; Kawamura-Konishi et al., 1998; Blackwood et al., 1998; de Lauzon et al., 1998; Romersberg et al., 1998; de Lauzon et al., 1999; Liu et al., 1999; Nimri and Keinan, 1999). In this strategy, the antibodies are designed either to bring into close proximity the cofactor and the substrate, or to bind

41

tightly the cofactor to enhance its reactivity. Thus, antibodies with metalloporphyrin cofactor appeared as an alternative approach to provide a route to catalysts tailored for specific oxidation reactions. In such models of hemoproteins, the heme should be mimicked by a synthetic metalloporphyrin, and the apoprotein should be mimicked by the antibody protein (Fig. 1). The main difficulty, thus, consisted in designing the ideal porphyrin hapten, which could induce in the binding site of the antibody, not only a binding site for the metalloporphyrin, but also an axial ligand of the Fe and a hydrophobic pocket to accommodate the substrate. Numerous teams have faced this problem in the last few years, and have raised antibodies against porphyrin derivatives (Fig. 2), in order to get porphyrin – antibody complexes having cytochrome P450- or peroxidase-like activities. 1.2. Antibody– porphyrin complexes with a peroxidase-like activity The active site of peroxidases, which contains a heme bound to the apoprotein by a histidine residue (Fig. 3), is relatively narrow (Everse et al., 1991; Marnett and Kennedy, 1995). Little space is left over the plane of the porphyrin, so that only the oxidant, H2O2 or ROOH can enter the active site and interact with the iron atom; whereas, the reducing cosubstrates only interact with the edge of the porphyrin (Everse et al., 1991; Marnett and Kennedy, 1995). Thus, at first sight, peroxidases appeared to be some of the easiest hemoproteins to be mimicked, and to induce the production of antibodies having a binding site with such characteristics, one simple strategy was to use as haptens quite unsophisticated flat porphyrins. Therefore, antibodies have been elicited against meso-carboxyaryl-substituted (Schwabacher et al., 1989; Harada et al., 1991; Takagi et al., 1995; Quilez et al., 1996; Khoda et al., 1997; Harada et al., 1997; de Lauzon et al., 1998; de Lauzon et al., 1999), nitrogensubstituted (Cochran and Schultz, 1990a,b; Feng et al., 1995; Kawamura-Konishi et al., 1996a,b; Kawamura-Konishi et al., 1998; Liu et al., 1999), tin (Sn)(Keinan et al., 1990, 1992) or palladium (Pd)-porphyrin (Savitsky et al., 1994a,b). Five of the obtained antibodies have shown, in the presence of their ironporphyrin cofactor, a significant peroxidase activity (Cochran and Schultz, 1990a,b; Feng et al., 1995;

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R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

Fig. 2. Structure and nomenclature of the various porphyrins mentioned in this article.

Fig. 3. Mechanism of the heterolytic cleavage of the O – O bond of hydroperoxides catalyzed by peroxidases.

Takagi et al., 1995; Quilez et al., 1996; KawamuraKonishi et al., 1998). The first one, 7G12, was obtained by Cochran and Schultz (1990a,b), using N-methylmesoporphyrin IX (N-CH3-MPIX) (Fig. 2), an inhibitor of ferrochelatase, as a hapten. The corresponding Fe(III)-MPIX – 7G12 complex was shown to catalyze the oxidation of several typical peroxidase cosubstrates, such as o-dianisidine, 2,2V-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) and pyrogallol, by H2O2. The structure of a mesoporphyrin IX – 7G12 complex was determined by X-ray diffraction studies (Romersberg et al., 1998) (Fig. 4). This remains the only three-dimensional structure of an antibody– porphyrin complex reported to date. It shows that approximately two-thirds of the porphyrin are interacting with the antibody pocket, three pyrrole rings being packed tightly against residues of the VH domain and two pyrrole rings packed against tyrosine residues of the VL domain. In addition, a methionine

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

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(para-carboxyphenyl)porphyrin (TpCPPH2) (Fig. 2) as a hapten. Not only did the corresponding FeTpCPP – antibody complexes catalyze the oxidation of ABTS and pyrogallol by H2O2, but the best catalyst for this reaction was found to be the complex associating Fe(TpCPP) with a recombinant antibody light (L) chain 13-1-L, which they named L-zyme.

Fig. 4. Mesoporphyrin IX in the binding site of the anti-N-methylmesoporphyrin IX antibody 7G12 (Romersberg et al., 1998).

H100c interacts specifically with one pyrrole ring and forces it to adopt a tilted conformation which should be favorable for the insertion of metal ions in the porphyrin ring and could explain the ferrochelatase activity of antibody 7G12. The same N-methylmesoporphyrin IX hapten was also used by two other groups, (Feng et al., 1995; Kawamura-Konishi et al., 1998) who, respectively, got two monoclonal antibodies, 2B4 and 9A5. When associated with the Fe(III)MPIX cofactor, those two antibodies were found to have a peroxidase activity: the 9A5 – Fe(III)-MPIX complex was found able to catalyze the oxidation of pyrogallol by H2O2 (Feng et al., 1995) and the 2B4 – Fe(III)-MPIX catalyzed that of o-dianisidine and ABTS but not that of pyrogallol nor that of hydroquinone by H2O2 (Kawamura-Konishi et al., 1998). Later, Feng et al. (1995) also used N-hydroxymethylmesoporphyrin IX (N-CH2OH MPIX) (Fig. 2) as a hapten, in which the oxygen atom of the hydroxymethyl group was supposed to mimic the oxygen atom of H2O2 when coordinated to the Fe atom of heme in peroxidase. They obtained one monoclonal antibody, 9A5, which was able to catalyze, in the presence of Fe(III)-mesoporphyrin IX, the oxidation of pyrogallol by H2O2. A third set of antibodies was obtained by Takagi et al. (1995) using meso-tetrakis

1.2.1. Antibodies elicited against iron(III)-a,a,a,bmeso-tetrakis(ortho-carboxyphenyl) porphyrin Finally, we obtained a fourth set of monoclonal anti-porphyrin antibodies, using as a hapten iron(III)a,a,a,h-meso-tetrakis(ortho-carboxyphenyl) porphyrin, a,a,a,h-Fe(ToCPP). This hapten was chosen after considering the mechanism of the peroxidase reaction. Indeed, in this mechanism, the key step is the heterolytic cleavage of the O – O bond of H2O2 or ROOH assisted by two amino acid residues, a histidine and an arginine (Fig. 3) (Marnett and Kennedy, 1995), which leads to a highly reactive Fe(V)-oxo species. This complex is further reduced to Fe(III) by two successive transfers of one electron from the reducing cosubstrate occurring through the porphyrin ring. a,a,a,h-Fe(ToCPP) was then chosen as a hapten not only to generate in antibodies a binding site for an Feporphyrin, but also in the hope of generating, opposite to the ortho-carboxylate substituents of the phenyl rings, amino acids, such as histidine or arginine, which would assist the heterolytic cleavage of the O –O bond of H2O2. In addition, the metal – porphyrin was used, expecting that it could induce in the antibody an amino acid residue that could bind the metal and mimic the histidine proximal ligand of Fe in peroxidases. Three monoclonal antibodies were found to recognize a,a,a,h-Fe(ToCPP). Two of them, 13G10 and 14H7, were able to bind it with Kd values of 2.9
109 and 5.5
109 M, respectively. Those values are the best ones ever reported for the binding of an Fe-porphyrin to an antibody (Schwabacher et al., 1989; Cochran and Schultz, 1990a,b; Keinan et al., 1990; Harada et al., 1991; Savitsky et al., 1994a; Takagi et al., 1995; Quilez et al., 1996; Harada et al., 1997), and are in the range of Kd values generally observed for natural heme – protein complexes (1012 –108 M) (Savitsky et al., 1994a). UV – visible spectroscopy studies, associated with the determination by competitive ELISA of the apparent dissociation constants (Kd) for various porphyrins

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have allowed to propose a possible binding site topology of antibodies 13G10 and 14H7 (de Lauzon et al., 1998). Both studies suggested first that the central metal atom of the hapten was not recognized by the antibody protein since a,a,a,h-Fe- and -Mn-ToCPP, ˆ oCPPH2, all bound as well as the free-base a,a,a,h-O to 13G10 with similar Kd (de Lauzon et al., 1998). This showed that no amino acid residue was induced in the antibody protein that was able to coordinate the Fe atom of the hapten. Consequently, both 13G10– and 14H7 – Fe(ToCPP) complexes were high-spin hexacoordinate complexes, in which two H2O molecules and no amino acid residues were bound to the Fe atom. Similar phenomena were observed by Feng et al. (1995) upon binding of Fe(MPIX) to antibodies 9A5 and 11D1, and a spectrum typical of a high-spin ferric porphyrin was also described by Cochran and Schultz (1990b) for the 7G12 –Fe(MPIX) complex. Second, it appeared that the ortho-carboxylate substituents of the phenyl rings of the hapten were crucial for the recognition of Fe(ToCPP) by the antibodies since the affinity of both 13G10 and 14H7 for this porphyrin was decreased by a factor of 102 and 103 when these substituents were esterified (a,a,a,h-Fe[ToCMePP]) or shifted to the meta-position (TmCPPH2). Finally, a comparison of the Kd for the four atropoisomers of ToCPPH2 (Fig. 2) (de Lauzon et al., 1998) showed that

mainly three of the ortho-carboxylate substituents of the phenyl rings, in a,a,h-positions were recognized by amino acids of the antibody protein. This led us to propose a possible binding site topology in which roughly two-thirds of the porphyrin macrocycle could be inserted in the binding pocket, with two adjacent a,a-carboxylates being more specifically bound to the protein. One phenyl bearing an a-carboxylate substituent could be outside the binding pocket, but the hcarboxylate, presumably the one bearing the linker to the carrier protein during immunization, could be on the edge of the pocket. The two a,a,a,h-Fe(ToCPP) – 13G10 and –14H7 complexes catalyzed the oxidation of ABTS by H2O2 and it was clearly demonstrated (Quilez et al., 1996) that the reaction did occur in the binding site of the antibodies. Both 13G10 – and 14H7 –porphyrin complexes exhibited catalytic efficiencies about fivefold higher than that obtained with free a,a,a,h-Fe(ToCPP) (Table 1). In this reaction, both antibody proteins exerted a protecting effect of toward the oxidative degradation of the porphyrin. Indeed, the rate of oxidation of ABTS by H2O2 remained constant for at least 1000 turnovers in the presence of both antibody– Fe(ToCPP) complexes as catalysts, whereas in the presence of Fe(ToCPP) alone, it slowed down after 15 min and the reaction stopped after only 40% con-

Table 1 Comparison of kinetic parameters for oxidation of various cosubstrates by H2O2 catalyzed by iron-porphyrin – antibody complexes Complex

Hapten

HRP (Kawamura-Konishi et al., 1998) FeIII-MPIXa – 7G12 (Cochran and Schultz, 1990a,b)

N-CH3-MPIX

FeIII-MPIX (Cochran and Schultz, 1990a,b) FeIII-MPIX – 9A5 FeIII-MPIX – 11D1 (Feng et al., 1995) FeIII-MPIX (Feng et al., 1995) FeIII-TpCPPc – 13-1-L (Takagi et al., 1995) FeIII-ToCPPd – 13G10 (de Lauzon et al., 1998) FeIII-ToCPP – 14H7 (Quilez et al., 1996) FeIII-ToCPP (Quilez et al., 1996) Fe-MPIX – 2B4 (Kawamura-Konishi et al., 1998) Fe-MPIX (Kawamura-Konishi et al., 1998) a

N-CH3-MPIX N-CH2OH-MPIX TpCPPH2 Fe(ToCPP) Fe(ToCPP) N-CH3-MPIX

MPIX, mesoporphyrin IX. ABTS, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). c TpCPP, meso-tetrakis(para-carboxyphenyl)porphyrin. d ToCPP, meso-tetrakis(ortho-carboxyphenyl)porphyrin. b

Substrate

kcat (min1)

Km (H2O2) (mM)

kcat/Km (M1 min1)

leucomalachite green o-dianisidine ABTSb pyrogallol o-dianisidine pyrogallol pyrogallol pyrogallol pyrogallol ABTS ABTS ABTS o-dianisidine o-dianisidine

306 394 – – 166 132 86 21 667 560 63 51 330 77

0.0005 24 – – 43 35 13 100 2.3 16 9 42 43 25

6.1
108 1.6
104 1.4
104 7.3
103 3.8
103 3.7
103 6.6
103 2.4
102 2.9
105 3.7
104 7.1
103 1.2
103 7.7
103 3.1
103

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version of ABTS. In addition, a,a,a,h-Fe(ToCPP) – 13G10 was found able not only to catalyze the oxidation of a variety of cosubstrates, such as o-dianisidine, pyrogallol, ABTS and TMB (tetramethyl-benzidine) by H2O2, but also to use a wide range of hydroperoxides ROOH other than H2O2 as substrates, including alkyl-, aralkyl- and fatty acid hydroperoxides (de Lauzon et al., 1999). The kinetic parameters for the oxidation of ABTS by H2O2 were measured as a function of pH. It appeared that the peroxidase activity of the a,a,a,hFe(ToCPP) –13G10 was optimal at pH below 5, with a kcat value of 560 min1 (Table 1) and that an amino acid with a pKa around 4.6 could participate in the reaction mechanism. It was, thus, proposed that a carboxylic acid side chain of the antibody could protonate one of the oxygen atoms of H2O2 and facilitate the release of a water molecule leading to the iron-oxo intermediate. Finally, since none of the reported antibodies afforded any axial proximal ligand for the iron atom, the influence of imidazole on our antibody – Fe-porphyrin ligand complexes was investigated. All of the 13G10– Fe-porphyrin complexes only bound one imidazole ligand on the iron atom, whereas imidazole was found to inhibit the peroxidase activity of the a,a,a,h-Fe(ToCPP) – 13G10 complex. It enhanced the peroxidase activity, by a factor five to six, of the complexes of antibody 13G10 with less-hindered iron(III)-a,a- and a,h-1,2-meso-di(ortho-carboxyphenyl) porphyrin [a,a- and a,h-1,2-Fe(DOCPP)] which then constituted a nice model of peroxidase (de Lauzon et al., 2002). This could be explained by the fact that in the case of the more-hindered Fe(ToCPP) porphyrin, imidazole was competing with H2O2 to bind to the iron on the less-hindered face of the porphyrin, whereas in the case of the less-hindered Fe(DoCPP) porphyrins, imidazole and H2O2 could bind simultaneously to the iron on the two opposite faces of the porphyrin, imidazole being able in this case to modulate the redox potential of the iron like the proximal histidine of peroxidases does.

lyzed by Fe-porphyrin –antibody complexes or by the corresponding free Fe(III)-porphyrins with those reported for horseradish peroxidase (Chance, 1943; Bakovic and Dunford, 1993; Kawamura-Konishi et al., 1998). In the case of Fe-porphyrin –Ab complexes, the kcat values range between 86 and 667 min1, the best value being obtained for the oxidation of pyrogallol by H 2 O 2 catalyzed by the Fe(TpCPP)– 13-1-L complex (Takagi et al., 1995). Those values are 2.4 – 6.3-fold higher than those reported for Fe(III)-porphyrins, which range from 21 to 166 min1, which shows that Fe-porphyrin – antibody complexes are better catalysts than the Fe(III)-porphyrins. Fe-porphyrin – antibody complexes also had a better affinity for H2O2 than free Fe(III)-porphyrins, as shown by the Km values for H2O2, which range between 2.3 and 35 mM for Feporphyrin –IgG complexes, when compared to values of 43 – 100 mM for free Fe-porphyrins. As a consequence, Fe(III)-porphyrin – antibody complexes exhibited higher efficiencies k cat /K m , ranging between 3.7
103 and 2.9
105 M1 min1, than the corresponding free Fe(III)-porphyrins, which led to kcat/Km values ranging between 2.4
102 and 3.8
103 M1 min1. It is noteworthy that the best efficiency was obtained for the L-zyme, which exhibited both the higher kcat value, 667 min1, and the lower Km value, 2.3 mM (Takagi et al., 1995). Unfortunately, those values cannot be compared to those corresponding to Fe(TpCPP) alone, which have not been reported. However, this kcat/Km value of about 2.9
105 M1 s1 remains far below those observed for peroxidases, which are among the most efficient enzymes, and exhibit kcat/Km values of about 6
108 M1 min1 (Chance, 1943; Bakovic and Dunford, 1993; Kawamura-Konishi et al., 1998). This is mainly due to the very good affinity of the enzyme for H2O2 as shown by a Km value of about 0.5 AM.

1.2.2. Comparison of the kinetic parameters for the peroxidase reaction catalyzed by various antibody– Fe-porphyrin complexes Table 1 compares the kinetic parameters for the oxidation of various coosubstrates by H2O2, cata-

1.3.1. Structure of cytochrome P450 As already mentioned, monooxygenases are widely distributed enzymes that activate dioxygen, using two electrons and two protons coming from nicotinamide adenine dinucleotide phosphate (NADPH), and cata-

1.3. Catalytic antibodies as models of cytochrome P450

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lyze the insertion of one O atom from O2 into a substrate with formation of water (Eq. (1)). RH þ O2 þ 2Hþ þ 2e ! ROH þ H2 O

oxidation of the Fe(III) (Eq. (2)). This highly oxidative species then transfers its O atom to the substrate (Eq. (3)).

ð1Þ

A great number of these enzymes contain cytochrome P450, which is the site for dioxygen activation. These cytochrome P450-dependent enzymes are involved in many steps of the biosynthesis and biodegradation of endogenous compounds, such as steroids, fatty acids, prostaglandins and leukotrienes and arginine (Mansuy et al., 1989). They also play a key role in the oxidative metabolism of exogenous compounds, such as drugs and other environmental products, allowing their elimination from living organisms. Cytochrome P450 have been the subject of many studies in the past 10 years, and much is known about their structure and functions (White and Coon, 1980; Ruckpaul and Rein, 1984; Mansuy et al., 1989; Guengerich and McDonald, 1990; Ortiz de Montellano, 1995; Poulos et al., 1995). Indeed, more than 500 P450 sequences are now available (Ortiz de Montellano, 1995), and the X-ray structures of eight cytochrome P450s have now been published: six bacterial P450s: P450cam (Poulos et al., 1985), P450BM3 (Ravichandran et al., 1993), P450Terp (Hasemann et al., 1994), P450eryF (Cupp-Vickery and Poulos, 1997), P450sca-2 (Ito et al., 1999), P450CYP1 19 (Yano et al., 2000); one from fungi: P450nor (Park et al., 1997); and one mammalian P450CYP2C5 (Williams et al., 2000). The active site of cytochrome P450 is divided in two parts: the heme-binding site, in which the heme iron is linked to the 50-kDa apoprotein by a proximal cysteinate, and, above the plane of the heme, a hydrophobic site for the binding of substrates (Fig. 1). 1.3.2. Mechanism of reactions catalyzed by cytochrome P450 The mechanism of the reactions catalyzed in vivo by cytochrome P450 has now been elucidated (White and Coon, 1980; Ruckpaul and Rein, 1984; Mansuy et al., 1989; Guengerich and McDonald, 1990; Mansuy and Battioni, 1994; Ortiz de Montellano, 1995; Poulos et al., 1995). The catalytic cycle involves the formation of a very reactive intermediate, a high-valent P450 FeV=O species derived from the binding of one oxygen atom of O2 to the iron, and a two-electron

P450FeIII þ O2 þ 2Hþ þ 2e ! P450FeV ¼ O þ H2 O ð2Þ P450FeV ¼ O þ SH ! P450FeIII þ SOH

ð3Þ

This complex can be directly obtained in vitro by reaction of cytochrome P450 with various single oxygen atom donors (AO), such as C6H5IO, H2O2 or NaIO4 (White and Coon, 1980; Ruckpaul and Rein, 1984; Mansuy et al., 1989; Guengerich and McDonald, 1990; Ortiz de Montellano, 1995) (Eq. (4)). P450FeIII þ AO ! P450FeV ¼ O þ A

ð4Þ

Accordingly, systems associating cytochrome P450 and AO are able to perform the same reactions as those catalyzed by cytochrome P450 in vivo, such as hydroxylation of alkanes and epoxidation of alkenes. The catalytic cycle, thus, described is called the short catalytic cycle. 1.3.3. Antiporphyrin antibodies as models of cytochrome P450 It appeared to be a very complex task to design models of cytochrome P450 using O2 as oxidant because the full in vivo catalytic cycle requires the presence not only of O2, but also of a reductant, NADPH, and the participation in the monooxygenase cluster of electron transfer cofactors, such as flavin adenine dinucleotide (FADH2) and flavin mononucleotide (FMNH2) and cytochrome P450 reductase to transfer electrons from NADPH to the heme iron. On the contrary, it seemed much easier to design models using single oxygen atom donors (AO) because the in vitro short cycle only requires the one pot association of cytochrome P450 and AO, and model systems, simply associating an O atom donor and an Fe(III)- or Mn(III)-porphyrin, have been found to be able to perform all the reactions typical of cytochrome P450, without the need for an axial ligand of the metal to mimic the cysteinate proximal ligand of heme in cytochrome P450 (Mansuy et al., 1989; Mansuy and Battioni, 1994). Associating AO donors and

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

antiporphyrin antibodies complexed with Fe(III)- or Mn(III)-porphyrins, thus, appeared as a reasonable strategy to build up models of cytochrome P450. An early report from Keinan et al. (1990) described the first attempt to use this simple strategy to get models of cytochrome P450. Monoclonal antibodies were raised against Sn(IV)-meso-tetrakis(para-carboxyvinylphenyl)porphyrin (Sn-TpCVPP) (Fig. 2). Five of those antibodies that bound the hapten with binding constants ranging between 2.5
108 and 1.6
107 M were selected and the ability of the antibody – Mn(III)-TpCVPP complexes to catalyze the epoxidation of styrene by iodosylbenzene was then investigated. The best results were obtained with lyophilized antibody – Mn-TpCVPP complexes as catalysts, which produced turnover numbers ranging between 424 and 537 turnovers per 17 h of reaction in CH2Cl2 at room temperature. Lyophilized Mn(III)TpCVPP alone was not catalytic (Keinan et al., 1990). However, those turnover numbers were only 30– 60% higher than that measured in the presence of a lyophilized mixture of a non relevant Ab and Mn(III)-TpCVPP (300 turnovers/17 h). In addition, since no asymmetric induction was observed in any of

47

these experiments, it seems unlikely that the reaction occurred inside the binding site of the antibodies but more probably it was caused by Mn-porphyrin molecules bound unspecifically on the surface of the antibody protein. A second short report by Liu et al. (1999) described the production of monoclonal antibodies against N-4-bromophenyl-mesoporphyrin IX, the N4-bromophenyl substituent being supposed to generate in the antibodies binding site a hydrophobic pocket to accomodate aromatic substrates. Two of the obtained antibodies, 7C7 and 4D4, when associated with ferric mesoporphyrin IX, catalyzed the epoxidation of styrene by NaOCl with respective turnovers of 0.48 and 0.98 min1 and the 4D4 –Femesoporphyrin IX catalyzed the N-demethylation of aminopyrine by H2O2 with a kcat of about 1 min1 (Liu et al., 1999). Finally, the most significant results were obtained recently by Nimri and Keinan (1999), who raised monoclonal antibodies against a water-soluble tin(IV) porphyrin containing an axial a-naphtoxy ligand (Fig. 5). Such a stable complex was designed to mimic the postulated transition state of the reaction of S-oxida-

Fig. 5. Mechanism of the oxidation of sulfides by metal-oxo-porphyrin complexes and the transition state analog, an a-naphtoxy-tin-porphyrin complex, used as a hapten for the generation of monoclonal antibodies having a monooxygenase-like activity (Nimri and Keinan, 1999).

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tion of aromatic sulfides (Fig. 5). One of the selected monoclonal antibodies (SN 37.4) was found able, in the presence of a Ru(II) porphyrin cofactor, to catalyze the stereoselective sulfoxidation of aromatic sulfides, such as thioanisole by iodosylbenzene. The S-enantiomer of thioanisole sulfoxide being obtained with a 43% enantiomeric excess. It is noteworthy that another strategy, which aims at modifying antisubstrate antibodies by covalent linkage of a Fe(III)-porphyrin in order to obtain artificial hemoproteins that are able to catalyze the selective oxidation of the substrate is under development (Mahy et al., 1998).

2. Anti-microperoxidase 8 antibodies 2.1. Strategy As already mentioned above, the antibody – porphyrin complexes described so far are not as efficient catalysts as their natural hemoprotein counterparts. The ones which display a peroxidase activity are characterized by kcat/Km values four to three orders of magnitude lower than those for natural peroxidases (Table 1) and the ones which display a monooxygenase-like activity do not catalyze very efficiently the oxidation of substrates, such as alkenes. One reason for this failure could be the absence, in those antibodies, of an amino acid, such as a histidine (peroxidases) or a cysteinate (cytochrome P450) which axially coordinates the iron and enhances its redox potential (Poulos, 1996; Rietjens et al., 1996). Accordingly, the addition of imidazole as a fifth ligand of the iron atom in 13G10 –Fe(DoCPP) complexes, greatly enhanced the peroxidase activity of those complexes (de Lauzon et al., 2002). To avoid this problem, we decided to use as a hapten microperoxidase 8 (MP8), a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c (Aron et al., 1986) (Fig. 6). Indeed, MP8 contains the heme prosthetic group together with the amino acid residues 14 – 21 of horse cytochrome c, and in particular His 18, whose imidazole group acts as the fifth axial ligand of the iron. In addition, it also contains four carboxylate substituents, two from the propionate side chains of the heme, and two from the C-terminal glutamate (Glu 24) of the

Fig. 6. Schematic view of the structure of microperoxidase 8 (MP8).

octapeptide, which could anchor it in the antibody binding site. Finally, MP8 itself possesses a peroxidase-like and a monooxygenase-like activity. Indeed, at pH lower than 9, the sixth coordination position of the iron is occupied by a water molecule. This weak ligand can be replaced by H2O2, which allows the formation of highly oxidized intermediates, responsible for the two types of catalytic reactions. First of all, MP8 is able to perform the oxidation of several typical peroxidase cosubstrates like o-dianisine (Baldwin et al., 1987), ABTS (Adams, 1990), guaiacol (Cunningham et al., 1991). Second, MP8 also catalyzes the para-hydroxylation of aniline (Rusvai et al., 1988), the S-oxidation of sulfides (Colonna et al., 1994), the monooxygenation of polycyclic aromatic compounds (Osman et al., 1996) and the N- and O-dealkylation of aromatic amines and ethers (Boersma et al., 2000). In addition, MP8 has also been recently shown to be able to oxidize N-monosubstituted hydroxylamines with formation of very stable iron(II) – nitrosoalkane complexes (Ricoux et al., 2000) and to catalyze the nitration of phenols by nitrite in the presence of H2O2 (Ricoux et al., 2001). Unfortunately, MP8 presents two main drawbacks. First, it dimerizes and aggregates at concentrations higher than 2 AM (Aron et al., 1986) and it is inactivated by oxydative degradation of the heme moiety (Cunningham et al., 1991). Then, the two first objectives were, through the binding of MP8 to antibodies, aimed to protect it against oxidative degradation of the heme moiety and to siteisolate it to keep it under a monomeric catalytically active form. The third objective was of course to induce a selectivity in the oxidation reactions catalyzed by MP8.

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

2.2. Materials and methods 2.2.1. Preparation of microperoxidase 8 Microperoxidase 8 (MP8) was prepared by sequential peptic and tryptic digestion of horse heart cytochrome c (Sigma) as previously described (Aron et al., 1986). The heme content was determined by the pyridine chromogen method (Aron et al., 1986). The purity of the sample was over 97% based on MALDITOF mass spectroscopy analysis.

49

anti-porphyrin antibodies were revealed with goat – antimouse antibodies labelled with peroxidase, using ABTS as a substrate. The binding constants were then determined as the concentration of porphyrin inhibiting 50% of the binding of the antibody to the immobilized antigen.

2.2.2. Preparation of monoclonal antibodies MP8 was covalently attached to keyhole limpet hemocyanin (KLH) and to bovine serum albumin (BSA) using glutaraldehyde as a coupling agent in 1 M bicarbonate buffer, pH 9.5, according to Tresca et al. (1995). The conjugates were then purified by column chromatography on Biogel P10. Hapten/protein ratios were determined spectrophotometrically using a molar absorption coefficient value (e) of 1.49
105 M1 cm1 at 407 nm for MP8. In the case of BSA, 6 mol of MP8 were bound per mol of protein, whereas in the case of KLH, 22 mol of MP8 were bound per 100,000 g of protein. Two 5-week-old female BALB/c mice were immunized with the hapten – KLH conjugate, and the mouse showing the best immune response 12 days after the third immunization was killed. Its splenocytes were fused with SP2O myeloma cells according to Ko¨hler and Milstein (1975). The resulting hybridomas were screened by ELISA for binding to the hapten – BSA conjugate using peroxidase-linked goat anti-mouse antibodies (de Lauzon et al., 1990). Positive hybridomas were cloned twice and produced in large amounts. Antibodies were then purified from hybridoma supernatants on a column of protein A and their homogeneity and purity were checked by SDS gel electrophoresis.

2.2.4. UV – visible spectroscopic studies The UV –visible characteristics of MP8 and of the IgG –MP8 complexes were determined in 0.1 M PBS, pH 7.4, using an UVIKON 940 spectrophotometer. For the titration of MP8 by antibody 3A3, MP8, 0.4 AM in 0.1 M PBS, pH 7.4, was incubated 20 min with antibody 3A3 at concentrations increasing from 0.05 to 1 AM and visible spectra were recorded between 380 and 420 nm (data not shown) and the absorbance at 396 nm was plotted against the 3A3/MP8 ratio. For the reactions of tertio-butylisonitrile (t-BuNC) with MP8 and 3A3 – MP8: t-BuNC (500 AM) was added to a solution of MP8 (0.86 AM) preincubated or not with 2 AM 3A3 in 0.1 M PBS, pH 7.4, and absolute spectra of the iron(III) –CN – tBu complexes were recorded on an UVIKON 940 spectrophotometer. The corresponding iron(II) – CN – tBu complexes were obtained by further reduction of these complexes by 1 mM sodium dithionite. The dissociation constants Kd of the Fe– CN –tBu complexes were determined from difference spectroscopy studies as follows: both reference and sample cuvette contained MP8 (0.86 AM) preincubated or not with 2 AM 3A3 in 0.1 M PBS, pH 7, and increasing concentrations of t-BuNC were added in the sample cuvette. Difference spectra were then recorded and the Kd values were calculated as the slope of the straight line representing the variations of 1/DA406 – 550 and 1/ DA423 – 550 as a function of 1/[t-BuNC] respectively for the iron(III) and the iron(II) complexes.

2.2.3. Determination of binding constants The binding constants were measured by competitive ELISA as follows: mixtures of a given antibody (at a concentration equal to its titer) and increasing amounts of porphyrin derivatives: 109 – 106 M were incubated overnight at 4 jC in 0.1 M PBS, pH 7.4, containing 0.1% casein. Fifty-microliter aliquots were then poured in microtiter wells previously coated with MP8 –BSA in PBS, pH 6.0 (100 ng/well), and saturated with casein (3%). After incubation for 2 h at RT,

2.2.5. Assay of peroxidase activity The peroxidase activity of MP8 and its complex with antibody 3A3 was assayed using as reducing cosubstrates o-dianisidine, ABTS, pyrogallol, guaı¨acol, p-cresol, TMB and hydroquinone. In a typical experiment, the oxidation of 50 AM reducing cosubstrate by 100 AM H2O2 was performed at 20 jC in 0.1 M PBS, pH 7.4, without catalyst or in the presence of either 0.1 AM MP8 alone or 0.1 AM MP8 and 0.1 AM 3A3 protein as catalysts. The absorbance was moni-

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tored by UVIKON 940 UV –visible recording spectrometer at, respectively, 500 nm (e= 7500 Ml cml) for o-dianisidine, 414 nm (e= 36000 Ml cml) for ABTS, 420 nm (e =3200 Ml cml) for pyrogallol, 490 nm (e =5500 Ml cml) for guaı¨acol, 300 nm (e =7300 Ml cml) for p-cresol, 650 nm (e =13600 Ml cml) for TMB, 295 nm (e = 2500 Ml cml) for hydroquinone and the rates of oxidation were measured using the corresponding e values. In the case of o-dianisidine, Lineweaver – Burk plots were established using concentrations of H2O2 ranging between 0.1 and 4 mM, and 0.1 AM MP8 was used either alone or in the presence of 0.1 AM 3A3 protein as a catalyst. 2.3. Results 2.3.1. Production of monoclonal antibodies and dissociation constants BALB/c mice were immunized with the MP8 – KLH conjugate and antibodies were produced using the hybridoma technology. Seven monoclonal antibodies were found to bind MP8, three were IgG1 (3A3, 8G9F11B8 and 8G9F11D8) and four IgG2a (8G4C11A12D6B4, 8G4C11A12D6G4, 6H11D6G3 and 6H11D6H3). After purification on protein A, apparent binding constants (Kd) of MP8 to the antibodies were measured by a competitive ELISA procedure as described in Section 2.2. Two of the IgG2a, 6H11D6G3 and 6H11D6H3, bound MP8 very poorly with respective binding constants of 103 and 104 M. The two other IgG2a (8G4C11A12D6B4 and 8G4C11A12D6G4) and two of the IgG1 (8G9F11B8 and 8G9F11D8) bound MP8 with binding constants of 105 – 106 M. Finally, the best antibody appeared to be 3A3, which bound MP8 with a binding constant of 107 M. We then concentrated our studies on this monoclonal antibody. First, affinities of this antibody for a series of heme derivatives were examined using the determination by competitive ELISA of apparent binding constants (Table 2). It first appeared that the octapeptide side chain of MP8 was not recognized by 3A3 since hemin which lacks this octapeptide bound 3A3 with an apparent Kd of 3
107 M, which was similar to that measured for MP8. Second, the iron atom of MP8 was not recognized by 3A3 as suggested by the similar Kd values found for hemin (Fe-PPIX), manganese-proto-

Table 2 Apparent binding constants (Kd) measured by competitive ELISA of 3A3 – porphyrin complexes Porphyrin

Apparent binding constant (Kd) [(M)
107]

MP8 Protoporphyrin IX (PPIX) Hemin Mn-Protoporphyrin IX PPIX – DME Hemin – DME ToCPPH2

1 3 3 5 5000 4000 800

porphyrin IX (Mn-PPIX) and free base protoporphyrin IX (PPIXH2): 3– 5
107. Finally, the results presented in Table 2 suggested that the carboxylate groups were essential for the recognition of the hapten by the antibodies. Indeed, when these substituents are esterified like in protoporphyrin IX – dimethyl ester (PPIX – DME) or in hemin– dimethyl ester (Fe-PPIX – DME), Kd is increased by a factor of about 103. Additionally, when these carboxylates are in orthoposition on the meso-phenyl substituents of a tetraarylporphyrin, Kd is increased by a factor 2
102. 2.3.2. UV – visible characteristics of the 3A3 – MP8 complex The binding of MP8 to the IgG 3A3 was studied by UV – visible spectroscopy. The spectrum of MP8 showed bands at 396, 490 and 526 nm (Fig. 7) characteristic of a high-spin hexacoordinate iron(III) species with His18 and H2O as axial ligands (Aron et al., 1986). Only minor changes were induced by the insertion of MP8 into 3A3: almost no shift and a slightly lower absorbance of the Soret band (Fig. 7) consistent with the binding of MP8 in a hydrophobic pocket with no change of the iron(III) spin state and no amino acid side chain from the antibody protein as a sixth axial ligand of the iron. The UV – visible titration of MP8 by 3A3 (Fig. 7, inset) showed that two molecules of MP8 were bound per molecule of IgG. 2.3.3. 3A3 –MP8 – Fe-t-butylisonitrile complexes Studies on the coordination of tertio-butyl-isonitrile on the iron of MP8 bound or not to antibody 3A3 were undertaken in order to get more precise informations on the topology of the binding site of the antibody and particularly to appreciate the size of the cavity left around the iron atom.

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

51

Fig. 7. UV – visible spectra of MP8 (- - -) and 3A3 – MP8 (——) 1 AM in 0.1 M PBS, pH 7.4. Inset: variations of the absorbance at 396 nm as a function of the 3A3/MP8 ratio for the addition of increasing amounts of 3A3 to 0.4 AM MP8 in 0.1 M PBS, pH 7.4.

The addition of a large excess (500 equivalents) of t-BuNC to a 0.86-AM solution of MP8 –FeIII in 0.1 M PBS, pH 7.4, led to the formation of a MP8FeIII – CNtBu complex (Eq. (5)) characterized by maxima of absorption at 406, 533 and 550 nm. Further reduction of this complex by 1 mM sodium dithionite led to the MP8FeII – CNtBu complex (Eq. (5)) absorbing at 423, 520 and 550 nm. That latter complex could also be obtained directly by addition of 500 equivalents of tBuNC to MP8 –FeII previously prepared by reduction of MP8– FeIII under anaerobic conditions.

3A3 –MP8FeII – CNtBu, characterized by spectra very similar to those of MP8FeIII –CNtBu and MP8FeII – CNtBu, with maxima of absorption, respectively, at 406, 533 and 550 nm and 423, 520 and 550 nm, and presenting a lower absorbance in the Soret band. The Kd values (Table 3) were determined for the four complexes from difference spectroscopy studies as described in Section 2.2. The Kd values for the 3A3 –MP8 complexes were about 10-fold lower than those observed for the MP8 complexes which showed

MP8FeIII þ tBuNCWMP8FeIII  CNtBu

Table 3 Dissociation constants (Kd) and Kr ratio for ferric and ferrous MP8Fe – CNtBu and 3A3 – MP8Fe – CNtBu complexes

 Wee MP8FeII

II

 CNtBuWMP8Fe þ tBuNC ð5Þ

Complex

When the same reactions were performed on solutions of MP8 (0.86 AM) previously preincubated with 2 AM 3A3 in 0.1 M PBS, pH 7.4, two new complexes were formed, 3A3 – MP8FeIII – CNtBu and

III

MP8Fe – CNtBu 3A3 – MP8FeIII – CNtBu MP8FeII – CNtBu 3A3 – MP8FeII – CNtBu

Kd (AM)

Kr

190 64 73 6.5

0.33 0.09

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that the affinity of the t-BuNC ligand for the iron atom was better in the case of the 3A3 – MP8 complex than in the case of MP8 alone whatever its oxidation state. Accordingly, the ratio between the two Kd values, Kr was found equal to 0.33 for the iron(III) complexes and 0.09 for the iron(II) complexes (Table 3). Marques et al. (1997) calculated the Kr values for a series of iron-complexes of heme –proteins compared to the same complexes of MP8 in order to appreciate the influence the protein on the ligand on the iron and to study the active site topology of hemoproteins. According to their results, such a Kr value <1 could be due to a steric hindrance of the distal face of MP8 by the antibody protein. 2.3.4. Peroxidase activity of the IgG – MP8 complexes The oxidation of several reducing cosubstrates including o-dianisidine, ABTS, pyrogallol, guaı¨acol, p-cresol, TMB and hydroquinone by H2O2 was used to assay the peroxidase activity of the 3A3 – MP8 complexes. Whereas these reactions were negligible in the absence of catalyst, they were catalyzed both by MP8 and 3A3 – MP8 (Fig. 8). For three of the cosubstrates

(pyrogallol, hydroquinone and TMB), the catalytic activity of the 3A3 – MP8 complex was similar to that of free MP8. For the four other cosubstrates (o-dianisidine, ABTS, guaı¨acol and p-cresol), 3A3 – MP8 was a better catalyst than free MP8. In those cases, both the initial rate of oxidation and the concentration of cosubstrate oxidized at the end of the reaction were higher with 3A3 – MP8 than with MP8 as catalyst (Fig. 8). Indeed, an initial rate enhancement between 1.6 and 2.8 and a 15– 70% higher concentration of cosubstrate oxidized at the end of the reaction were obtained with 3A3 – MP8 as catalyst (Fig. 8). This showed the protecting effect of the antibody towards the oxidative degradation of the porphyrin ring by H2O2. The oxidation of o-dianisidine was particularly studied as a function of the H2O2 concentration. The 3A3 – MP8 complex, as well as MP8 alone, led to peroxidation reactions displaying saturation kinetics with respect to H2O2 and the enzymatic kinetic parameters were determined from Lineweaver – Burk plots (Table 4). Close Km values were observed for MP8 (0.4 mM) and for 3A3 – MP8 (0.45 mM), which showed that H2O2 had a similar affinity for the MP8 –

Fig. 8. Kinetics of the oxidation of reducing cosubstrates (50 AM) by H2O2 (100 AM) in the presence of 0.1 AM MP8 (- - -) or 3A3 – MP8 (——) in 0.1 M PBS, pH 7.4, at 20 jC.

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Table 4 Comparison of kinetic parameters for the oxidation of o-dianisidine by H2O2 catalyzed by porphyrin – antibody complexes Complex

Hapten

Km (mM)

kcat (min1)

Fe-MPIX – 2B4 (Kawamura-Konishi et al., 1998) Fe-MPIX (Kawamura-Konishi et al., 1998) Fe-MPIX – 7G12 (Cochran and Schultz, 1990a,b) Fe-MPIX (Cochran and Schultz, 1990a,b) Fe – MP8 – 3A3 (This work) Fe – MP8 (This work)

N-CH3-MPIX – N-CH3-MPIX – MP8 –

43 25 24 43 0.45 0.4

330 77 394 166 885 590

antibody complex and for MP8 alone. The 3A3 – MP8 exhibited slightly higher kcat (885 minl) and kcat/Km values (2
106 Ml minl) than MP8 alone (kcat=590 minl and kcat/Km=1.45
106 Ml minl) (Table 4). 2.4. Discussion The aforementioned results show that the immunization of mice with a MP8 – KLH conjugate has led to the production of seven monoclonal antibodies recognizing MP8 (four IgG2a and three IgG1). The one having the best affinity for MP8 was the IgG1 3A3 which bound this heme octapeptide with an apparent Kd measured by competitive ELISA of 107 M. Such a value is in the range of the apparent Kd values already described for IgG – metalloporphyrin complexes (de Lauzon et al., 1998; Mahy et al., 1998). UV –visible studies show that the obtained 3A3 – MP8 complex is a high-spin hexacoordinate iron(III) complex characterized by a spectrum very similar to that of MP8 – FeIII with only a lower intensity of the soret band at 396 nm (Fig. 8). Such a phenomenon could be due to a simple hydrophobic interaction between MP8 and the antibody protein without any amino acid side chain acting as a sixth axial ligand of the iron. In addition, a titration of MP8 by 3A3 showed that at saturation two molecules of MP8 were bound per antibody molecule (Fig. 8, inset). Preliminary studies have been undertaken to determine the binding site topology of antibody 3A3, using the determination by competitive ELISA of the apparent dissociation constants (Kd) for various heme derivatives (Table 2). Several conclusions can be drawn from Table 2. First, the central metal atom of the hapten is not recognized by the antibody since hemin, Mn-protoporphyrin IX and free-base proto-

E=kcat/Km (M1 min1) 7.7
103 3.1
103 1.6
104 3.9
103 2
106 1.45
106

E (Ab)/E (porph) 2.5 4.1 1.5

porphyrin IX (PPIXH2), all bound to 3A3 with Kd of 3– 5
107 M. This confirms the above-mentioned results of the UV – visible studies which indicate that no amino acid residue that was able to coordinate the Fe atom of the hapten was induced in the antibody binding site. The second important observation from Table 2 is that the octapeptide side chain of MP8 was not recognized by 3A3 since both hemin which lacks this octapeptide and MP8 bound 3A3 with an apparent Kd of about 107 M. Third, the carboxylate substituents of the hapten play a key role in the recognition of MP8 by the antibody. When these substituents are esterified (PPIX – DME or Fe-PPIX – DME) or when these carboxylates are in ortho-position on the mesophenyl substituents of a tetraarylporphyrin, Kd is increased, respectively, by a factor of 103 and 2
102. In addition, to appreciate the size of the cavity left around the iron atom, the affinities of the tertio-butylisonitrile ligand for the iron of MP8 or for that of 3A3 –MP8, in the reduced and in the oxidized state were compared. The corresponding ratio between the two Kd values, Kr values were found, respectively, equal to 0.33 and 0.09 (Table 3). Such values which are far below 1 are in favor of a steric hindrance brought by the antibody protein on the distal face of MP8 (Marques et al., 1997). We, thus, propose a possible active site topology for antibody 3A3 (Fig. 9), in which MP8 binds to the antibody thanks to interactions of the carboxylates from the heme propionates and from the C-terminal glutamate of the octapeptide, at the opposite of the octapeptide chain, and the protein brings a partial steric hindrance of the distal face of MP8. The peroxidase activity of the 3A3 – MP8 complex was analyzed and compared to that of MP8 alone. It appeared that 3A3 –MP8 was a better catalyst than MP8 alone for the oxidation of four cosubstrates (o-

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Fig. 9. Possible binding site topology of anti-microperoxidase 8 antibodies.

dianisidine, ABTS, guaı¨acol and p-cresol) (Fig. 7). These results are similar to those obtained by Kawamura-Konishi et al. (1998) with the 2B4 – MPIX –Fe complex as catalyst. For the four cosubstrates, not only was the initial rate of oxidation slightly larger but also the percentage of cosubstrate oxidized at the end of the reaction was 15– 70% larger with 3A3 –MP8 which indicates that the antibody has a protecting effect towards the oxidative degradations of MP8. The kinetic parameters for the oxidation of odianisidine by H2O2 catalyzed by either 3A3 –MP8 or MP8 are compared with those already reported in the literature for the oxidation of the same cosubstrate by H2O2 catalyzed by porphyrin – antibody complexes (Table 4). The affinity of H2O2 for the MP8 – antibody complex and for MP8 alone was very similar as shown by the Km values observed for MP8 (0.4 mM) and for 3A3 – MP8 (0.45 mM) and 3A3 – MP8 exhibited slightly higher kcat (885 minl) and kcat/Km values (2
106 Ml minl ) than MP8 alone (kcat=590 minl and kcat/Km=1.45
106 Ml minl) (Table 4). Those two observations suggested that on the distal face of MP8, there was no amino acid from the antibody participating to the

catalysis of the heterolytic cleavage of the O – O bond of H2O2, which would have caused larger rate and efficiency enhancements. However, when compared to the values reported in the literature for other antibody – porphyrin complexes, the kcat and kcat/Km values obtained in the case of 3A3 – MP8 constitute the best ones ever observed for the oxidation of o-dianisidine by H2O2 catalyzed by Fe-porphyrin –antibody complexes. In particular, the kcat/Km values are two to three orders of magnitude higher than those observed for other Feporphyrin – antibody complexes, which arises both from a 100-fold better affinity of H2O2 for 3A3 – MP8 than for other Fe-porphyrin – antibody complexes and from an about two- to threefold higher kcat due to the axial coordination of the proximal histidine His 18 on the iron atom of MP8. 2.5. Conclusion Our results show that the immunization of mice with MP8 –KLH conjugates leads to the production of antibodies recognizing MP8 and to MP8 –antibody complexes which possess an interesting peroxidase

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

activity. This approach has particularly been successful to fulfil two of the original objectives: to obtain monoclonal antibodies that protect MP8 against oxidative destruction by H2O2 and to get kcat and kcat/Km values that are higher than those already published for other Fe-porphyrin – antibody complexes, mainly thanks to the proximal histidine which axially coordinates the iron atom of MP8 and regulates its redox potential. However, this activity could even be higher if there was in the antibody binding site, on the distal face of the heme, an amino acid participating to the catalysis of the heterolytic cleavage of the O – O bond of H2O2. Further work including 3D structure determination followed by site directed mutagenesis will probably be necessary for this. Finally, since the antibody protein brings a steric hindrance on the distal face of MP8, the use of the 3A3 – MP8 complexes for the selective oxidation of substrates, such as sulfides, alkanes and alkenes will now be undertaken.

References Adams, P.A., 1990. The peroxidasic activity of the heme octapeptide microperoxidase 8 (MP8). The kinetic mechanism of the catalytic reduction of H2O2 by MP8 using 2,2V-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a reducing substrate. J. Chem. Soc., Perkin Trans., 2, 1407 – 1414. Antonini, E., Brunori, M., 1971. In: Neuberger, A., Tatum, E.L. (Eds.), Hemoglobin and Myoglobin in their Reactions with Ligands. North-Holland, Amsterdam. Aron, J., Baldwin, D.A., Marques, H., Pratt, J.M., Adams, P.A., 1986. Hemes and hemoproteins: 1. Preparation and analysis of heme containing octapeptide (microperoxidase 8) and identification of the monomeric form in aqueous solution. J. Inorg. Biochem. 27, 227 – 243. Avalle, B., Thomas, D., Friboulet, A., 1998. Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolizing beta-lactams. FASEB J. 12, 1055 – 1060. Bakovic, M., Dunford, H.B., 1993. Kinetics of the oxidation of pcoumaric acid by prostaglandin H synthase and hydrogen peroxide. Biochemistry 32, 833 – 840. Baldwin, D.A., Marques, H., Pratt, J.M., 1987. Hemes and hemoproteins: 5. Kinetics of the peroxidasic activity of microperoxidase 8, model for the peroxidase enzymes. J. Inorg. Biochem. 30, 203 – 217. Benkovic, S.J., 1992. Catalytic antibodies. Annu. Rev. Biochem. 61, 29 – 54. Blackburn, G.M., Garcßon, A., 2000. Catalytic antibodies. In: Kelly, D.R. (Ed.), Biotechnology, 2nd ed., vol. 8b. Wiley-VCH, Weinheim, pp. 403 – 490.

55

Blackburn, G.M., Datta, A., Denham, H., Wentworth Jr., P., 1998. Catalytic antibodies. Adv. Phys. Org. Chem. 31, 249 – 292. Blackwood Jr., M.E., Rush III, T.S., Romesberg, F., Schultz, P.G., Spiro, T.G., 1998. Alternative modes of substrate distortion in enzyme and antibody-catalyzed ferrochelation reactions. Biochemistry 37, 779 – 782. Boersma, M.G., Primus, J.L., Koerts, J., Veeger, C., Rietjens, I.M., 2000. Heme-(hydro)peroxide mediated O- and N-dealkylation. A study with microperoxidase. Eur. J. Biochem. 267, 6673 – 6678. Chance, B., 1943. The kinetics of the enzyme-substrate compound of peroxidase. J. Biol. Chem. 151, 553 – 577. Cochran, A.G., Schultz, P.G., 1990a. Antibody catalyzed porphyrin metallation. Science 249, 781 – 783. Cochran, A.G., Schultz, P.G., 1990b. Peroxidase activity of an antibody – heme complex. J. Am. Chem. Soc. 112, 9414 – 9415. Colonna, S., Gagerro, N., Carrea, G., Pasta, P., 1994. The microperoxidase-11 catalysed oxidation of sulfides is enantioselective. Tetrahedron Lett. 35, 9103 – 9104. Crowder, M.W., Stewart, J.D., Roberts, V.A., Bender, C.J., Tevelrakh, E., Peisach, J., Getzoff, E.D., Gaffney, B.T., Benkovic, S.J., 1995. Spectroscopic studies on the designed metal-binding sites of the 43C9 single chain antibody. J. Am. Chem. Soc. 117, 5627 – 5634. Cunningham, I.D., Bachelor, J.L., Pratt, J.M., 1991. Kinetic study of the H2O2 oxidation of phenols, naphthols and anilines catalysed by the haem octapeptide microperoxidase 8. J. Chem. Soc., Perkin Trans., 2, 1839 – 1843. Cupp-Vickery, J.R., Poulos, T.L., 1997. Structure of cytochrome P450eryF: substrate, inhibitors, and model compounds bound in the active site. Steroids 62, 112 – 116. de Lauzon, S., Desfosses, B., Moreau, M., Le Trang, N., Rajkovski, K., Cittanova, N., 1990. Comparison of monoclonal antibodies to estradiol obtained from structurally different immunogens. Hybridoma 9, 481 – 491. de Lauzon, S., Quilez, R., Lion, L., Desfosses, B., Desfosses, B., Lee, I., Sari, M.A., Benkovic, S.J., Mansuy, D., Mahy, J.P., 1998. Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin. Eur. J. Biochem. 257, 121 – 130. de Lauzon, S., Desfosses, B., Mansuy, D., Mahy, J.P., 1999. Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin. FEBS Lett. 443, 229 – 234. de Lauzon, S., Mansuy, D., Mahy, J.P., 2002. Coordination chemistry of iron(III)-porphyrin – antibody complexes: influence on the peroxidase activity of the axial coordination of an imidazole on the iron atom. Eur. J. Biochem. 269, 1 – 11. Everse, J., Everse, K.E., Grisham, M.B., 1991. Peroxidases in Chemistry and Biology, vol. 2. CRC, Boca Raton. Feng, Y., Liu, Z., Gao, G., Gao, S.J., Liu, X.Y., Yang, T.S., 1995. Study of the abzyme with catalytic peroxidase activity. Ann. N. Y. Acad. Sci. 750, 271 – 276. Guengerich, F.P., McDonald, T.L., 1990. Mechanisms of cytochrome P-450 catalysis. FASEB J. 4, 2453 – 2459. Harada, A., Okamoto, K., Kamachi, M., 1991. Cooperative bind-

56

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57

ing of porphyrin by anti-porphyrin antibodies. Chem. Lett., 953 – 956. Harada, A., Fukushima, H., Shiotsuki, K., Yamaguchi, H., Oka, F., Kamachi, M., 1997. Peroxidation of pyrogallol by antibody – metalloporphyrin complexes. Inorg. Chem. 36, 6099 – 6102. Hasemann, C.A., Ravichandran, K.G., Peterson, J.A., Deisenhofer, J., 1994. Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution. J. Mol. Biol. 236, 1169 – 1185. Hilvert, D., 2000. Critical analysis of antibody catalysis. Annu. Rev. Biochem. 69, 751 – 793. Hsieh, L.C., Yonkovich, S., Kochersperger, L., Schultz, P.G., 1993. Controlling chemical reactivity with antibodies. Science 260, 337 – 339. Hsieh, L.C., Stephans, J.C., Schultz, P.G., 1994. An efficient antibody-catalyzed oxygenation reaction. J. Am. Chem. Soc. 116, 2167. Ito, S., Matsuoka, T., Watanabe, I., Kagasaki, T., Serizawa, N., Hata, T., 1999. Crystallization and preliminary X-ray diffraction analysis of cytochrome P450sca-2 from Streptomyces carbophilus involved in production of pravastatin sodium, a tissue-selective inhibitor of HMG-CoA reductase. Acta Crystallogr., D. Biol. Crystallogr. 55, 1209 – 1211. Iverson, B.L., Lerner, R.A., 1990. Sequence-specific peptide cleavage catalyzed by an antibody. Science 243, 1184 – 1187. Izadar, L., Friboulet, A., Remy, M.H., Roseto, A., Thomas, D., 1993. Monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity. Proc. Natl. Acad. Sci. U. S. A. 90, 8876 – 8880. Kawamura-Konishi, Y., Hosomi, N., Neya, S., Sugano, S., Funasaki, N., Suzuki, H., 1996a. Kinetic characterization of antibody catalyzed insertion of metal ion into porphyrin. J. Biochem. 119, 857 – 862. Kawamura-Konishi, Y., Neya, S., Funasaki, N., Suzuki, H., 1996b. Specific orientation of porphyrin at the binding site of catalytic antibody. Biochem. Biophys. Res. Commun. 225, 537 – 544. Kawamura-Konishi, Y., Asano, A., Yamasaki, M., Tashiro, H., Suzuki, H., 1998. Peroxidase activity of an antibody – ferric porphyrin complex. J. Mol. Catal., B Enzym. 4, 181 – 190. Keinan, E., Sinha, S.C., Sinha-Bagchi, A., Benory, E., Ghozi, M.C., Eshhar, Z., Green, B.S., 1990. Towards antibody-mediated metallo-porphyrin chemistry. Pure Appl. Chem. 62, 2013 – 2019. Keinan, E., Benory, E., Sinha, S.C., Sinha-Bagchi, A., Eren, D., Eshhar, Z., Green, B.S., 1992. Catalytic antibodies: circular dichroism and ultraviolet-visible studies of antibody – metalloporphyrins interactions. Inorg. Chem. 31, 5433 – 5438. Khoda, K., Kakehi, M., Ohtsuji, Y., Takagi, M., Imanaka, T., 1997. Studies of high thermostability and peroxidase activity of recombinant antibody L chain – porphyrin Fe(III) complex. FEBS Lett. 407, 280 – 284. Ko¨hler, G., Milstein, C., 1975. Continuous culture of fused cells secreting antibodies of predefined specificity. Nature 256, 495 – 497. Lerner, R.A., Benkovic, S.J., Schultz, P.G., 1991. At the crossroads of chemistry and immunology: catalytic antibodies. Science 252, 659 – 667. Liu, X., Chen, S., Feng, Y., Gao, G., Yang, T., 1999. Study of

abzymes with cytochrome P450 activity. Ann. N. Y. Acad. Sci. 750, 273 – 275. Mahy, J.P., Desfosses, B., de Lauzon, S., Quilez, R., Desfosses, B., Lion, L., Mansuy, D., 1998. Appl. Biochem. Biotechnol. 75, 103 – 127. Mansuy, D., Battioni, P., 1989. Alkane functionnalisation by cytochrome P-450 and by model systems using O2 or H2O2. In: Hill, C.L. (Ed.), Activation and Functionnalisation of Alkanes. Wiley, New York, pp. 195 – 218. Mansuy, D., Battioni, P., 1994. Cytochrome P450 model systems. In: Sheldon, R.A. (Ed.), Metalloporphyrins in Catalytic Oxidations. Marcel Dekker, New York, pp. 99 – 132. Mansuy, D., Battioni, P., Battioni, J.P., 1989. Chemical model systems for drug-metabolizing cytochrome-P-450-dependent monooxygenases. Eur. J. Biochem. 184, 267 – 285. Marnett, L.J., Kennedy, T.A., 1995. Comparison of the peroxidase activity of hemoproteins and cytochrome P450 (EC 1 14 14 1). In: Ortiz de Montellano, P.R. (Ed.), Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed. Plenum, New York, pp. 49 – 80. Marques, H.M., Shongwe, M.S., Munro, O.Q., Egan, T.J., 1997. The haem peptides from cytochrome c. Exploring the basic chemistry of the iron porphyrins and modelling aspects of the haemoproteins. S. Afr. Tydskr. Chem. 50, 166 – 180. Nimri, S., Keinan, E., 1999. Antibody – metalloporphyrin catalytic assembly mimics natural oxidation enzymes. J. Am. Chem. Soc. 121, 8978 – 8982. Ortiz de Montellano, P.R., 1995. Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed. Plenum, New York. Osman, A.M., Koerts, J., Boersma, M.G., Boeren, S., Veeger, C., Rietjens, I., 1996. Microperoxidase/H2O2-catalysed aromatic hydroxylation proceeds by a cytochrome-P450 type oxygen transfer reaction mechanism. Eur. J. Biochem. 240, 232 – 238. Park, S.Y., Shimizu, H., Adachi, S., Shiro, Y., Iizuka, T., Nakagawa, A., Tanaka, I., Shoun, H., Hori, H., 1997. Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor. FEBS Lett. 412, 346 – 350. Poulos, T.L., 1996. The role of proximal ligand in heme enzymes. J. Biol. Inorg. Chem. 1, 356 – 359. Poulos, T.L., Finzel, B.C., Gunsalus, I.C., Wagner, G.C., Kraut, J., 1985. The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J. Biol. Chem. 260, 16122 – 16130. Poulos, T.L., Cupp-Vickery, J., Li, M., 1995. In: Ortiz de Montellano, P.R. (Ed.), Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed. Plenum, New York, pp. 125 – 150. Quilez, R., de Lauzon, S., Desfosses, B., Mansuy, D., Mahy, J.P., 1996. Artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy substituted tetraarylporphyrin hapten and exhibiting a high affinity for iron-porphyrins. FEBS Lett. 395, 73 – 76. Ravichandran, K.G., Boddupalli, S.S., Hasermann, C.A., Peterson, J.A., Deisenhofer, J., 1993. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450s. Science 261, 731 – 736. Reymond, J.L., 1999. Catalytic antibodies for organic synthesis. Top. Curr. Chem. 200, 59 – 93.

R. Ricoux et al. / Journal of Immunological Methods 269 (2002) 39–57 Ricoux, R., Boucher, J.L., Mansuy, D., Mahy, J.P., 2000. Formation of iron(II)-nitrosoalkane complexes: a new activity of microperoxidase 8. Biochem. Biophys. Res. Commun. 278, 217 – 223. Ricoux, R., Boucher, J.L., Mansuy, D., Mahy, J.P., 2001. Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals. Eur. J. Biochem. 268, 3783 – 3788. Rietjens, I.M., Osman, A.M., Veeger, C., Zakharieva, O., Antony, J., Grodzicki, M., Trautwein, A.X., 1996. On the role of the axial ligand in heme based catalysis of the peroxidase and cytochrome P450 type. J. Biol. Inorg. Chem. 1, 372 – 376. Roberts, V.A., Iverson, B.L., Iverson, S.A., Benkovic, S.J., Lerner, R.A., Getzoff, E.D., Tainer, J.A., 1990. Antibody remodeling: a general solution to the design of a metal coordination site in an antibody binding pocket. Proc. Natl. Acad. Sci. U. S. A. 87, 6654 – 6658. Romersberg, F.E., Santarsiero, B.D., Spiller, B., Yin, J., Barnes, D., Schultz, P.G., Stevens, R.C., 1998. Structural and kinetic evidence for strain in biological catalysis. Biochemistry 37, 14404 – 14409. Ruckpaul, K., Rein, H., 1984. Cytochrome P450. Akademie Verlag, Berlin. Rusvai, E., Vegh, M., Kramer, M., Horvat, I., 1988. Hydroxylation of aniline mediated by heme-bound oxy-radicals in a heme peptide model system. Biochem. Pharmacol. 37, 4574 – 4577. Savitsky, A.P., Demcheva, M.V., Mantrova, E.Y., Ponomarev, G.V., 1994a. Monoclonal antibodies against metalloporphyrins. Specificity of interaction with structurally different metalloporphyrins. FEBS Lett. 355, 314 – 316. Savitsky, A.P., Nelen, M.I., Yatsmirsky, A.K., Demcheva, M.V., Ponomarev, G.V., Sinikov, I.V., 1994b. Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III)-coproporphyrin. Appl. Biochem. Biotechnol. 47, 317 – 327. Schultz, P.G., Lerner, R.A., 1995. From molecular diversity to catalysis: lessons from the immune system. Science 269, 1835 – 1842.

57

Schwabacher, A.W., Weinhouse, M.I., Auditor, M.M., Lerner, R.A., 1989. Metalloselective anti-porphyrin monoclonal antibody. J. Am. Chem. Soc. 111, 2344 – 2346. Shokat, K.M., 1988. An antibody-mediated redox reaction. Angew. Chem. Int. Ed. Engl. 27, 1172 – 1174. Stevenson, J.D., Thomas, N.R., 2000. Catalytic antibodies and other biomimetic catalysts. Nat. Prod. Rep. 17, 535 – 577. Takagi, M., Khoda, K., Hamuro, T., Harada, A., Yamaguchi, H., Harada, A., Kamachi, M., Imanaka, T., 1995. Thermostable peroxidase activity with a recombinant antibody L chain – porphyrin Fe(III) complex. FEBS Lett. 375, 273 – 276. Thomas, N.R., 1994. Hapten design for the generation of catalytic antibodies. Appl. Biochem. Biotechnol. 47, 345 – 372. Thomas, N.R., 1996. Catalytic antibodies—reaching adolescence? Nat. Prod. Res. 13, 479 – 511. Tresca, J.P., Ricoux, R., Pontet, M., Engler, R., 1995. Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay. Ann. Biol. Clin. (Paris) 53, 227 – 231. Wade, W.S., Koh, J.S., Han, N., Hoekstra, D.M., Lerner, R.A., 1993a. Engineering metal coordination sites into the antibody light chain. J. Am. Chem. Soc. 115, 4449 – 4456. Wade, W.S., Ashley, J.A., Jahangiri, G.K., Mc Elhaney, G., Janda, K.D., Lerner, R.A., 1993b. A highly selective metal-activated catalytic antibody. J. Am. Chem. Soc. 115, 4906 – 4907. Wentworth, P., Janda, K.D., 1998. Catalytic antibodies. Curr. Opin. Chem. Biol. 2, 138 – 144. White, R.E., Coon, M.J., 1980. Oxygen activation by cytochrome P-450. Annu. Rev. Biochem. 49, 315 – 356. Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F., McRee, D.E., 2000. Microsomal cytochrome P4502C5: comparison to microbial P450s and unique features. J. Inorg. Biochem. 81, 183 – 190. Yano, J.K., Koo, L.S., Schuller, D.J., Li, H., Ortiz de Montellano, P.R., Poulos, T.L., 2000. Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus. J. Biol. Chem. 275, 31086 – 31092.