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An artificial peroxidase-like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porphyrin [Fe(ToCPP)] hapten. In this antibody, about two-thirds of the porphyrin moiety is inserted in the binding site, its ortho-COOH substituents being recognized by amino-acids of the protein, and a carboxylic acid side chain of the protein acts as a general acid base catalyst in the heterolytic cleavage of the O-O bond of H2O2, but no amino-acid residue is acting as an axial ligand of the iron. We here show that the iron of 13G10-Fe(ToCPP) is able to bind, like that of free Fe(ToCPP), two small ligands such as CN-, but only one imidazole ligand, in contrast to to the iron(III) of Fe(ToCPP) that binds two. This phenomenon is general for a series of monosubstituted imidazoles, the 2- and 4-alkyl-substituted imidazoles being the best ligands, in agreement with the hydrophobic character of the antibody binding site. Complexes of antibody 13G10 with less hindered iron(III)-tetraarylporphyrins bearing only one [Fe(MoCPP)] or two meso-[ortho-carboxyphenyl] substituents [Fe(DoCPP)] also bind only one imidazole. Finally, peroxidase activity studies show that imidazole inhibits the peroxidase activity of 13G10-Fe(ToCPP) whereas it increases that of 13G10-Fe(DoCPP). This could be interpreted by the binding of the imidazole ligand on the iron atom which probably occurs in the case of 13G10-Fe(ToCPP) on the less hindered face of the porphyrin, close to the catalytic COOH residue, whereas in the case of 13G10-Fe(DoCPP) it can occur on the other face of the porphyrin. The 13G10-Fe(DoCPP)-imidazole complex thus constitutes a nice artificial peroxidase-like hemoprotein, with the axial imidazole ligand of the iron mimicking the proximal histidine of peroxidases and a COOH side chain of the antibody acting as a general acid-base catalyst like the distal histidine of peroxidases does.

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 x 10(-9) M and 5.5 x 10(-9) M have been determined for the two IgG1-Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgG1-Fe(ToCPP) complexes were high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-Fe(ToCPP) complex was a low-spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1-Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron-porphyrin had no effect on its catalytic activity. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.