Mahy, J.-P.

FEBS Lett. 1996, 395, 73-76, 10.1016/0014-5793(96)01006-X

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)α,α,α,β‐mesotetrakis‐orthocarboxyphenyl‐porphyrin (Fe(ToCPP))‐KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG, 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 K d values of 2.9 × 10−9 M and 5.5 × 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 highspin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2α‐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 (3ethylbenzothiazoline‐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. k cat values of 100 min−1 and 63 min−1 and k cat/K m. values of 105 M−1 s−1 and 119 M−1 s−1 have been found respectively for the two IgG1‐Fe(ToCPP) complexes.

Mahy, J.-P.

Eur. J. Biochem. 2002, 269, 470-480, 10.1046/j.0014-2956.2001.02670.x

An artificial peroxidase‐like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III)–α,α,α,β‐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.

Golinelli-Pimpaneau, B.

PLoS One 2012, 7, e51128, 10.1371/journal.pone.0051128

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

Mahy, J.-P.

FEBS Lett. 1999, 443, 229-234, 10.1016/S0014-5793(98)01703-7

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)‐α,α,α,β‐meso‐tetrakis(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°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 H2O2 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 K m(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.