Mahy, J.-P.

Eur. J. Biochem. 1998, 257, 121-130, 10.1046/j.1432-1327.1998.2570121.x

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin {α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin]}, and which exhibit in the presence of this α,α,α,β‐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 α,α,α,β‐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 α,α,α,β‐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 α, α, β position are recognized by both 13G10 and 14H7 with the two in the α, β 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 α,α,α,β‐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 α,α,α,β‐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.

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.

Mahy, J.-P.

Eur. J. Biochem. 2004, 271, 1277-1283, 10.1111/j.1432-1033.2004.04032.x

In order to estimate the size of the cavity remaining around the heme of the 3A3–microperoxidase 8 (MP8) hemoabzyme, the formation of 3A3–MP8–Fe(II)‐nitrosoalkane complexes upon oxidation of N‐monosubstituted hydroxylamines was examined. This constituted a new reaction for hemoabzymes and is the first example of fully characterized Fe(II)–metabolite complexes of antibody–porphyrin. Also, via a comparison of the reactions with N‐substituted hydroxylamines of various size and hydrophobicity, antibody 3A3 was confirmed to bring about a partial steric hindrance on the distal face of MP8. Subsequently, the influence of the antibody on the stereoselectivity of the S‐oxidation of sulfides was examined. Our results showed that MP8 alone and the antibody–MP8 complex catalyze the oxidation of thioanisole by H2O2 and tert‐butyl hydroperoxide, following a peroxidase‐like two‐step oxygen‐transfer mechanism involving a radical–cation intermediate. The best system, associating H2O2 as oxidant and 3A3–MP8 as a catalyst, in the presence of 5% tert‐butyl alcohol, led to the stereoselective S‐oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer. This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes.