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.

DeGrado, W.F.; Lombardi, A.

C. R. Chim. 2007, 10, 703-720, 10.1016/j.crci.2007.03.010

A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.

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.

Mahy, J.-P.

C. R. Chim. 2007, 10, 684-702, 10.1016/j.crci.2006.12.014

Catalytic antibodies with a metalloporphyrin cofactor or “hemoabzymes”, used as models for hemoproteins like peroxidases and cytochrome P450s, represent a promising route to catalysts tailored for selective oxidation reactions. The first strategy has been to produce anti-porphyrin antibodies, raised against various N-substituted- and meso-carboxyaryl-porphyrins, which led to monoclonal antibodies exhibiting, in the presence of the corresponding iron-porphyrin cofactor, a significant peroxidase activity. We ourselves obtained an artificial hemoprotein by associating a monoclonal antibody, 13G10, and its iron(III)-α,α,α,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin (Fe(ToCPP)) hapten, which exhibited a significant peroxidase activity. Biological studies suggested that in this antibody, a carboxylic acid side chain of the protein participated in the catalysis, but no amino acid residue acting as an axial ligand of the iron was detected. Therefore, to provide the iron atom with an axial ligand, we raised antibodies against microperoxidase 8, a heme octapeptide containing a histidine bound to the iron atom. This strategy was successful, as an antibody–microperoxidase 8 complex (3A3–MP8) led to the best kcat/Km ever reported for antibody–porphyrin complexes. The ability of the 3A3–MP8 complex to catalyze the selective oxidation of substrates was studied and it was found able to catalyze the regioselective nitration of aromatics by NO2−/H2O2 as well as the stereoselective oxidation of sulfides like thioanisole by H2O2. Other strategies based on antibodies have to be developed to obtain more efficient biomimetic systems for cytochrome P450s. A first one could involve the modification of anti-substrate antibodies by covalent linkage of an iron(III)-porphyrin close to the binding site of the substrate, to obtain an artificial hemoprotein able to catalyze its regioselective oxidation.

Ward, T.R.

C. R. Chim. 2007, 10, 678-683, 10.1016/j.crci.2007.02.020

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N-protected dehydroamino acids using streptavidin as host protein. Introduction of an (R)-proline spacer between the biotin anchor and the diphosphine moiety affords a versatile ligand Biot-(R)-Pro-1 which displays good (S)-selectivities in the presence of streptavidin (91% ee). The resulting artificial metalloenzyme [Rh(Biot-(R)-Pro-1)(COD)]+ ⊂ WT-Sav displays increased stability against organic solvents.