12 publications

12 publications

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

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.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 105 M-1 * s-1

Coordination Chemistry of Iron(III)-Porphyrin-Antibody Complexes Influence on the Peroxidase Activity of the Axial Coordination of an Imidazole on the Iron Atom

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.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 15200 M-1 * s-1

Coordination Chemistry Studies and Peroxidase Activity of a New Artificial Metalloenzyme Built by the “Trojan Horse” Strategy

Mahy, J.-P.

J. Mol. Catal. A: Chem. 2010, 317, 19-26, 10.1016/j.molcata.2009.10.016

In the general context of green chemistry, a considerable research effort is devoted to the elaboration of new artificial metalloproteins that catalyze, under mild conditions, the oxidation of a wide range of organic compounds, using cheap and environmentally friendly oxidants. A new artificial hemoprotein was obtained by the so-called “Trojan horse” strategy involving the non-covalent insertion of a cationic iron–porphyrin–estradiol cofactor into an anti-estradiol antibody. UV–vis titrations showed the formation of a 1/2 antibody/cofactor complex with a dissociation constant KD = 4.10−7 M. UV–vis determination of the Fe-imidazole binding constants showed that the protein provided a weak steric hindrance around the iron–porphyrin cofactor. The antibody–estradiol–iron–porphyrin complex displayed a peroxidase activity and catalyzed the oxidation of ABTS by H2O2 with about double the efficiency of the iron–porphyrin–estradiol alone. Kinetic studies revealed that this was due to a faster formation of the intermediate high valent iron–oxo species in the presence of the antibody protein. Consequently, the association of an anti-estradiol antibody with an iron–porphyrin–estradiol cofactor leads to a new artificial hemoprotein with an interesting peroxidase activity and the “Trojan horse” strategy appears as a valuable method to generate artificial metalloenzymes that could act as biocatalysts for selective oxidations.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 7A3
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: k1 = 574 M-1 * min-1

Enzyme Repurposing of a Hydrolase as an Emergent Peroxidase Upon Metal Binding

Fujieda, N.; Ward, T.R.

Chem. Sci. 2015, 6, 4060-4065, 10.1039/c5sc01065a

Adding a metal cofactor to a protein bearing a latent metal binding site endows the macromolecule with nascent catalytic activity.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 35
ee: ---
PDB: ---
Notes: ---

Flavohemoglobin: A Semisynthetic Hydroxylase Acting in the Absence of Reductase

Kaiser, E.T.

J. Am. Chem. Soc. 1987, 109, 606-607, 10.1021/ja00236a062

n/a


Metal: Fe
Ligand type: Porphyrin
Host protein: Hemoglobin
Anchoring strategy: ---
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Helichrome: Synthesis and Enzymatic Activity of a Designed Hemeprotein

Kaiser, E.T.; Sasaki, T.

J. Am. Chem. Soc. 1989, 111, 380-381, 10.1021/ja00183a065

n/a


Metal: Fe
Ligand type: Porphyrin
Host protein: Artificial construct
Anchoring strategy: Covalent
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: Only 60 amino acids

Hemoabzymes: Towards New Biocatalysts for Selective Oxidations

Mahy, J.-P.

J. Immunol. Methods 2002, 269, 39-57, 10.1016/S0022-1759(02)00223-5

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 M−1 min−1. 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 α-naphtoxy 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 10−7 M. The corresponding 3A3–MP8 complex was found to have a good peroxidase activity characterized by a kcat/Km value of 2×106 M−1 min−1, 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.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 3A3
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 33000 M-1 * s-1

Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces Lividans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins

Mahy, J.-P.

Bioconjug. Chem. 2008, 19, 899-910, 10.1021/bc700435a

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-α4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 µM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV–visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.


Metal: Fe
Ligand type: Porphyrin
Host protein: Xylanase A (XynA)
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 1083 M-1 * s-1

Manganese Terpyridine Artificial Metalloenzymes for Benzylic Oxygenation and Olefin Epoxidation

Lewis, J.C.

Tetrahedron 2014, 70, 4245-4249, 10.1016/j.tet.2014.03.008

New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.


Metal: Mn
Ligand type: Poly-pyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Max TON: 19.2
ee: ---
PDB: 3EMM
Notes: ---

Metal: Mn
Ligand type: Poly-pyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Reaction: Epoxidation
Max TON: 19.8
ee: ---
PDB: 3EMM
Notes: ---

Peroxidase Activity of an Antibody-Heme Complex

Schultz, P.G.

J. Am. Chem. Soc. 1990, 112, 9414-9415, 10.1021/ja00181a065

The specificity and diversity of the immune system have recently been exploited in the generation of antibodies that catalyze a wide variety of chemical reactions.1·2 Several general strategies for the design of catalytic antibodies have emerged, including the use of antibody binding energy to enhance the chemical reactivity of a cofactor or to position a cofactor and a substrate in close proximity.3,4 An intriguing target for antibody-cofactor catalysis is the oxidative reactions characteristic of heme proteins. Here we report that antibodies specific for A-methylmesoporphyrin IX bind iron(III) mesoporphyrin IX and that the resulting complex catalyzes the oxidation of several substrates. These studies are a first step toward the development of selective antibody-heme monooxygenase catalysts.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody7G12-A10-G1-A12
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 200-500
ee: ---
PDB: ---
Notes: ---

Studies of the Reactivity of Artificial Peroxidase-Like Hemoproteins Based on Antibodies Elicited Against a Specifically Designed ortho-Carboxy Substituted Tetraarylporphyrin

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.


Metal: Fe
Ligand type: Porphyrin
Host protein: Antibody 13G10
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: TOF = 4.7 min-1

Towards Antibody-Mediated Metallo-Porphyrin Chemistry

Keinan, E.

Pure Appl. Chem. 1990, 62, 2013-2019, 10.1351/pac199062102013

An attempt was made to mimic cytochrome P-450-like activity using antibodies elicited against metallo-porphyrins. Monoclonal antibodies raised against a water-soluble Sn(1V) porphyrin complex (1) exhibited Specificity for a variety of monomeric metalloporphyrins, as well as for the b-0x0-Fe(III) porphyrin dimer 2. Some antibodies were found to be more selective for the monomer 1 than for the dimer 2, suggesting an "edge-on" recognition of the planar porphyrin molecule. The catalytic activity of the antibody-metalloporphyrin complexes was investigated using the epoxidation of styrene by iodosobenzene as a model reaction. Three biphasic media were studied for this reaction: reverse micelles, microemulsions, and solid catalyst in organic solvent. The most promising results were obtained with solid catalyst (obtained via lyophilization of equimolar amounts of Mn(TCP)Cl and specific antibody) in dry CHzClz at room temperature, as indicated by the high turnover numbers of the catalyst. A difference in the relative activity of the various monoclonal antibodies (MABs) was noted. The anti-1 antibodies displayed ca. 30-60% higher activity compared to a nonrelevant MAB.


Metal: Mn
Ligand type: Porphyrin
Host protein: Antibody
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 549
ee: ---
PDB: ---
Notes: ---