Tài liệu Báo cáo khoa học: Coordination chemistry of iron(III)±porphyrin±antibody complexes In¯uence

Coordination chemistry of iron(III)±porphyrin±antibody complexes

In¯uence on the peroxidase activity of the axial coordination of an imidazole

on the iron atom

Solange de Lauzon1

, Daniel Mansuy1 and Jean-Pierre Mahy2

1

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Rene Descartes,

Paris, France; 2

Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, ICMO, BaÃt. 420,

Universite Paris-Sud XI, Orsay, France

An arti®cial peroxidase-like hemoprotein has been obtained

by associating a monoclonal antibody, 13G10, and its

iron(III)±a,a,a,b-meso-tetrakis(ortho-carboxyphenyl)por￾phyrin [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 res￾idue 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. Com￾plexes 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 arti®cial 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.

Keywords: catalytic antibody; peroxidase; arti®cial hemo￾protein; porphyrin; imidazole.

The production of monoclonal antibodies raised against

transition state analogs has proven to be a powerful strategy

to obtain antibodies that are able to catalyze a wide range of

reactions [1±8]. However, as most of these catalytic

antibodies have modest catalytic ef®ciencies, several other

strategies have been envisioned. A ®rst strategy involves the

production of antibodies directed toward the idiotype of

antienzymes antibodies. This strategy has led to antibodies

that display an acetylcholine esterase activity, with the

highest ef®ciency (1.35 ´ 105 M)1

ás

)1

) ever reported for

catalytic antibodies [9], or a b-lactamase activity [10]. A

second strategy is based on the association of antibodies

with cofactors such as inorganic cofactors [11,12], natural

cofactors [13], metal ions [14±17], or metal cofactors [18±40].

In particular, antibodies raised against porphyrin deriva￾tives have received in the last few years considerable

attention as models for hemoproteins of biological impor￾tance such as cytochromes P450 [41] and heme peroxidases

[42]. Antibodies have thus been elicited against meso￾carboxyaryl substituted- [19,23,28,31±33,36,38], N-substi￾tuted- [20,21,27,29,30,34,39], and Sn- [22,24] or Pd- [25,26]

porphyrins. Five of the obtained antibodies [21,27,28,31,34]

were found to have a signi®cant peroxidase activity

with kcat/Km values ranging between 3.8 ´ 103 and

2.9 ´ 105 M)1

ámin)1

. Three metalloporphyrin±antibody

complexes were found to have a cytochrome P450-like

activity: two had a weak catalytic activity for the epoxida￾tion of styrene [22,39] and, more recently, a monoclonal

antibody raised against a water soluble Sn(IV) porphyrin

containing an axial a-naphthoxy ligand, was found to be

able, in the presence of a Ru(II) porphyrin cofactor, to

catalyze the stereoselective sulfoxidation of aromatic sul-

®des by iodosylbenzene [43]. In previous papers [31,36,38],

we reported the production of two monoclonal antibodies,

Correspondence to J.-P. Mahy, Laboratoire de Chimie Bioorganique

et Bioinorganique, FRE 2127 CNRS, ICMO, Baà t. 420, UniversiteÂ

Paris-Sud XI, 91405 Orsay Cedex, France.

Fax: + 36 1 01 69 15 72 81, E-mail: [email protected]

Abbreviations: ToCPP, meso-tetrakis(ortho-carboxyphenyl)porphyrin;

DoCPP, meso-di(ortho-carboxyphenyl)diphenylporphyrin; MoCPP,

meso-mono(ortho-carboxyphenyl) triphenylporphyrin; ABTS,

2,2¢-azinobis(3-ethylbenzothiazoline-6 sulfonic acid); ImH, imidazole;

KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin;

ELISA, Enzyme linked immunosorbent assay.

Enzymes: cytochrome P-450 (EC 1.14.14.1); horseradish peroxidase

(EC 1.11.1.7).

(Received 27 July 2001, revised 7 November 2001, accepted 14

November 2001)

Eur. J. Biochem. 269, 470±480 (2002) Ó FEBS 2002