Tài liệu Báo cáo khoa học: 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 ppt

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 Lauzon
1
, Daniel Mansuy
1
and Jean-Pierre Mahy
2
1
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Â
Rene
Â
Descartes,
Paris, France;
2
Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, ICMO, Ba
Ã
t. 420,
Universite
Â
Paris-Sud XI, Orsay, France
An arti®cial p eroxidase-like h emoprotein 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 H
2
O
2
, but no amino-acid res-
idue is acting as an axial ligand of the iron.We here show that
theironof13G10±Fe(ToCPP)isabletobind,likethatof
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 w ith less hindered i ron(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 increa ses that of 1 3G10±
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 p eroxidases and a

COOH side chain of the an tibody acting as a general acid-
base catalyst like the distal histidine of peroxidases does.
Keywords: catalytic antibody; p eroxidase; arti®cial hemo-
protein; porphyrin; imidazole.
The production of monoclonal antibodies raised against
transition state a nalogs has proven to be a powerful strategy
to obtain antibodies that are able to catalyze a wide range o f
reactions [1±8]. However, as most o f these cata lytic
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 ´ 10
5
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 a s 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 s uch 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 a ctivity
with k
cat
/K
m
values ranging between 3.8 ´ 10
3
and
2.9 ´ 10
5
M
)1
ámin
)1
. Three metalloporphyrin±antibody
complexes were f ound to have a cytochrome P450-like
activity: two had a weak catalytic activity for the epoxida-
tion o f 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 o f a romatic s ul-
®des by iodosylbenzene [43]. In previous papers [31,36,38],
we reported the production of two monoclonal antibodies,
Correspondence to J P. M ahy, Laboratoire de Chimie Bioorganique
et Bioinorganique, FRE 2127 CNRS, ICMO, Baà t. 420, Universite
Â
Paris-Sud XI, 91405 Orsay Cedex, France.

Fax: + 36 1 01 69 15 72 81, E-mail:
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
13G10 and 14H7, which not only bound the hapten,
iron(III)-a,a,a,b-meso-tetrakis(ortho-carboxyphenyl)por-
phyrin [Fe(ToCPP)] (Fig. 1) w ith a high af®nity
(K
d
 10
)9
M
), but also exhibited in i ts presence an
interesting peroxidase activity with k
cat
 540 min
)1
and
k
cat
/K
m

 3.2 ´ 10
4
M
)1
ámin
)1
[38]. Measurements of the
binding constants for various porphyrins [36], together with
pH dependence studies of the kinetics of t he peroxidase
reaction [38] associated with chemical modi®cations of the
antibody protein [36,38] have shown that: (a) approximately
two-thirds of the porphyrin moiety was inserted in the
antibody pocket, three of the ortho-carboxylate substituents
of the meso-phenyl rings being recognized by th e side chains
of amino acids of the antibody [36]; (b) in the case of 13G10,
one carboxylic acid residue of the protein could participate
in the catalysis of the heterolytic cleavage of the O±O bond
of peroxides [38]; (c) unfortunately, no amino-acid residue
was coordinating the iron atom. We have thus undertaken
studies of the coordination chemistry of the iron(III) of
Fe(ToCPP) bound or not to antibody 13G10 with two
objectives: ®rst, to get more precise information about the
topology of the binding site of the antibody and particularly
to appreciate the size of the cavity left around the iron atom
and which kind of ligands it can accommodate; second, to
measure the in¯uence of an axial ligand of the iron atom
such as imidazole on the catalytic activity of the Fe(ToCPP)
±IgG complex.
In the present paper, we report the results obtained by
absorption spectroscopy studies which show that: (a) the

iron atom is able to bind two CN
±
ligands in Fe(ToCPP)
alone as well as in its complex with antibody 13G10; (b) in
contrast, whereas the iron(III) of Fe(ToCPP) alone is able to
bind two imidazole ligands, that of the Fe(ToCPP))13G10
complex is able to bind only one imidazole ligand; (c) the
binding of one imidazole to the iron atom inhibits the
peroxidase activity of the Fe(ToCPP))13G10 complex
whereas it e nhances that of the complexes of 13G10 with
iron(III)-mono- and di-ortho-carboxyphenyl substituted
tetraaryl porphyrins, Fe(MoCPP) and Fe(DoCPP). Finally,
this paper shows that the association of an anti-porphyrin Ig
13G10 with a F e(III)-di-ortho-carboxyphenyl-porphyrin
and imidazole provides an accurate arti®cial peroxidase-
like hemoprotein, with the axial imidazole ligand of the iron
mimicking the proximal histidine o f peroxidases and a
COOH side ch ain of the antibody acting as a general acid-
base catalyst like the distal histidine of peroxidases does.
EXPERIMENTAL PROCEDURES
Chemicals
Sodium azide and sodium isothiocyanate were from Sigma.
Potassium cyanide, imidazole, 1-methylimidazole, 1-benzy-
limidazole, 2-methylimidazole, 4-methylimidazole, and
2-ethylimidazole were from Fluka. 2,2¢-azinobis(3-eth yl-
benzothiazoline-6 sulfonic acid) (ABTS), and H
2
O
2
from

Sigma.
Synthesis of iron(III)±
ortho
-carboxyphenyl
substituted tetraarylporphyrins
The synthesis of the four atropoisomers of Fe(ToCPP) as
well as those of Fe(MoCPP) and of Fe(DoCPP) has been
made in three steps as described in a previous paper [36].
The ortho-carboxymethyl substituted tetraaryl porphyrins
were ®rst synthesized by reaction at room temperature of
ortho-carbomethoxybenzaldehyde with pyrrole in CH
2
Cl
2
in the presence of BF
3
-etherate as catalyst according to an
already described procedure [36,43]. The atropoisomers
have then been separated on a silicagel column and
Fig. 1. Structure and nomenclature of the
various porphyrins used in this work.
Ó FEBS 2002 Peroxidase-like Fe±porphyrin±antibody complexes (Eur. J. Biochem. 269) 471
identi®ed by absorption,
1
H NMR and mass spectroscopies
[36]. The iron atom was then inserted b y reaction of the
isolated atropoisomers with Fe(CO)
5
in the presence of I
2

in
toluene at room temperature to avoid isome rization [44].
Finally, the ortho-carboxy substituted t etraarylporphyrin
isomers were subsequently obtained by saponi®cation of the
ortho-methyl ester substituents in 2
M
KOH in 80% EtOH
at room temperature [45].
Production of monoclonal antibodies
The generation of monoclonal antibodies has been
reported i n d etail in previous p apers [31,36,38]. Fe(ToC-
PP) was activated by N-hydroxysuccinimide and cova-
lently attached to keyhole limpet hemocyanin and BSA in
phosphate buffered saline p H 7.5. The conjugates were
then puri®ed by chromatography on Biogel P10 and four
5-week-old, female BALB/c mice w ere i mmunized con -
ventionally with the Fe(ToCPP)±KLH conjugate. The
spleen cells of the m ouse showing the best immune
response were fused with PAI myeloma cells acc ording to
Ko
È
hler & Milstein [46]. The supernatants from the
hybridoma cells were screened by ELISA for binding to
the h apten±BSA c onjugate p eroxidase linked goat anti-
(mouse Ig) Ig [47]. Positive hybridoma were cloned twice
and propagated in ascites. Antibodies were then puri®ed
from ascite ¯uid by protein A af®nity chromatography
and their purity and homogeneity were checked by SDS
gel electrophoresis.
Absorption spectroscopy measurements

Absorption spectra were recorded at 19  0.1 °Cusingan
UVIKON 860 UV/visible spectrophotometer as follows.
The sample cuvette contained either 2 l
M
Fe(ToCPP) or
Fe(ToCPP) preincubated with 3 l
M
13G10 in 50 m
M
phosphate buffer pH 7.0, the reference cuvette only co n-
tained 50 m
M
phosphate buffer pH 7.0. Equal amounts of
ligand L (L  imidazole, mono-substituted imidazole, CN
±
,
SCN
±
,N
3
±
)(2
M
in the same buffer) were then added in both
cuvettes and difference spectra were recorded between 350
and 650 nm.
In most cases, the spectral evolution observed involved
the formation of well de®ned isobestic points indicating the
presence of two absorbing species. The reaction could then
be represented by:

PFe
III
 nL  PFe
III
L
n
1
where P  ToCPP, 13G10±(ToCPP) and L  imidazole,
mono-substituted imidazole, CN
±
,SCN
±
,N
3
±
. According to
Brault & Rougee [48], it could then be analyzed by means of
the standard equation
1aDA  1aDA
I
 K
d
aDA
I
 1aL
n
2
where DA  A ) A
0
, DA

I
 A ) A
I
and A
0
, A
I
,andA
are the absorbances of the initial, ®nal and mixed species,
respectively. The linearity of the graph representing 1/DA as
a function of 1/[L]
n
wasthenassayedwithn  1andn  2
and K
d
and A
I
could be determined graphically. I t is
noteworthy that when n  1, C
50
 K
d
,whereaswhenn  2,
C
50
 K
1a2
d
,withC
50

representing the concentration of
ligand for which half o f the starting Fe(ToCPP) or
Fe(ToCPP))13G10 complex has been converted into
(ToCPP)Fe(L)
n
or 13G10±(T oCPP) Fe(L)
n
.
Assay of peroxidase activity
To assay the peroxidase activity of the various iron(III)±
ortho-carboxy substituted tetraarylporphyrins and their
complexes with antibody 13G10, the oxidation of ABTS
by H
2
O
2
was performed at 19  0.1 °Cin0.1
M
citrate/
0.2
M
phosphate buffer, pH 5, containing 0.2% dimethyl-
sulfoxide. The absorbance was monitored at 414 nm using
an UVIKON 860 UV/visible spectrophotometer. T he initial
rates of oxidation were determined from the slope at the
origin of the curve representing the variations of the
absorbance at 414 nm as a function of time, using an e
value of 28 000
M
)1

ácm
)1
[21].
In a ®rst set of experiments, ABTS (0.2 m
M
) was oxidized
by H
2
O
2
(0.7 m
M
) in the presence of 0.4 l
M
Fe(ToCPP) or
Fe-a,a-1,2- or -a,b-1,2-(DoCPP) preincubated or not with
0.6 l
M
13G10 as catalysts.
In a second set of experiments, ABTS (0.03 m
M
)was
oxidized by H
2
O
2
(5 m
M
) in the presence of 0.4 l
M

Fe(MoCPP) preincubated 60 min with 0.2 l
M
13G10 or
0.2 l
M
Fe(ToCPP) or Fe-a,a-1,2- or -a,b-1,2 -(DoCPP)
preincubated with 0 .4 l
M
13G10.
The in¯uence of imidazole on the kinetic parameters of
the oxidation of ABTS by H
2
O
2
in the presence of Fe±
porphyrin±antibody complexes was examined as follows.
The catalysts were ®rst prepared by preincubation of 0.4 l
M
Fe(ToCPP) or a,a-1,2-Fe(DoCPP) or a,b-1,2-Fe(DoCPP)
with 0.6 l
M
13G10 for 60 min at 19 °C. For the reactions
with imidazole, a further 15 min incubation at 19 °Cwith
50 m
M
imidazole was done; 0.2 m
M
ABTS was then added
and the reaction was s tarted by the addition of H
2

O
2
at
concentrations ranging b etween 0 and 10 m
M
. The initial
rates of oxidation were then measured as above mentioned
and the k
cat
and K
m
were calculated in all the cases from
Lineweaver-Burk plots.
RESULTS
Binding of cyanide to Fe(ToCPP) and to 13G10±Fe(ToCPP)
The reactions of SCN
±
,N
3
±
and CN
±
with the iron(III) of
Fe(ToCPP) and its complex with antibody 13G10 were
examined by UV/visible spectroscopy in 0.1
M
phosphate
buffer, pH 7 at 19  0.1 °C as described in experimental
procedures. SCN
±

and N
3
±
failed to react with both
complexes [data not shown] but, when increasing amounts
of potassium cyanide, up to 11 m
M
,wereaddedtoa2l
M
solution of Fe(ToCPP), the initial spectrum characteristic
of a high spin iron(III) species was gradually replaced, with
isobestic points at 4 07, 479 and 548 nm, by a new
spectrum with maxima of absorption at 417 and 549 nm
(Fig. 2A). Such a spectrum is similar to that already
described for tetraaryl-Fe
III
±CN complexes [49]. As in
addition, 1/DA
417
varied linearly with 1/[CN
±
] ( Fig. 2A,
inset), it is clear that the ®rst reaction observed was the
binding of CN
±
ligand to the iron(III) of Fe(ToCPP)
(Eqn 3), with a calculated K
d
value of 3.70  0.06 m
M

(Table 1).
FeToCPPCN
À
ToCPPFe
III
À CN 3
472 S. de Lauzon et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Further addition of potassium cyanide, up to 50 m
M
,
resulted in the appearence, with isobestic points at 422, 486
and 586 nm, of a new spectrum with peaks at 426, 565 and
600 nm (Fig. 2B), characteristic of a [(tetraarylporphy-
rin)Fe
III
(CN]
2
]
±
species [49]. Accordingly, when 1/ DA
426
was
plotted vs. 1/[CN
±
], a straight line was obtained and a K
d
value of 19.5  0.3 m
M
could be determined graphically
(Table 1).

ToCPPFe
III
À CN  CN
À
ToCPPFe
III
CN
2

À
4
When the same e xperiment was carried o ut with the
Fe(ToCPP))13 G10 comp lex (2 l
M
), a ®rst species absorb-
ing at 420 and 555 nm was formed with isobestic points at
409, 477 and 552 nm for concentrations of CN
±
below
6m
M
(Fig. 3A). A second species absorbing at 429, 541 and
608 nm was obtained, with isobestic points at 428, 485 and
594 nm, for concentrations of CN
±
higher than 10 m
M
(Fig. 3B). As, respectively, 1/DA
420
and 1/DA

429
varied
linearly as a function of 1/[CN
±
] (Fig. 3, insets), it is clear
that those two species were, respectively, the 13G10±
(ToCPP)Fe±CN and 13G10-[(ToCPP)Fe(CN)
2
]
±
complexes.
Accordingly, when (ToCPP)Fe-CN and [(ToCPP)
Fe(CN)
2
]
±
were inserted into the antibody protein, the
characteristic bands in their visible spectrum were shifted
toward higher wavelengths to g ive spectra that were similar
to those obtained upon direct reaction of potassium cyanide
with 13G10±Fe(ToCPP) (data not shown). This shows
that the binding of the two cyanide ligands to the iron
atom of 13G10±Fe(ToCPP) actually occurred inside the
binding pocket of the antibody. The value of the dissoci-
ation constant calculated for 13G10±(ToCPP)Fe±CN
(0.39  0.01 m
M
) was about 10-fold lower than th at calcu-
lated for (ToCPP)Fe±CN (3.70  0.06 m
M

)(Table1),
whereas that of 13G10±[[ToCPP]Fe[CN]
2
]
±
(16.90 
0.13 m
M
) was only slightly lower than that of
Fig. 2. Addition of cyanide to Fe(ToCPP).
(A) Spectral evolution observed for the
addition of 0±11 m
M
CN
±
to 2 l
M
Fe(ToCPP)
in 0.1
M
phosphate buer, pH 7 at 19 °C.
Inset: corresponding values of 1/DA
417
plotted
against 1/[CN
±
]. (B) Spectral evolut ion
observed for the addition of 11±50 m
M
CN

±
to
2 l
M
Fe(ToCPP) in 0.1
M
phosphate buer,
pH 7 at 19 °C. Inset: correspondin g values of
1/DA
417
plotted against 1/[CN
±
].
Table 1. Visible characteristics and C
50
values of the complexes of
Fe(ToCPP) and 13G10- Fe(ToCPP) with cyanide in 50 m
M
phosphate
buer, pH 7.0 at 20 °C.
Complex
Visible bands
kmax (nm)
C
50
a
(m
M
)
(ToCPP)Fe

III
±CN 417, 549 3.70  0.06
13G10±(ToCPP)Fe
III
±CN 420, 555 0.39  0.01
((ToCPP)Fe
III
(CN]
2
)
±
426, 565, 600 19.5  0.3
(13G10±(ToCPP)Fe
III
(CN)
2
)
±
429, ±, ± 16.90  0.13
a
When only one CN
±
is bound to Fe, C
50
 K
d
and when two CN
±
are bound to Fe, C
50

 K
d
1/2
.
Fig. 3. Addition of cyanide to the
Fe(ToCPP))13G10 complex. (A) Spectral
evolution observed for the addition of 0±6 m
M
CN
±
to 2 l
M
13G10±Fe(ToCPP) in 0.1
M
phosphate buer, pH 7 at 19 °C. Inset:
corresponding values of 1/DA
420
plotted
against 1/[CN
±
]. (B) Spectral evolut ion
observed for the addition of 10±50 m
M
CN
±
to 2 l
M
13G10±Fe(ToCPP) in 0.1
M
phos-

phate buer, pH 7 at 19 °C. Inset:
corresponding values of 1/DA
420
plotted
against 1/[CN
±
].
Ó FEBS 2002 Peroxidase-like Fe±porphyrin±antibody complexes (Eur. J. Biochem. 269) 473
[(ToCPP)Fe(CN)
2
]
±
(19.5  0.3 m
M
) (Table 1). The bind-
ing of the ®rst CN
±
ligand to the iron was thus more easy in
the hydrophobic binding pocket of the antibody than the
binding of the second one, most probably because of the
steric hindrance brought by the protein around the iron
atom of the porphyrin.
Binding of monosubstituted imidazoles to Fe(ToCPP)
and to its complex with antibody 13G10
Upon addition of increasing amounts of imidazole (ImH),
up to 14 m
M
,toa2l
M
solution of Fe(ToCPP), the initial

spectrum of the high spin iron(III)±(ToCPP) was gradually
replaced, with isobestic points at 408,475, 551 and 600 nm,
by a new spectrum with maxima of absorption at 417, 549
and 580 nm (Fig. 4). Such a spectrum is similar to that
already described for tetraaryl-Fe
III
[ImH]
2
complexes [50].
As on addition, 1/DA
417
varied linearly with 1/[ImH]
2
but
not with 1/[ImH] (Fig. 5, inset), it is clear that the reaction
observed was the binding of two ImH ligands to the
iron(III) of Fe(ToCPP) (Eqn 5), with a C
50
( K
1a2
d
) value
of 2.70  0.04 m
M
(Table 2).
FeToCPP2ImH ToCPPFe
III
ImH
2
5

When the same experiment was carried out with the
Fe(ToCP P))13G10 complex (2 l
M
), a species absorbing at
419, 552 and 587 nm was formed, with isobestic points at
407 and 538 nm for concentrations of ImH up to 200 m
M
(Fig. 5). As 1/DA
419
varied linearly with 1/[ImH] but not
with 1/[ImH]
2
(Fig. 5, inset), it is clear that contrary to free
Fe(ToCPP), the 13G10±Fe(ToCPP) complex was only able
to bind one imidazole ligand (Eqn 6).
13G10 ÀToCPPFe
III
 ImH
 13G10 ÀToCPPFe
III
ImH6
In addition, the C
50
( K
d
) value calculated in this case
(21.3  0.3 m
M
) is about 10-fold higher than that obtained
for the formation of the (ToCPP)Fe

III
(ImH)
2
complex.
The same reaction was performed with several mono-
substituted imidazoles. In all the cases, the iron of free
Fe(ToCPP) was able to bind two monosubtituted imidazole
ligands whereas that of Fe(ToCPP) complexed with anti-
body 13G10 was able to bind only one, the absorption
spectra being similar to those obtained with non substituted
imidazole (Table 2).
In the case of 1-substituted imidazoles, the C
50
( K
d
)
values observed with 13G10±Fe(ToCPP) were higher than
the C
50
( K
1a2
d
) values observed in the case of free
Fe(ToCPP). Indeed for 1-methyl- and 1-benzylimidazole,
C
50
( K
d
) values of, respectively, 4.30  0.06 m
M

and
14.5  0.2 m
M
were observed in the case of 13G10±
Fe(ToCPP) whereas C
50
( K
1a2
d
) values of, respectively,
2.60  0.04 m
M
and 0.63  0.01 m
M
were observed in the
case of free Fe(ToCPP) (Table 2).
In the case of 2 - and 4-substituted imidazoles, the iron o f
free Fe(ToCPP) was also able to bind two ligands, w ith
much higher C
50
( K
1a2
d
) values (10- to 90-fold) than
those calculated for imidazole and 1-substituted imidazoles
(Table 2). Indeed, C
50
values of 26.2  0.04 m
M
, 56.0 

0.8 m
M
and 54.0  0.8 m
M
could be calculated for
4-methyl-, 2-methyl- a nd 2-ethylimidazole, respectively
Fig. 4. Addition of imidazole to iron(III)-a,a,a,b- meso-tetrakis(ortho-
carboxyphenyl)porphyrin (Fe(ToCPP)). Spec tral evolution observed
for the ad dition of 0±14 m
M
ImH to 2 l
M
Fe(ToCPP) in 0.1
M
phosphate buer, pH 7 at 19 °C. In set: c orrespondin g values o f
1/DA
417
plotted against 1/[ImH] and 1/[ImH ]
2
.
Fig. 5. Ad dition of imida zole to the Fe(ToCPP))13G10 complex.
Spectral evolution observed for t he addition of 0±200 m
M
ImH to
2 l
M
Fe(ToCPP) in 0.1
M
phosphate buer, pH 7 at 19 °C. Inset:
corresponding values of 1/DA

419
plotted against 1/[ImH] and 1/[ImH]
2
.
474 S. de Lauzon et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Table 2). Those values were also 10±15-fold higher than the
C
50
( K
d
) values found for the 13G10±(ToCPP)Fe)4-
methyl- (2.80  0.06 m
M
), 2-methyl (4.10  0.08 m
M
)
and 2-ethylimidazole (3.10  0.05 m
M
)(Table2).
Binding of imidazole to various iron(III)-
ortho
-carboxy
substituted tetraarylporphyrins and to their complexes
with antibody 13G10
We also examined the binding of imidazole to iron(III)-
mono- and di-ortho-carboxyphenyl substituted tetraaryl-
porphyrins, Fe(MoCPP) and Fe(DoCPP) (Fig. 1), which
were previously shown to form complexes with antibody
13G10 with, respectively, a 50-fold lower and an almost
equal af®nity than Fe(ToCPP) [36]. The addition of

increasing amounts of imidazole to 2 l
M
solutions of a,b-
1,2-Fe(DoCPP) or Fe(MoCPP) in 50 m
M
phosphate buffer
pH 7.4, at 19 °C, led to the formation of the corresponding
porphyrin±Fe
III
(ImH)
2
complexes, characterized by absorp-
tion spectra with bands around 420, 550 and 580 nm
(Table 3). However, owing to the low solubility of those
complexes in the reaction medium, their K
d
values could not
be determined accurately. When the same reaction was
performed with a,a-1,2-Fe(DoCPP)-, a,b-1,2-Fe(DoCPP)-
or Fe(MoCPP))13G10 complexes, new complexes, absorb-
ing around 420, 550 and 580 nm were obtained (Table 3). In
all the cases, 1/DA
420
was a linear function of 1/[ImH], which
showed that in those cases, as in the case of Fe(ToCPP),
only one imidazole ligand bou nd to the iron atom. In
addition, the C
50
( K
d

) values c ould b e calculated
(Table 3) and it appeared that the C
50
values obtained in
the case of 13G10-a,a-1,2-Fe(DoCPP) and 13G10-a,b-1,2-
Fe(DoCPP) were threefold and twofold lower than that
obtained with 13G10±Fe(ToCPP). In contrast, a much
higher C
50
value was found for 13G10±Fe(MoCPP)
(236  4m
M
)(Table3).
In¯uence of imidazole on the peroxidase activity
of various iron(III)-
ortho
-carboxy substituted
tetraarylporphyrins and their complexes
with antibody 13G10
The in¯uence of the binding of imidazole to the iron atom
of iron(III)-ortho-carboxy s ubstituted tetraarylporphyrins
and their complexes with antibody 13G10 on their
peroxidase activity was studied. The rate of oxidation of
0.2 m
M
ABTS by 0.7 m
M
H
2
O

2
was then measured at
19 °C in the presence of increasing concentrations of
imidazole, using as catalyst e ither 0.3 l
M
iron(III)±por-
phyrin or 0.3 l
M
iron(III)±porphyrin previously incubated
with 0.6 l
M
13G10. The reactions were performed in
50 m
M
phosphate buffer pH 5 as it had previously been
shown that the peroxidase activity of the 13G10±Fe(ToC-
PP) complex was optimal around this pH value [38]. We
®rst of all checked by absorption spectroscopy that both
Fe(ToCPP) and 13G10±Fe(ToCPP) still bound imidazole
at pH 5 with C
50
values similar to those calculated at pH 7
(data not shown). The peroxidase activity of Fe(ToCPP)
alone was then assayed in the presence of concentrations of
imidazole increasing from 0 to 150 m
M
(Fig. 6). With this
catalyst, the rate of oxidation of ABTS by H
2
O

2
increased
from an initial value of 0.16 l
M
ABTS oxidized per min to
Table 2. Visible characteristics and C
50
values of the complexes of Fe(ToCPP) and 13G10-Fe(ToCPP) with various monosubstituted imidazoles in
50 m
M
phosphate buer, pH 7.0 at 20 °C.
L
P  ToCPP P  13G10-ToCPP
n
Visible bands
kmax (nm)
C
50
a
(m
M
) n
Visible bands
kmax (nm)
C
50
a
(m
M
)

ImH 2 417, 549, 580 (sh) 2.70  0.04 1 419, 552, 587(sh) 21.3  0.3
1-CH
3
-Im 2 418, 550, 580 (sh) 2.60  0.04 1 419, 548, 580(sh) 4.30  0.06
1-Bz-Im 2 417, 546, 582 (sh) 0.63  0.01 1 419, 548, 590(sh) 14.5  0.2
4-CH
3
-Im 2 418, 554, 582 (sh) 26.2  0.4 1 420, ±, - 2.80  0.06
2-CH
3
-Im 2 418, ±, ± 56.0  0.8 1 421, ±, - 4.10  0.05
2-C
2
H
5
-Im 2 419, ±, ± 54.0  0.8 1 421, ±, - 3.10  0.05
a
n and K
d
for Fe
III
(L)
n
complexes were determined as described in Experimental procedures: when n  1, C
50
 K
d
and when n  2,
C
50


Kd1/2
.
Table 3. Visible characteristics and C
50
values of the complexes of various iron(III)-ortho- carboxy-substituted-tetraarylporphyrins with imidazole in
50 m
M
phosphate buer, pH 7.0 at 20 °C in the presence or not of antibody 13G10.
Porphyrin
(Porphyrin)Fe
III
(ImH)
2
Visible bands
kmax (nm)
13G10-(Porphyrin)Fe
III
(ImH)
Visible bands
kmax (nm)
C
50
a
(mM)
a,a,a,b-Fe(ToCPP) 417, 549, 580(sh) 419, 552, 587(sh) 21.3  0.3
a,b-1,2-Fe(DoCPP) 417, 552, 581(sh) 417, 548, 580(sh) 13.0  0.2
a,a-1,2-Fe(DoCPP) ± 419, 547, 580(sh) 7.7  0.1
Fe(MoCPP) 420, 545, 582(sh) 420, 549, 580(sh) 236  4
a

n and K
d
were determined as described in Experimental Procedures, C
50
 K
d
.
Ó FEBS 2002 Peroxidase-like Fe±porphyrin±antibody complexes (Eur. J. Biochem. 269) 475
a plateau value of 0.72 l
M
ABTS oxidized per min for a
concentration of imidazole of 50 m
M
(Fig. 6). For con-
centrations o f imidazole higher than 1 00 m
M
the r ate of
oxidationofABTSstartedtodecrease(Fig.6).With
13G10±Fe(ToCPP) a s catalyst, the rate of oxidation of
ABTS sharply decreased from 1.62 to 0.79 l
M
ABTS
oxidized per min. in the p resence of concentrations o f
imidazole increasing from 0 to 20 m
M
and then decreased
more slowly to reach a plateau value of about 0.40 l
M
ABTS oxidized per min. for a concentration of imidazole
of 150 m

M
(Fig. 6). Thus, w hereas the addition of imid-
azole to Fe(ToCPP) was found to increase its peroxidase
activity with a A
50
of 16 m
M
, it inhibited the peroxidase
activity of the 1 3G10±Fe(ToCPP) with an I
50
of about
19 m
M
. In addition, the activity of Fe(ToCPP) was even
higher than that of 13G10±Fe(ToCPP) for concentrations
of imidazole higher than 50 m
M
, as shown by the curves
representing the variations of the rates of oxidation of
ABTS by H
2
O
2
observed, respectively, for those two
catalysts (Fig. 6).
The peroxidase activity of the two atropoisomers of
a,a-anda,b-1,2-Fe(DoCPP) (Fig. 1), which were previously
found to have also a high af®nity for antibody 13G10 [36],
was also assayed in the presence of increasing concentra-
tions of imidazole and compared to that of the a,a-and

a,b-1,2-Fe(DoCPP ))13G10 complexes. With both a,b-and
a,a-1,2-Fe(DoCPP), the rate of oxidation of ABTS by H
2
O
2
increased with increasing concentrations of imidazole, from
initial values of, respectively, 0.08 and 0.10 l
M
ABTS
oxidized per min to respective plateau values of 0.47 and
0.36 l
M
ABTS oxidized per min in the presence of more
than 50 m
M
imidazole ( Fig. 6). When the reaction was
performed in the presence of a,a-anda,b-1,2-Fe(DoC-
PP))13G10 complexes, the rate of oxidation of ABTS by
H
2
O
2
increased sharply in the presence of increasing
amounts of imidazole, from an initial value of 0.37 l
M
ABTS oxidized per min to respective maximum values of
3.68 and 2.43 l
M
ABTS oxidized per min in the presence of
more than 50 m

M
imidazole (Fig. 6). Thus, contrary to
what occurred with the 13G10±Fe(ToCPP) complex, t he
addition of imidazole to a,a-anda,b-1,2-Fe(DoC-
PP))13G10 complexes was found to increase largely their
peroxidase activity with respective A
50
values of 15 and
25 m
M
.
The kinetic parameters for the oxidation of 0.2 m
M
ABTS by H
2
O
2
, in the presence of either Fe(ToCPP) or
Fe(ToCPP)- and Fe(DoCPP))13G10 complexes as cata-
lyst, were measured at pH 5 without imidazole and in the
presence of 50 m
M
imidazole (Table 4). It appeared that
in all the cases the addition of 50 m
M
imidazole had a
major effect on the k
cat
value: in the case of Fe(ToCPP))
13G10, it ca used a de crease of the k

cat
value by a factor of
% 4, from 109  10 min
)1
to 32  3min
)1
whereas in
contrast, with both a,a-anda,b-1 ,2-Fe(DoCPP))13G10
complexes, it caused an increase the k
cat
value by a
factor of % 5±6, respectively, from 32  3min
)1
to 152 
10 min
)1
and from 16  2min
)1
to 96  9min
)1
.The
addition of 50 m
M
imidazole had a more moderate effect
on the K
m
value that only slightly decreased from
29  3m
M
to 19  2m

M
inthecaseofFe(ToCPP))
13G10, whereas it decreased by a factor 3, respectively,
from 34  3m
M
to 10  1m
M
and from 18 
2m
M
to 7  1m
M
with a,a-anda,b-1,2-Fe(DoCPP))
13G10. As a consequence, the addition of 50 m
M
imida-
zole caused a two fold decrease of the k
cat
/K
m
value from
3.8  0.7 ´ 10
3
M
)1
ámin
)1
to 1.7  0.4 ´ 10
3
M

)1
ámin
)1
inthecaseofFe(ToCPP))13G10 as catalyst, whereas on
the contrary, it caused an about 15-fold increase of the
k
cat
/K
m
value, respectively, from 0.9  0.2 ´ 10
3
M
)1
ámin
)1
to 15.2  2.5 ´ 10
3
M
)1
ámin
)1
and 0.9  0.2 ´
Fig. 6. In¯uence of the addition of imidazole on the peroxidase activity
of Fe(ToCPP), a,a-1,2- and -a,b-1,2-F e(DoCPP) and their complexes
with 13G10. Variations of the initial rate of oxidation of 0.2 m
M
ABTS
by 0.7 m
M
H

2
O
2
as a function of the concentration of imidazole in
the presence 0.4 lm catalyst: (s) Fe(ToCPP) (d) 13G10±Fe(ToCPP)
(h) a,a-1,2-Fe(DoCPP) (j)13G10-a,a-1,2-Fe(DoCPP) (n) a,b-1,2-
Fe(DoCPP) (m) 13G10-a,b-1,2-Fe(DoCPP).
Table 4. In¯uence of imidazole on the kinetic parameters of the oxidation of ABTS by H
2
O
2
catalyzedbyFe(ToCPP)±andFe(DoCPP))13G10
complexes at pH 5.
Catalyst
Without ImH + 50 m
M
ImH
k
cat
(min
)1
)
K
m
(m
M
)
k
cat/
K

m
(
M
)1
ámin
)1
)
k
cat
(min
)1
)
K
m
(m
M
)
k
cat/
K
m
(M
)1
ámin
)1
)
Fe(ToCPP) 68  737 4 1.8  0.3 ´ 10
3
71  7 8.5  1 8.3  1.5 ´ 10
3

Fe(ToCPP))13G10 109  10 29  3 3.8  0.7 ´ 10
3
32  319 2 1.7  0.4 ´ 10
3
a,a-1,2-Fe(DoCPP))
13G10
32  334 3 0.9  0.2 ´ 10
3
152  10 10  1 15.2  2.5 ´ 10
3
a,b-1,2-Fe(DoCPP))
13G10
16  218 2 0.9  0.2 ´ 10
3
96  97 1 13.7  2.8 ´ 10
3
476 S. de Lauzon et al. (Eur. J. Biochem. 269) Ó FEBS 2002
10
3
M
)1
ámin
)1
to 1.7  0.4 ´ 10
3
M
)1
ámin
)1
with a,a-1,2-

Fe(DoCPP)- an d a,b-1 ,2-Fe(DoCPP))13G10 as catalysts
(Table 4).
DISCUSSION
Binding of cyanide to the iron(III) of Fe(ToCPP)
and Fe(ToCPP))13G10
First of all, the aforementioned results show that the
iron(III) of the Fe(ToCPP))13G10 complex is able to bind,
like that of free Fe(ToCPP), two cyanide ligands. Indeed,
like in the case of free Fe(ToCPP), the addition of increasing
amounts of cyanide to Fe(ToCPP))13G10 leads to the
formation of two successive complexes (Fig. 3): a ®rst one
absorbing at 420 nm for CN
±
concentrations below 6 m
M
and a second one absorbing at 429 nm for CN
±
concentra-
tions higher than 10 m
M
. The 13G10±(ToCPP)Fe±CN and
13G10±[(ToCPP]Fe(CN)
2
]
±
structures were strongly sug-
gested for those two complexes as: (a) their spectra of
absorption were similar to those previously reported f or
(porphyrin)Fe±CN and [(porphyrin)Fe(CN)
2

]
±
complexes
[49], their maxima of absorption being only 3 nm redshifted;
(b) both 1/DA
420
and 1/DA
429
varied linearly as a function of
1/[CN
±
] (Fig. 3, insets); (c) when (ToCPP)Fe-CN and
[(ToCPP)Fe(CN)
2
]
±
were reinserted into apo-13G10, spec-
tra similar to those already oberved for 13G10±(ToCPP)Fe±
CN and 13G10±[(ToCPP)Fe(CN)
2
]
±
were obtained, which
showed that the binding of the two cyanide ligands on the
iron did occur inside the binding pock et o f the antibody.
This is totally diff erent f rom what w as reported b y
Kawamura-Konishi et al. [29] for the anti-(N-methyl mes-
oporphyrin IX) Ig 2B4. Indeed, in this case, the iron(III) of
the 2B4±Fe(mesoporphyrin IX) complex was found to be
able to bind only one CN

±
ligand, which was interpreted as a
side-on binding of the porphyrin inside the antibody pocket,
leaving only one of its faces accessible to ligands. Conse-
quently, it is more likely that in our case, a n edge-on binding
of Fe(ToCPP) occurs inside the binding pocket of 13G10
(Fig. 7) that allows 2 CN
±
ligands to bind on the iron atom,
one on each face of the porphyrin. This hyp othesis is in
agreement with the binding site topology w hich we
proposed recently, and in which approximately two-thirds
of the porphyrin moiety was inserted in the antibody pocket,
three of the ortho-carboxylate substituents of the me so-
phenyl rings being bound to side chains of amino acids such
as arginine [36]. In this respect, it is noteworthy that the
X-ray structure of a complex of iron(III)±mesoporphyrin IX
with an anti-(N-methyl-mesoporphyrin IX) Ig [37] showed
that in this case also, approximately two-thirds of the
porphyrin moiety was inside the antibody pocket with three
pyrrole rings packed tightly against r esidues of the V
H
domain and two pyrrole rings packed against tyrosine
residues of the V
L
domain.
Binding of imidazoles to the iron(III) of Fe(ToCPP)
and Fe(ToCPP))13G10
The second part of our results concerns the binding of
imidazole d erivatives on the iron of Fe(ToCPP) either alone

in solution or inside the binding pocket of antibody 13G10.
It then appears that Fe(ToCPP) alone forms bis-imidazole
complexes with imidazole and its deriva tives: 1-methyl-,
1-benzyl-, 2-methyl-, 2-ethyl- and 4-methylimidazole. This
was shown particularly by: (a) the UV/visible spectra
obtained after addition of imidazole (Fig. 4) or its deriva-
tives (Table 2) to Fe(ToCPP), which were very similar to
those already reported for (tetraarylporphyrin)Fe
III
(ImH)
2
complexes [50]; (b) the linear dependence of 1/DA
417
as a
function of 1/[ImH]
2
when increasing amounts of imidazole
were added to Fe(ToCPP); and (c) the C
50
( K
1a2
d
) found
for the complexes of Fe(ToCPP) with imidazole derivatives
(Table 2), which were in good agreement with the equilib-
rium constant b
2
measured for the formation of (porphy-
rin)Fe(ImH)
2

complexes [51]. This of course is not very
surprising as it has been reported extensively in the literature
that Fe-tetraarylporphyrins were able to form bis-imida-
zole±iron(III) complexes [50±53] and the X-ray structure of
some of these complexes has been determined [52,53]. More
surprising, ho wever, i s the ®nding that the iron of the
Fe(ToCPP))13G10 complex is able to bind only one
imidazole ligand as shown by the fact that 1/DA
419
varies
linearly as a function of 1/[ImH] but not as a function of
1/[ImH]
2
(Fig. 5, inset). This suggests that there is not
enough space left around the iron atom inside the antibody
pocket to accommodate two imidazole ligands and, as the
C
50
value calculated in this case (21.3  0.3 m
M
) is about
10-fold higher than that calculated in the case of Fe(ToCPP)
(2.70  0.04 m
M
) (Table 2), that even the formation of the
mono-imidazole complex of 13G10±Fe(ToCPP) is more
dif®cult than the formation of the bis-imidazole complex of
Fe(ToCPP). A likely explanation for this is that one
imidazole ligand is able to bind on the less hindered face
of the porphyrin bearing only the b-carboxyphenyl group

Fig. 7. Various possibilities for the binding of ligands on the iron
of Fe(porphyrin))13G1 0 complexes: (A) binding of H
2
O
2
on the iron of
13G10-(Fe(ToCPP), (B) bind ing of two CN
±
ligands on the iron
of 13G10-(Fe(ToCPP), (C) binding of H
2
O
2
and imidazole on the iron of
13G10-(Fe(ToCPP), (D) binding of H
2
O
2
and imidazole on the iron
of 13G10-a,a-1,2-Fe(DoCPP).
Ó FEBS 2002 Peroxidase-like Fe±porphyrin±antibody complexes (Eur. J. Biochem. 269) 477
but a second imidazole is then unable to bind on the other
more hindered f ace of the porphyrin that bears the three
a-carboxyphenyl groups and is stacked against the antibody
protein (Fig. 7).
This hypothesis is furth er sustained by t he C
50
values
measured for the binding of various substituted imidazoles
on the iron of Fe(ToCPP) and 13G10±Fe(ToCPP)

(Table 2). First, with 1 -substituted imidazoles, which bear
an hydrophobic substituent on the nitrogen atom opposite
to th at which binds to th e iron, the C
50
values measured were
lower than those measured for imidazole both with F e(ToC-
PP) and 13G10±Fe(ToCPP). Indeed, for Fe(ToCPP), C
50
values of 2.60  0.04 m
M
and 0 .6 3  0.01 m
M
were
found, respectively, for 1-CH
3
- and 1-benzylimidazole and
2.70  0.04 m
M
for imidazole whereas for 13G10±Fe(ToC-
PP), C
50
values of 4.30  0.06 and 14.5  0.2 m
M
were
found, respectively, for 1-CH
3
- and 1-benzylimidazole and
21.3  0.3 m
M
for imidazole. This could be explained by a

greater hydrophobicity of the two 1-substituted imidazoles
with respect to that of imidazole. Second, like in the case of
imidazole, the C
50
values are higher with the antibody±
Fe(ToCPP) complex than in the case of Fe(ToCPP) alone,
which con®rms that the binding of one 1-substituted
imidazole on the iron of 13G10±Fe(ToCPP) is even more
dif®cult than the binding of two 1-substituted imidazoles on
the iron of free Fe(ToCPP). In addition, the in¯uence of the
nature of the substituent was different in both cases. In the
case of Fe(ToCPP), it did not cause any steric hindrance for
the binding and 1-methylimidazole had the same af®nity for
the iron th an imidazole, whereas 1-benzylimidazole had a
better af®nity than imidazole. In constrast, in the case of
13G10±Fe(ToCPP), the C
50
value for 1-benzylimidazole was
about fourfold higher than that for 1-methylimidazole,
which could arise from a more important steric interaction
of the 1-benzyl substituent with the antibody protein than
that with the 1-methyl substituent.
In the case of 2- and 4-substituted imidazoles, which bear
an alkyl substituent on the carbon next to the nitrogen atom
binding the iron, the C
50
values obtained for Fe(ToCPP)
were 20- to 4 0-fold higher than the one for i midazole
(Table 2). This was due to an important steric interaction
between the 4- and 2-alkyl substituent with the plane of

the porphyrin [48,52]. For 13G10±Fe(ToCPP), much lower
C
50
( K
d
) values, of, respectively, 2.80  0.06 m
M
,
4.10  0.05 m
M
, and 3.10  0.0 5 m
M
for 4-methyl-,
2-methyl-, and 2-ethylimidazole were observed (Table 2).
This could be explained by the sum of three effects: (a) a
higher hydrophobic c haracter of 2- and 4-substituted
imidazoles with respect to imidazole; (b) the absence of
steric interaction between the 2- a nd 4-alkyl substituent and
the protein; and (c) ®nally, in the antibody±Fe(ToCPP)
complex, as only one 2- or 4-substituted imidazole was
bound to the iron, the steric hindrance due to the substituent
could be balanced by a distortion of the porphyrin ring and
a shift of the iron atom outside the plane of the porphyrin.
Binding of imidazole to the iron(III) of Fe(ToCPP),
a,a-1,2- a,b-1,2-Fe(DoCPP), Fe(MoCPP)
and to their complexes with antibody 13G10
The UV/visible s tudies reported above s howed that the
addition of increasing amounts of imidazole to a,a-1,2- and
a,b-1,2-Fe(DoCPP ) and to F e(MoCPP ), and to their
complexes with 13G10 led to results that were similar to

those observed f or Fe(ToCPP): iron(III)±bis-imidazole
complexes were formed in the case of free Fe±porphyrins
whereas mono imidazole±iron(III) complexes were formed
in the case of Fe±porphyrin±antibody complexes (Table 3).
In addition, in the particular c ase of a,b-1,2- and a,a-1,2-
Fe(DoCPP))13G10 complexes, the C
50
values found were,
respectively, twofold and threefold lower than that found
for Fe(ToCPP) (Table 3). This could be due to an easier
access of the imidazole to the iron in those complexes of
13G10 w ith t wo less hindered d i-ort ho-carboxyphenyl
substituted tetraarylporphyrins.
In¯uence of imidazole on the peroxidase activity
of the Fe±porphyrin±antibody complexes
In heme peroxidases, such as horseradish peroxidase, the
iron atom is bound to the apoprotein by a proximal
histidine [42] and it has been reported that this axial ligand
has an important role in the modulation of the redox
potential of the iron [54] and thus has a great in¯uence on
the catalytic activity of those enzymes. Because our studies
on the binding of imidazole to the iron(III) of our
Fe±porphyrin±antibody complexes have shown that, in all
the cases, only one imidazole was able to bind to the iron
atom, this suggested that the association of Fe±porphyrin±
antibody complexes with imidazole could constitute a very
good biomimetic system for peroxidases. Consequently, the
peroxidase activity of the iron(III)-ortho-carboxy substi-
tuted tetraarylporphyrins and their complexes with anti-
body 13G10 was measured in the presence of varying

concentrations of imidazole ( Fig. 6, Table 4).
First of all, the addition of increasing amounts of
imidazole, from 0 to 150 m
M
, caused a slight increase of
the peroxidase activity of Fe(ToCPP), a,a-1,2- and a,b-1,2-
Fe(DoCPP) ( Fig. 6). This w as not surprising as it was
previously reported that imidazole increased the ability of
iron(III)± and Mn(III)±porphyrin complexes to catalyze the
oxidation o f substrates such as sul®des [55], alkanes and
alkenes [56,57] by H
2
O
2
. Second, when increasing amounts
of imidazole were added to the Fe±porphyrin±antibody
complexes, two different effects were observed depending
upon the nature of the porphyrin. In the case of 13G10±
Fe(ToCPP), the progressive addition of up to 150 m
M
imidazole was found to inhibit strongly the peroxidase
activity with an I
50
of about 19 m
M
(Fig. 6). As this value is
close to that of the C
50
calculated for the formation of the
13G10±(ToCPP)Fe±ImH complex, it is clear that this

inhibition was due to the binding of the imidazole ligand
on the iron atom. In addition, measurement of the kinetic
parameters for the oxidation of ABTS by H
2
O
2
catalyzed by
Fe(ToCPP))13G10 in the presence of 50 m
M
imidazole,
showed that the k
cat
/K
M
value was decreased by a factor of
% 2 (Table 4). Contrary to what was observed in the case of
13G10±Fe(ToCPP), the addition of increasing amounts
of imidazole to a,a-1,2- and a,b-1,2-Fe(DoCPP))13G10
complexes led to a large increase of the peroxidase activity
with A
50
values of about 15 and 25 m
M
, respectively, the
activity being optimal for a concentration of imidazole of
50 m
M
(Fig. 6). Such a concentration o f imidazole was
found to cause a 15-fold increase of the k
cat

/K
m
value with
both a,a-1,2-Fe(DoCPP)- and a,b-1,2-Fe(DoCPP))13G10
complexes.
478 S. de Lauzon et al. (Eur. J. Biochem. 269) Ó FEBS 2002
We propose a likely explanation for t he above men-
tioned results based on the possible active site topology of
antibody 13G10 presented in Fig. 7 and which also takes
into account our previously published observations [36,38]:
(a) about two-thirds of the porphyrin moiety is inserted
inside the antibody active site, a nd (b) a carboxylic acid
residue of the protein participates to the catalysis of the
heterolytic cleavage of the O±O bond of H
2
O
2
.Inthecase
of 13G10±Fe(ToCPP) it is likely that there is room enough
in the antibody active site to allow the binding of two CN
±
ligands on the iron atom (Fig. 7B). In contrast, there is
probably not enough space t o accommodate two imidazole
ligands on the iron atom, and the only one that enters the
active site bind on the less hindered face of the porphyrin
which bears only one ortho-COOH substituent (Fig. 7C).
The inhibition of the peroxidase activity could then arise
from the fact that this imidazole binds on the same face of
the porphyrin as the catalytic COOH residue, thus
preventing it from acting as a g eneral acid±base catalyst,

whereas H
2
O
2
can only bind on the opposite, more
hindered face of the porphyrin. This probably does not
occur in the case of the complexes of antibody 13G10 with
the less hindered a,a-1,2- and a,b-1,2-Fe(DoCPP), as the
addition of increasing amounts of imidazole to those
complexes causes an increase of their peroxidase activity. It
is then likely that in those complexes, the imidazole can
bind on either face of the porphyrin a nd, in the more
favorable conformation, H
2
O
2
binds to the iron on the
same face of the porphyrin as the catalytic COOH residue,
the imidazole binding to the iron on the opposite face of
the porphyrin (Fig. 7D). An optimal catalytic effect can
then be obtained as the COOH residue can act as a general
acid±base catalyst and the imidazole ligand can modulate
the redox potential of the iron atom [54].
Finally, the present work has led t o a new arti®cial
hemoprotein or h emoabzyme that displays an interesting
peroxidase-like a ctivity. The complex is composed of a
robust protein, a monoclonal anti-porphyrin I g, with an
iron(III)-DoCPP cofactor and imidazole as an axial ligand
of the iron which, respectively, mimick the heme cofactor
and the axial histidine ligand of the iron in peroxidases,

whereas a COOH side chain of the antibody acts as a
general acid±base catalyst in the same manner as the distal
histidine of peroxidases.
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