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

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-

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

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

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

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

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

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

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

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

)(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

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

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

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

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 

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

) 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
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
)


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

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

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

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

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

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-

]
±
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

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

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)

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

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

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

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,

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