We here show that the iron of 13G10—FeToCPP is able to bind, like that of free FeToCPP, two small ligands such as CN , but only one imidazole ligand, in contrast to to the ironII of FeTo
Trang 1Coordination chemistry of iron(IIl)—porphyrin—antibody complexes Influence on the peroxidase activity of the axial coordination of an imidazole
on the iron atom
Solange de Lauzon’, Daniel Mansuy' and Jean-Pierre Mahy?
‘Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université René Descartes,
Paris, France; ?Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, ICMO, Bat 420,
Université Paris-Sud XI, Orsay, France
An artificial peroxidase-like hemoprotein has been obtained
by associating a monoclonal antibody, 13G10, and its
iron(III)—«,«,«, 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 H2O>, 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(II) 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(IID-—
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 Keywords: catalytic antibody; peroxidase; artificial 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 efficiencies, several other
strategies have been envisioned A first 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
Correspondence to J.-P Mahy, Laboratoire de Chimie Bioorganique
et Bioinorganique, FRE 2127 CNRS, ICMO, Bat 420, Université
Paris-Sud XI, 91405 Orsay Cedex, France
Fax: + 36 1 01 69 15 72 81, E-mail: jpmahy@icmo.u-psud.fr
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)
highest efficiency (1.35 x 10° m7''s7') 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 significant peroxidase activity with #¿/K„ values ranging between 3.8 x 10° and 29x 100M *min ' 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 o-naphthoxy ligand, was found to be able, in the presence of a Ru(II) porphyrin cofactor, to catalyze the stereoselective sulfoxidation of aromatic sul- fides by iodosylbenzene [43] In previous papers [31,36,38],
we reported the production of two monoclonal antibodies,
Trang 2© FEBS 2002
13G10 and 14H7, which not only bound the hapten,
iron(IIT)-a,«,«, B-meso-tetrakis(ortho-carboxyphenyl)por-
phyrin [Fe(ToCPP)] (Fig 1) with a high affinity
(Ky = 10° m), but also exhibited in its presence an
interesting peroxidase activity with k., = 540 min™’ and
Keat|/ Km = 3.2 X 10” M ':min' [38] Measurements of the
binding constants for various porphyrins [36], together with
pH dependence studies of the kinetics of the peroxidase
reaction [38] associated with chemical modifications 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 the 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(II) of
Fe(ToCPP) bound or not to antibody 13G10 with two
objectives: first, 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 1ron atom
and which kind of ligands 1t can accommodate; second, to
measure the influence 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 1s able to bind two CN ligands in Fe(ToCPP)
alone as well as 1n its complex with antibody 13G10; (b) in
contrast, whereas the iron(II) 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
R'!'! 5
Peroxidase-like Fe—porphyrin—antibody complexes (Eur J Biochem 269) 471
peroxidase activity of the Fe(ToCPP)—13G10 complex whereas it enhances that of the complexes of 13G10 with Iron(lIII)-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_ Fe(III)-di-ortho-carboxyphenyl-porphyrin and imidazole provides an accurate artificial peroxidase- like hemoprotein, with the axial 1midazole 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
EXPERIMENTAL PROCEDURES
Chemicals
Sodium azide and sodium isothiocyanate were from Sigma Potassium cyanide, imidazole, 1-methylimidazole, 1-benzy-
2-ethylimidazole were from Fluka 2,2’-azinobis(3-ethyl- benzothiazoline-6 sulfonic acid) (ABTS), and H,O, from Sigma
Synthesis of iron(IIl)—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 first synthesized by reaction at room temperature of ortho-carbomethoxybenzaldehyde with pyrrole in CH>Cl
in the presence of BF3-etherate as catalyst according to an already described procedure [36,43] The atropoisomers have then been separated on a silicagel column and
R's
Rs
mt
1
meso-tetrakis(ortho-carboxyphenyl)porphyrin (ToCPPH)) a,a,0,8 isomer: R; = R'; = R'"'; = R's = COOH, other substituents = H meso-di(ortho-carboxyphenyl)-diphenylporphyrin (DoCPPHạ)
a,a-1,2 isomer: Rị = R'¡ = COOH, other substituents = H
Fig 1 Structure and nomenclature of the
various porphyrins used in this work
meso-mono(ortho-carboxypheny])-triphenylporphyrin (MoCPPH,)
R, = COOH, other substituents = H
Trang 3identified by absorption, 'H NMR and mass spectroscopies
[36] The iron atom was then inserted by reaction of the
isolated atropoisomers with Fe(CO)s in the presence of I, in
toluene at room temperature to avoid isomerization [44]
Finally, the ortho-carboxy substituted tetraarylporphyrin
isomers were subsequently obtained by saponification of the
ortho-methyl ester substituents in 2 Mm KOH in 80% EtOH
at room temperature [45]
Production of monoclonal antibodies
The generation of monoclonal antibodies has been
reported in detail in previous papers [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 pH 7.5 The conjugates were
then purified by chromatography on Biogel P10 and four
5-week-old, female BALB/c mice were immunized con-
ventionally with the Fe(ToCPP)-KLH conjugate The
spleen cells of the mouse showing the best immune
response were fused with PAI myeloma cells according to
Kohler & Milstein [46] The supernatants from the
hybridoma cells were screened by ELISA for binding to
the hapten—BSA conjugate peroxidase linked goat anti-
(mouse Ig) Ig [47] Positive hybridoma were cloned twice
and propagated in ascites Antibodies were then purified
from ascite fluid by protein A affinity chromatography
and their purity and homogeneity were checked by SDS
gel electrophoresis
Absorption spectroscopy measurements
Absorption spectra were recorded at 19 + 0.1 °C using an
UVIKON 860 UV/visible spectrophotometer as follows
The sample cuvette contained either 2 HM Fe(ToCPP) or
Fe(ToCPP) preincubated with 3 um 13G10 in 50 mm
phosphate buffer pH 7.0, the reference cuvette only con-
tained 50 mm phosphate buffer pH 7.0 Equal amounts of
ligand L (L = imidazole, mono-substituted imidazole, CN ,
SCN , N; ) (2M 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 defined isobestic points indicating the
presence of two absorbing species The reaction could then
be represented by:
PFe!! + nL = PFel(L), (1)
where P = ToCPP, 13G10{ToCPP) and L = imidazole,
mono-substituted imidazole, CN ,SCN , N3 According to
Brault & Rougee [48], it could then be analyzed by means of
the standard equation
L/AA = 1/AAx + Kz/AA„ x 1/41" (2)
where AA = A — Ap, AA,, = A - A, and Ap, Az, and A
are the absorbances of the initial, final and mixed species,
respectively The linearity of the graph representing 1/AA as
a function of 1/[L]" was then assayed with n = 1 and n = 2
and Kg and A,, could be determined graphically It is
noteworthy that when n = 1, Cs) = Kg, whereas when n = 2,
Co = Kỳ , with Cso representing the concentration of
ligand for which half of the starting Fe(ToCPP) or
Fe(ToCPP)-I3GIO complex has been converted into (ToCPP)Fe(L), or 13G10{ToCPP) Fe(L),
Assay of peroxidase activity
To assay the peroxidase activity of the various iron(IID-— ortho-carboxy substituted tetraarylporphyrins and_ their complexes with antibody 13G10, the oxidation of ABTS
by HO, was performed at 19 + 0.1 °C in 0.1 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 The 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 & value of 28 000 x”!*em' [21]
In a first set of experiments, ABTS (0.2 mm) was oxidized
by HO; (0.7 mm) in the presence of 0.4 tum Fe(ToCPP) or Fe-a,o-1,2- or -o,8-1,2-(DoCPP) preincubated or not with 0.6 um 13G10 as catalysts
In a second set of experiments, ABTS (0.03 mm) was oxidized by H,O, (5 mm) in the presence of 0.4 um Fe(MoCPP) preincubated 60 min with 0.2 um 13G10 or 0.2 um Fe(ToCPP) or Fe-w,s-l,2- or -+,-l,2-(DoCPP) preincubated with 0.4 um 13G10
The influence of imidazole on the kinetic parameters of the oxidation of ABTS by HO; in the presence of Fe— porphyrin—antibody complexes was examined as follows The catalysts were first prepared by preincubation of 0.4 um Fe(ToCPP) or o,0-1,2-Fe(DoCPP) or z,B-1,2-Fe(DoCPP) with 0.6 um 13G10 for 60 min at 19 °C For the reactions with imidazole, a further 15 min incubation at 19 °C with
50 mm imidazole was done; 0.2 mm ABTS was then added and the reaction was started by the addition of H,O, at concentrations ranging between 0 and 10 mm The initial rates of oxidation were then measured as above mentioned and the k,,; and K,, 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; and CN’ with the iron(HI) of Fe(ToCPP) and its complex with antibody 13G10 were examined by UV/visible spectroscopy in 0.1 Mm phosphate buffer, pH 7 at 19 + 0.1 °C as described in experimental procedures SCN” and N3 failed to react with both complexes [data not shown] but, when increasing amounts
of potassium cyanide, up to 11 mmo, were added to a 2 um solution of Fe(ToCPP), the initial spectrum characteristic
of a high spin iron(II] species was gradually replaced, with isobestic points at 407, 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'"-CN complexes [49] As in addition, 1/AA4,7 varied linearly with 1/[CN ] (Fig 2A, inset), it is clear that the first reaction observed was the binding of CN ligand to the iron(II) of Fe(ToCPP) (Eqn 3), with a calculated Kg value of 3.70 + 0.06 mm (Table 1)
Fe(ToCPP) + CN” = (ToCPP)Fe™’—CN (3)
Trang 4© FEBS 2002
A
Abs 0.2 +
(A) Spectral evolution observed for the
in 0.1 mM phosphate buffer, pH 7 at 19 °C
Inset: corresponding values of 1/AA4)7 plotted il | i
against 1/[CN ] (B) Spectral evolution
observed for the addition of 11-50 mm CN to J/
2 uM Fe(ToCPP) in 0.1 m phosphate buffer,
pH 7 at 19 °C Inset: corresponding values of 0
1/AAgj7 plotted against 1/[CN ] 350 420
Table 1 Visible characteristics and Cs g values of the complexes of
Fe(ToCPP) and 13G10- Fe(ToCPP) with cyanide in 50 mm phosphate
buffer, pH 7.0 at 20 °C
Visible bands Cao”
Complex Amax (nm) (mM)
(ToCPP)Fe "'-CN 417, 549 3.70 + 0.06
13G10-(ToCPP)Fe ''—CN 420, 555 0.39 + 0.01
((ToCPP)Ee ““(CN)_ 426, 565, 600 19.5 + 0.3
(13G10-(ToCPP)Fe '"(CN),) 429,—,-— 16.90 + 0.13
“When only one CN is bound to Fe, Cs) = Ka and when two CN"
are bound to Fe, Cs) = K,!’
Further addition of potassium cyanide, up to 50 mM,
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’ (CN],] species [49] Accordingly, when 1/AA47¢ was
plotted vs I/[CN ], a straight line was obtained and a Kg
value of 19.5 + 0.3 mm could be determined graphically
(Table 1)
(ToCPP)Fe' — CN+ CN™ = [(ToCPP)Fe'" (CN),]~
(4)
Abs 0.150 1
Fig 3 Addition of cyanide to the
Fe(ToCPP)—13G10 complex (A) Spectral
evolution observed for the addition of 0-6 mm
CN to 2 um 13G10—Fe(ToCPP) in 0.1 m
phosphate buffer, pH 7 at 19 °C Inset:
corresponding values of 1/AA4 9 plotted
against 1/[CN ] (B) Spectral evolution
observed for the addition of 10-50 mm CN™
to 2 um 13G10—Fe(ToCPP) in 0.1 m phos-
phate buffer, pH 7 at 19 °C Inset:
corresponding values of 1/AA4 9 plotted °
0.075 +
Peroxidase-like Fe—porphyrin—antibody complexes (Eur J Biochem 269) 473
807
703 eo:
50:
102 s0:
0.1 4 203
0 0,010,02 0,03 0,04 0,05 1ICNˆ] (mM"b
A (nm)
When the same experiment was carried out with the Fe(ToCPP)—13G10 complex (2 um), a first species absorb- ing at 420 and 555 nm was formed with isobestic points at
409, 477 and 552 nm for concentrations of CN below
6 mM (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 mm
(Fig 3B) As, respectively, 1/AAgo9 and l/A⁄44;so varied
linearly as a function of 1/[CN ] (Fig 3, insets), it 1s clear that those two species were, respectively, the 13G10— (ToCPP)Fe—CN and 13G10-[(ToCPP)Fe(CN),z] complexes Accordingly, when (ToCPP)Fe-CN and [(ToCPP) Fe(CN),] were inserted into the antibody protein, the characteristic bands in their visible spectrum were shifted toward higher wavelengths to give 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 mm) was about 10-fold lower than that calcu- lated for (ToCPP)Fe-CN (3.70 + 0.06 mm) (Table 1), whereas that of 13G104[ToCPP]Fe[CN]] (16.90 + 0.13 mm) was only slightly lower than that of
Abs 0.1507
1 0 TTTTTTT TT TT T TT rTTTrrrn
4 6 8 10
1/[CN7] (mM)
10 1 rT | Tor fof | corr 1 rorer oT
0,02 0,04 0,06 0,08
1⁄ICN-](mM-Ù
420
against 1/[CN ]
650 350 420
A (nm)
Trang 5
—
+
4 507
30-4
20- 10-
` 1/[ImH] (mM-Ù
—
¬
Ấ n3
J¬
74
5
3 ee
1/1ImH]2 (mM-Z
539 sto ( )
A (nm)
Fig 4 Addition of imidazole to iron(IID)-0,0,0,B- meso-tetrakis(ortho-
carboxyphenyl)porphyrin (Fe(ToCPP)) Spectral evolution observed
for the addition of 0-14 mm ImH to 2 um Fe(ToCPP) in 0.1 mM
phosphate buffer, pH 7 at 19 °C Inset: corresponding values of
1/AAqy7 plotted against 1/[ImH] and 1/[ImHÍ
((ToCPP)Fe(CN).; (19.5 + 0.3 mm) (Table 1) The bind-
ing of the first CN” ligand to the iron was thus more easy 1n
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 mm, to a 2 uM solution of Fe(ToCPP), the initial
spectrum of the high spin 1ron(IIJ)—-(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 1s similar to that
already described for tetraaryl-Fe" [ImH], complexes [50]
As on addition, 1/AA44)7 varied linearly with 1/[ImH] but
not with 1/[ImH] (Fig 5, inset), 1t is clear that the reaction
observed was the binding of two ImH Igands, to the
iron(II) of Fe(ToCPP) (Eqn 5), with a Csp (= Ki ^ value
of 2.70 + 0.04 mm (Table 2)
Fe(ToCPP) + 2ImH = (ToCPP)Fe”°(ImH), (5)
When the same experiment was carried out with the
Fe(ToCPP)—13G10 complex (2 um), 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 mm
(Fig 5) As 1/AAgjo varied linearly with 1/[ImH] but not
with 1/[ImH]° (Fig 5, inset), it is clear that contrary to free
4
< 4 Abs =
3
2
2
1
1
401 0 5 10 15 20 25 30 35 40
1/{ImH] x 102¢mM7)
™
é
1/[ImH}2 x 102 (mM-2)
Fig 5 Addition of imidazole to the Fe(ToCPP)—13G10 complex Spectral evolution observed for the addition of 0-200 mm ImH to
2 uM Fe(ToCPP) in 0.1 m phosphate buffer, pH 7 at 19 °C Inset: corresponding values of 1/AAqj9 plotted against 1/[ImH] and 1/[ImHP
Fe(ToCPP), the 13G10—Fe(ToCPP) complex was only able
to bind one imidazole ligand (Eqn 6)
13G10 —
= 13G10 -
(ToCPP)Fe" + ImH (ToCPP)Fe”” (ImH) (6)
In addition, the Cso (= Kạ) value calculated 1n this case (21.3 + 0.3 mM) 1s about 10-fold higher than that obtained for the formation of the (ToCPP)Fe””(ImH); 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 l-substituted imidazoles, the Csp (= Kg) values observed with 13G10-Fe(ToCPP) were hipher than the Cso (= K, MZ ) values observed in the case of free Fe(ToCPP) Indeed for l-methyl- and 1-benzylimidazole, Cso (= Kg) values of, respectively, 4.30 + 0.06 mm and 14.5 + 0.2 mm were observed in the case of 13G10— Fe(ToCPP) whereas C5) (= Ki *) values of, respectively, 2.60 + 0.04 mm and 0.63 + 0.01 mm were observed in the case of free Fe(ToCPP) (Table 2)
In the case of 2- and 4-substituted imidazoles, the iron of free Fe(ToCPP) was also, able to bind two ligands, with much higher Cso (= Ki *) values (10- to 90-fold) than those calculated for imidazole and 1-substituted imidazoles (Table 2) Indeed, Cs9 values of 26.2 + 0.04 mm, 56.0 + 0.8mm and 54.0 + 0.8mm could be calculated for 4-methyl-, 2-methyl- and 2-ethylimidazole, respectively
Trang 6© FEBS 2002 Peroxidase-like Fe-porphyrin—antibody complexes (Eur J Biochem 269) 475
Table 2 Visible characteristics and Cs values of the complexes of Fe(ToCPP) and 13G10-Fe(ToCPP) with various monosubstituted imidazoles in
50 mm phosphate buffer, pH 7.0 at 20 °C
Visible bands Cao” Visible bands Ca”
ImH 2 417, 549, 580 (sh) 2.70 + 0.04 I 419, 552, 587(sh) 213 +03
I-CH:-Im 2 418, 550, 580 (sh) 2.60 + 0.04 l 419, 548, 580(sh) 4.30 + 0.06
1-Bz-Im 2 417, 546, 582 (sh) 0.63 + 0.01 I 419, 548, 590(sh) 145402
4-CH3-Im 2 418, 554, 582 (sh) 26.2 + 0.4 1 420, -, - 2.80 + 0.06 2-CH3-Im 2 418, -, - 56.0 + 1 421, -, - 4.10 + 0.05 2-C›H:-Im 2 419, -, - 54.0 + 1 421, -, - 3.10 + 0.05
@» and Kg for Fel! (L), complexes were determined as described in Experimental procedures: when n = 1, Csp = Kg and when n = 2,
— Kd1/2
(Table 2) Those values were also 10—15-fold higher than the
Csp (= Kg) values found for the 13G104ToCPP)Fe—4-
methyl- (2.80 + 0.06 mm), 2-methyl (4.10 + 0.08 mm)
and 2-ethylimidazole (3.10 + 0.05 mm) (Table 2)
Binding of imidazole to various iron(IIl)-ortho-carboxy
substituted tetraarylporphyrins and to their complexes
with antibody 13G10
We also examined the binding of imidazole to iron(IID-
mono- and di-ortho-carboxyphenyl substituted tetraaryl-
porphyrins, Fe7MoCPP) 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 affinity than Fe(ToCPP) [36] The addition of
increasing amounts of imidazole to 2 um solutions of o,B-
1,2-Fe(DoCPP) or Fe(MoCPP) in 50 mm phosphate buffer
pH 7.4, at 19 °C, led to the formation of the corresponding
porphyrin—Fe'"'(ImH)> 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 Kg values could not
be determined accurately When the same reaction was
performed with o,0-1,2-Fe(DoCPP)-, «,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/AA429 was a linear function of 1/[ImH], which
showed that in those cases, as in the case of Fe(ToCPP),
only one imidazole ligand bound to the iron atom In
addition, the Cso (= Kg) values could be calculated
(Table 3) and it appeared that the Csg values obtained in
the case of 13G10-a,0-1,2-Fe(DoCPP) and 13G10-ø,B-1,2- Fe(DoCPP) were threefold and twofold lower than that obtained with 13G10—Fe(ToCPP) In contrast, a much higher Csg value was found for 13G10—-Fe(MoCPP) (236 + 4 mm) (Table 3)
Influence of imidazole on the peroxidase activity
of various iron(III)-ortho-carboxy substituted tetraarylporphyrins and their complexes with antibody 13G10
The influence of the binding of imidazole to the iron atom
of iron(III)-ortho-carboxy substituted tetraarylporphyrins and their complexes with antibody 13G10 on_ their peroxidase activity was studied The rate of oxidation of 0.2mm ABTS by 0.7 mm H;O, was then measured at 19°C in the presence of increasing concentrations of imidazole, using as catalyst either 0.3 um iron(IID-por- phyrin or 0.3 HM 1ron(IHI)-porphyrin previously incubated with 0.6 um 13G10 The reactions were performed in
50 mm 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 first of all checked by absorption spectroscopy that both Fe(ToCPP) and 13G10—Fe(ToCPP) still bound imidazole
at pH 5 with Cso 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 mm (Fig 6) With this catalyst, the rate of oxidation of ABTS by HO: increased from an initial value of 0.16 jum ABTS oxidized per min to
Table 3 Visible characteristics and Cs values of the complexes of various iron(III)-ortho- carboxy-substituted-tetraarylporphyrins with imidazole in
50 mm phosphate buffer, pH 7.0 at 20 °C in the presence or not of antibody 13G10
(Porphyrin)Fe"!ImH)?
Visible bands Porphyrin Amax (nm)
13G10-(Porphyrin)Fe”"(ImH)
%,,œ,B-Fe(ToCPP) 417, 549, 580(sh)
%,B-1,2-Fe(DoCPP) 417, 552, 581(sh)
at,o- 1,2-Fe(DoCPP) —
Fe(MoCPP) 420, 545, 582(sh)
419, 552, 587(sh) 21.3 + 0.3
417, 548, 580(sh) 13.0 + 0.2
420, 549, 580(sh) 236 + 4
*n and Kg were determined as described in Experimental Procedures, Csy = Kg.
Trang 7S& 475
¬ `
27 j : +
60 80 100 120 140 160
[ImH] (mM)
Fig 6 Influence of the addition of imidazole on the peroxidase activity
of Fe(ToCPP), o,«-1,2- and -a,B-1,2-Fe(DoCPP) and their complexes
with 13G10 Variations of the initial rate of oxidation of 0.2 mm ABTS
by 0.7 mm H>O, as a function of the concentration of imidazole in
the presence 0.4 um catalyst: (O) Fe(ToCPP) (@) 13G10—Fe(ToCPP)
(LJ) o,o-1,2-Fe(DoCPP) (MI) 13G10-o,0-1,2-Fe(DoCPP) (A) o,B-1,2-
Fe(DoCPP) (A) 13G10-a,B-1,2-Fe(DoCPP)
a plateau value of 0.72 um ABTS oxidized per min for a
concentration of imidazole of 50 mm (Fig 6) For con-
centrations of imidazole higher than 100 mm the rate of
oxidation of ABTS started to decrease (Fig 6) With
13G10—Fe(ToCPP) as catalyst, the rate of oxidation of
ABTS sharply decreased from 1.62 to 0.79 um ABTS
oxidized per min in the presence of concentrations of
imidazole increasing from 0 to 20 mm and then decreased
more slowly to reach a plateau value of about 0.40 um
ABTS oxidized per min for a concentration of imidazole
of 150 mm (Fig 6) Thus, whereas the addition of imid-
azole to Fe(ToCPP) was found to increase its peroxidase
activity with a Aso of 16 mm, it inhibited the peroxidase
activity of the 13G10—Fe(ToCPP) with an Jsq of about
19 mm In addition, the activity of Fe(ToCPP) was even
higher than that of 13G10—Fe(ToCPP) for concentrations
of imidazole higher than 50 mm, as shown by the curves
representing the variations of the rates of oxidation of
ABTS by H>,O, observed, respectively, for those two
catalysts (Fig 6)
The peroxidase activity of the two atropoisomers of
a,o- and o,B-1,2-Fe(DoCPP) (Fig 1), which were previously
found to have also a high affinity for antibody 13G10 [36], was also assayed in the presence of increasing concentra- tions of imidazole and compared to that of the o,a0- and
a, B- 1 ,2-Fe(DoCPP)—13G10 complexes With both œ,B-and
increased with increasing concentrations of imidazole, from initial values of, respectively, 0.08 and 0.10 um ABTS oxidized per min to respective plateau values of 0.47 and 0.36 um ABTS oxidized per min in the presence of more than 50 mm imidazole (Fig 6) When the reaction was performed in the presence of o,o- and «,B-1,2-Fe(DoC- PP)—13G10 complexes, the rate of oxidation of ABTS by HO; increased sharply in the presence of increasing amounts of imidazole, from an initial value of 0.37 um ABTS oxidized per min to respective maximum values of 3.68 and 2.43 um ABTS oxidized per min in the presence of more than 50 mm imidazole (Fig 6) Thus, contrary to what occurred with the 13G10—Fe(ToCPP) complex, the
PP)—13G10 complexes was found to increase largely their peroxidase activity with respective As values of 15 and
25 mM
The kinetic parameters for the oxidation of 0.2 mm ABTS by H,Os;, 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 1n the presence of 50 mm imidazole (Table 4) It appeared that
in all the cases the addition of 50 mm imidazole had a
major effect on the k,,, value: in the case of Fe(ToCPP)— 13G10, it caused a decrease of the k,,; value by a factor of
4 from 109 + 10 min” to 32 + 3 min whereas in contrast, with both o,o- and œ,B-1,2-Fe(DoCPP)—-I3GI0 complexes, it caused an increase the k,,, value by a
factor of + 5-6, respectively, from 32 + 3 min ` to 152 +
10 min ` and from l6 + 2 min ' to 96 + 9 min The addition of 50 mm imidazole had a more moderate effect
on the K,, value that only slightly decreased from
29 + 3 mm to 19 + 2 mM 1m the case of Fe(ToCPP)-— 13G10, whereas it decreased by a factor 3, respectively, from 34+ 3mm to 10 + 1mm and from 18 + 2mm to 7 + 1 mmM with oo and o,B-1,2-Fe(DoCPP)— 13G10 As a consequence, the addition of 50 mm imida- zole caused a two fold decrease of the k,.;/K,, value from 3.8 + 07x10 “min” to 1.7 + 04x 10° mM min!
in the case of Fe(ToCPP)—13G10 as catalyst, whereas on the contrary, it caused an about 15-fold increase of the kcauL K,a value, respectively, from 0.9 + 0.2 x 10” M “min `
to 152 + 25x 10M 'mn ` and 09 + 02x
Table 4 Influence of imidazole on the kinetic parameters of the oxidation of ABTS by H;O; catalyzed by Fe(ToCPP)- and Fe(DoCPP)—-13G10 complexes at pH 5
Catalyst (min ') (mM) (M min `) (min `) (mM) (M'*min ')
Fe(ToCPP) 68 +7 37 + 4 1.8 + 0.3 x 10° 71] + 7 8.5 + 1 8.3 + 1.5~x 10° Fe(ToCPP)—13G10 109 + 10 29 + 3 3.8 + 0.7 x 10° 32 + 3 19 +2 1.7 + 0.4x 10”
%„œ-1,2-Fe(DoCPP)~ 32 + 3 34 + 3 0.9 + 0.2x 10° 152 + 10 10 +1 15.2 +2.5x10° 13G10
a, B- 1,2-Fe(DoCPP)-— l6 + 2 I§+ 2 0.9 + 0.2x 10° 96 + 9 7+] 13.7 + 2.8 x10”
13G10
Trang 8© FEBS 2002
Fe(DoCPP)- and œ,B-I,2-Fe(DoCPP)—-I3GI10 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(II) 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 first one
absorbing at 420 nm for CN’ concentrations below 6 mm
and a second one absorbing at 429 nm for CN’ concentra-
tions higher than 10 mm The 13G10-(ToCPP)Fe—-CN and
13G10-{(ToCPP]|Fe(CN),] structures were strongly sug-
gested for those two complexes as: (a) their spectra of
absorption were similar to those previously reported for
(porphyrin)Fe-CN and [(porphyrin)Fe(CN),]" complexes
[49], their maxima of absorption being only 3 nm redshifted;
(b) both 1/AAgo9 and 1/AA4>9 varied linearly as a function of
LICN ] (Fig 3, insets); (c) when (ToCPP)Fe-CN and
[((ToCPP)Fe(CN).| were reinserted into apo-13G10, spec-
tra similar to those already oberved for 13G10—(ToCPP)Fe—
CN and 13G10-[(ToCPP)Fe(CN)»s]° were obtained, which
showed that the binding of the two cyanide ligands on the
iron did occur inside the binding pocket of the antibody
This is totally different from what was reported by
Kawamura-Konishi et al [29] for the anti-(N-methyl mes-
oporphyrin LX) Ig 2B4 Indeed, in this case, the iron(II) 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, an 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 hypothesis is in
agreement with the binding site topology which 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 meso-
phenyl rings being bound to side chains of amino acids such
as arginine [36] In this respect, it 1s noteworthy that the
X-ray structure of a complex of 1ron(I]])-mesoporphyrin LX
with an anti-(V-methyl-mesoporphyrin [X) 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 residues of the Vy
domain and two pyrrole rings packed against tyrosine
residues of the V; 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 derivatives 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 derivatives: 1l-methyl-,
Peroxidase-like Fe-porphyrin—antibody complexes (Eur J Biochem 269) 477
B 13G1U-ke(1oCPP) +2CN
A 13G10-Fe(ToCPP) + HO;
C 13G10-Ee(ToCPP) + H;O; + InH D 13G10-Fe(DoCPP) + HO; + ImH
Fig 7 Various possibilities for the binding of ligands on the iron
of Fe(porphyrin)—13G10 complexes: (A) binding of H,O), on the iron of 13G10-(Fe(ToCPP), (B) binding of two CN ligands on the iron
of 13G10-(Fe(ToCPP), (C) binding of H,O, and imidazole on the iron of 13G10-(Fe(ToCPP), (D) binding of H,QO, and imidazole on the iron
of 13G10-0,0-1,2-Fe(DoCPP)
l-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’ (ImH), complexes [50]; (b) the linear dependence of 1/AA4;7 as a function of 1/[ImH] when increasing amounts of imidazole were added to Fe(ToCPP); and (c) the C59 (= Ki °) found for the complexes of Fe(ToCPP) with imidazole derivatives (Table 2), which were in good agreement with the equilib- rium constant B> measured for the formation of (porphy- rin)FeImH), complexes [51] This of course is not very surprising as it has been reported extensively 1n the literature that Fe-tetraarylporphyrins were able to form bis-imida- zolesron(III) complexes [50—53] and the X-ray structure of some of these complexes has been determined [52,53] More surprising, however, is the finding that the iron of the Fe(ToCPP)—13G10 complex is able to bind only one imidazole ligand as shown by the fact that 1/AA4)9 varies linearly as a function of |/[ImH] but not as a function of 1/[ImHf (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's) value calculated in this case (21.3 + 0.3 mm) is about 10-fold higher than that calculated 1n the case of Fe(ToCPP) (2.70 + 0.04 mm) (Table 2), that even the formation of the mono-imidazole complex of 13G10—Fe(ToCPP) is more difficult 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
Trang 9but a second imidazole is then unable to bind on the other
more hindered face of the porphyrin that bears the three
a-carboxyphenyl groups and is stacked against the antibody
protein (Fig 7)
This hypothesis is further sustained by the Cso 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 that which binds to the iron, the Cs) values measured were
lower than those measured for imidazole both with Fe(ToC-
PP) and 13G10—Fe(ToCPP) Indeed, for Fe(ToCPP), Cs
values of 2.60 + 0.04 mm and 0.63 + 0.01 mm were
found, respectively, for 1-CH3- and 1-benzylimidazole and
2.70 + 0.04 mm for imidazole whereas for 13G10—Fe(ToC-
PP), Cso values of 4.30 + 0.06 and 14.5 + 0.2 mm were
found, respectively, for 1-CH3- and 1-benzylimidazole and
21.3 + 0.3 mm for imidazole This could be explained by a
greater hydrophobicity of the two I-substituted imidazoles
with respect to that of imidazole Second, like in the case of
imidazole, the Cso values are higher with the antibody—
Fe(ToCPP) complex than in the case of Fe(ToCPP) alone,
which confirms that the binding of one 1-substituted
imidazole on the iron of 13G10—Fe(ToCPP) is even more
difficult than the binding of two 1-substituted imidazoles on
the iron of free Fe(ToCPP) In addition, the influence 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 affinity for
the iron than imidazole, whereas 1-benzylimidazole had a
better affinity than imidazole In constrast, in the case of
13G10—Fe(ToCPP), the Cs 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 l-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 Cso values obtained for Fe(ToCPP)
were 20- to 40-fold higher than the one for imidazole
(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
Cso (= Ka) values, of, respectively, 2.80 + 0.06 mm,
4.10 + 0.05 mm, and 3.10 + 0.05mM 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 character of 2- and 4substituted
imidazoles with respect to imidazole; (b) the absence of
steric interaction between the 2- and 4-alkyl substituent and
the protein; and (c) finally, 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),
œ,œ-1,2- œ,B-1,2-Fe(DoCPP), Fe(MoCPP)
and to their complexes with antibody 13G10
The UV/visible studies reported above showed that the
addition of increasing amounts of imidazole to o,o-1,2- and
a,B-1,2-Fe(DoCPP) and to Fe(MoCPP), and to their
complexes with 13G10 led to results that were similar to those observed for Fe(ToCPP): iron(II)-bis-imidazole complexes were formed in the case of free Fe-porphyrins whereas mono imidazoleiron(II]) complexes were formed
in the case of Fe-porphyrin—antibody complexes (Table 3)
In addition, in the particular case of œ,B-l,2- and œ,œ-l,2- Fe(DoCPP)—13G10 complexes, the Cs9 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 with two less hindered di-ortho-carboxyphenyl substituted tetraarylporphyrins
Influence 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 influence on the catalytic activity of those enzymes Because our studies
on the binding of imidazole to the iron(II) 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(HI)-or/ho-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 mm, caused a slight increase of the peroxidase activity of Fe(ToCPP), «,o-1,2- and o,B-1,2- Fe(DoCPP) (Fig 6) This was not surprising as it was previously reported that imidazole increased the ability of 1ron(HI- and Mn(III-porphyrin complexes to catalyze the oxidation of substrates such as sulfides [55], alkanes and alkenes [56,57] by H2O 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 mm imidazole was found to inhibit strongly the peroxidase activity with an Js9 of about 19 mm (Fig 6) As this value is close to that of the Cso 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 H2O; catalyzed by Fe(ToCPP)-13G10 in the presence of 50 mm imidazole, showed that the A.,,/Ky, 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 o,o-1,2- and a,B-1,2-Fe(DoCPP)-13G10 complexes led to a large increase of the peroxidase activity with Asg values of about 15 and 25 mm, respectively, the activity being optimal for a concentration of imidazole of
50 mm (Fig 6) Such a concentration of imidazole was found to cause a 15-fold increase of the Keat/Km value with both o,0-1,2-Fe(DoCPP)- and «,B-1,2-Fe(DoCPP)-13G10 complexes
Trang 10© FEBS 2002
We propose a likely explanation for the 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, and (b) a carboxylic acid
residue of the protein participates to the catalysis of the
heterolytic cleavage of the O-O bond of H;O: In the case
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 to 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 general acid-base catalyst,
whereas HO, 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 o,0-1,2- and «,6-1,2-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 and, in the more
favorable conformation, HO, 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 to a new artificial
hemoprotein or hemoabzyme that displays an interesting
peroxidase-like activity The complex is composed of a
robust protein, a monoclonal anti-porphyrin Ig, with an
iron(IID-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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