Tài liệu Báo cáo khóa học: New activities of a catalytic antibody with a peroxidase activity ppt

New activities of a catalytic antibody with a peroxidase activity
Formation of Fe(II)–RNO complexes and stereoselective oxidation of sulfides
Re
´
my Ricoux, Edyta Lukowska, Fabio Pezzotti and Jean-Pierre Mahy
Laboratoire de Chimie Bioorganique et Bioinorganique, Institut de Chimie Mole
´
culaire et des Mate
´
riaux d’Orsay,
Universite
´
Paris-Sud XI, Orsay, France
In order to estimate the size of the cavity remaining around
the heme of the 3A3–microperoxidase 8 (MP8) hemo-
abzyme, the formation of 3A3–MP8–Fe(II)-nitrosoalkane
complexes upon oxidation of N-monosubstituted hydroxyl-
amines was examined. This constituted a new reaction for
hemoabzymes and is the first example of fully characterized
Fe(II)–metabolite complexes of antibody–porphyrin. Also,
via a comparison of the reactions with N-substituted hyd-
roxylamines of various size and hydrophobicity, antibody
3A3 was confirmed to bring about a partial steric hindrance
on the distal face of MP8. Subsequently, the influence of
the antibody on the stereoselectivity of the S-oxidation of
sulfides was examined. Our results showed that MP8 alone
and the antibody–MP8 complex catalyze the oxidation of
thioanisole by H
2
O
2

and tert-butyl hydroperoxide, following
a peroxidase-like two-step oxygen-transfer mechanism
involving a radical–cation intermediate. The best system,
associating H
2
O
2
as oxidant and 3A3–MP8 as a catalyst, in
the presence of 5% tert-butyl alcohol, led to the stereo-
selective S-oxidation of thioanisole with a 45% enantiomeric
excess in favour of the R isomer. This constitutes the highest
enantiomeric excess reported to date for the oxidation of
sulfides catalyzed by hemoabzymes.
Keywords: artificial hemoproteins; abzymes; nitrosoalcanes;
microperoxidase 8; S-oxidation.
Catalytic antibodies with a metalloporphyrin cofactor, or
ÔhemoabzymesÕ, are not as efficient a category of catalysts as
their natural hemoprotein counterparts. The hemoabzymes,
which display a peroxidase activity, are characterized by
k
cat
/K
m
values that are three to four orders of magnitude
lower than those for natural peroxidases [1]. The relatively
low efficiency of these porphyrin–antibody complexes is
probably the result, at least in part, of the fact that no
proximal ligand of the iron has been induced in these
antibodies. To avoid this problem, we decided to use, as
a hapten, microperoxidase 8 (MP8), a heme octapeptide

where the imidazole side-chain of histidine 18 acts as a
proximal ligand of the iron atom. A set of six monoclonal
antibodies was thus obtained: the best peroxidase activity –
that found with the complex of MP8 and one of those
antibodies, 3A3 – was characterized by a k
cat
/K
m
value of
2 · 10
6
M
)1
Æmin
)1
, the best ever reported for an antibody–
porphyrin complex [2]. Active-site topology studies sugges-
ted that the binding of MP8 occurred through interactions
of its carboxylate substituents with amino acids of the
antibody, and that the protein provided a partial steric
hindrance of the distal face of the heme [2]. In addition,
it was shown recently that 3A3–MP8 was a more efficient
catalyst for the nitration of phenol by NO
2
–
/H
2
O
2
than

MP8 alone, and that the antibody protein not only
protected MP8 against oxidative degradations but also
induced a regioselectivity of the reaction in favor of the
formation of 2-nitrophenol [3]. Consequently, it was tempt-
ing to examine whether the hemoabzyme 3A3–MP8 was
able to catalyze the selective oxidation of other substrates.
In the present study, compounds containing sulfur were
chosen as substrates, as they play an important role in
medicine and agriculture. It has been reported that  10–
15% of medicinal and veterinary products and  33% of
synthetic organic pesticides contain sulfur [4]. The activity
of organosulfur compounds is often modified by oxidative
metabolism. Indeed, enzymatic oxidation produces sulf-
oxide metabolites that are chemically more reactive than
the starting substrate, and which are responsible for their
direct toxicity [5]. Numerous peroxidases catalyze the
in vitro S-oxygenation of alkyl-aryl-sulfides, with some-
times a good enantioselectivity resulting from the inter-
action of sulfides with a chiral environment in the heme’s
active site [6]. The prevailing sulfoxide has the R absolute
configuration in the presence of chloroperoxidase (CPO)
[6], lactoperoxidase (LPO) [7] and myeloperoxidase (MPO)
Correspondence to J P. Mahy, Laboratoire de Chimie Bioorganique
et Bioinorganique, UMR 8124 CNRS, Institut de Chimie
Mole
´
culaire et des Mate
´
riaux d’Orsay, Baˆ timent 420,
Universite

´
Paris-Sud XI, 91405, Orsay cedex, France.
Fax: + 33 1 69 15 72 81, Tel.: + 33 1 69 15 74 21,
E-mail:
Abbreviations: CcP, cytochrome c peroxidase; CH
3
COOEt, ethyl-
acetate; CiP, Coprinus cinereus peroxydase; CPO, chloroperoxidase;
HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin;
LPO, lactoperoxidase; mCPBA, meta-chloroperbenzoic acid; MP8,
microperoxidase 8; MPO, myeloperoxidase; NOS, nitric acid
synthase; RNO, Fe(II)–nitrosoalkane complex; tBuOH, tert-butyl
alcohol; tBuOOH, tert-butyl hydroperoxide.
Enzymes: catalase (EC 1.11.1.6); horseradish peroxidase, myelo-
peroxidase, lactoperoxidase (EC 1.11.1.7); chloroperoxidase
(EC 1.11.1.10); cytochrome c peroxidase (EC 1.11.1.6).
(Received 8 December 2003, revised 21 January 2004,
accepted 6 February 2004)
Eur. J. Biochem. 271, 1277–1283 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04032.x
[8], as catalysts, and the S configuration in the presence
of horseradish peroxidase (HRP) [9], Coprinus cinereus
peroxydase (CiP) [7], cytochrome c peroxidase (CcP) [10]
and soybean peroxygenase [11]. The S-oxidation of organic
sulfides by peroxidases could involve two types of mech-
anisms (Scheme 1). The first mechanism is a Ôone-step
oxygen transfer mechanismÕ, with an oxygen atom being
directely transferred from Compound I to the sulfur atom
of the organic sulfide; the second mechanism is the Ôtwo-
step oxygen-transfer mechanismÕ which involves a radical–
cation intermediate.

In the present report, we first used coordination chemistry
to examine the topology of the binding site of the anti-MP8
Ig, 3A3, especially to evaluate the size of the cavity
remaining around the iron atom. For this purpose, we
studied the formation of Fe(II)-nitrosoalkane (RNO)
complexes upon the oxidation of N-substituted hydroxyl-
amines by the 3A3–MP8 complex. The first was a rather
small and hydrophilic hydroxylamine bearing a branched
alkyl group, isopropylhydroxylamine 1 [R ¼ (CH
3
)
2
-CH-],
whereas the second, N-(1-p-chlorophenylpropyl)hydroxyl-
amine-2 [R ¼ (p-ClPh-CH
2
)(CH
3
)CH-], was more bulky
and hydrophobic. A comparison of the results obtained
with both hydroxylamines confirmed that the antibody 3A3
brought a partial steric hindrance on the distal face of MP8.
Consequently, the influence of the antibody on the stereo-
selectivity of the S-oxidation of sulfides was examined. For
this study, the oxidation of thioanisole by different oxidants
was performed in the presence of either MP8 alone or with
the antibody–MP8 complex acting as a catalyst. The results
described here show that, in the presence of 3A3 antibody,
the S-oxidation of thioanisole by H
2

O
2
occurs with a 45%
enantiomeric excess in favour of the R isomer. This
constitutes the highest enantiomeric excess reported to date
for the oxidation of sulfides catalyzed by porphyrin–
antibody complexes.
Materials and methods
Preparation of MP8
MP8 was prepared by sequential peptic and tryptic digestion
of horse-heart cytochrome c(Sigma), as described previously
[12]. The heme content was determined using the pyridine
chromogen method [12]. The purity of the sample was greater
than 97%, based on MALDI-TOF mass spectrometry.
Preparation of monoclonal antibodies
MP8 was covalently attached to keyhole limpet hemo-
cyanin (KLH) and to BSA, using glutaraldehyde as a
coupling agent, in 1
M
bicarbonate buffer, pH 9.5, according
to Tresca et al. [13]. The conjugates were then purified by
column chromatography on Biogel P10. Hapten–protein
ratios were determined spectrophotometrically using a molar
absorption coefficient value (e)of1.49· 10
5
M
)1
Æcm
)1
at

407 nm for MP8. In the case of BSA, 6 mol of MP8 were
bound per mol of protein, whereas in the case of KLH,
22 mol of MP8 were bound per 100 000 g of protein. Two,
5-week-old, female BALB/c mice were immunized with the
hapten–KLH conjugate, and the mouse showing the best
immune response 12 days after the third immunization was
killed. Its splenocytes were fused with SP
2
O myeloma cells, as
described by Ko
¨
hler & Milstein [14]. The resulting hybrido-
mas were screened by ELISA for binding to the hapten–BSA
conjugate, using peroxidase-linked goat anti-mouse Ig [15].
Positive hybridomas were cloned twice and produced in large
quantities. Antibodies were then purified from hybridomas
supernatants on a column of protein A, and their homogen-
eity and purity were checked by SDS gel electrophoresis.
All animal experimentation was carried out in accordance
with the European Communities Council Directive of 24
November 1986 (86/609/EEC).
Hydroxylamines
N-isopropylhydroxylamine (hydroxylamine 1)waspre-
pared by reducing the corresponding 2-nitropropane with
Zn in the presence of ammonium chloride, according to a
procedure described previously [16]. The characteristics of
the product were found to be identical to those reported
in the literature [17]. N-(1-p-chlorophenylpropyl)hydroxyl-
amine (hydroxylamine 2) was prepared in two steps. First,
the condensation of p-chlorobenzaldehyde with nitroethane

under acidic conditions generated the corresponding 1-p-
chlorophenyl-2-nitropropene [18], which was then reduced
into N-substituted hydroxylamine using a lithium-alumin-
ium hydride, as described previously [19].
Reaction of MP8 and 3A3–MP8 with N-monosubstituted
hydroxylamines
Forty microlitres of a 10
)2
M
solution of N-monosubsti-
tuted hydroxylamine in CH
3
OH was added to a cuvette
containing 1 mL of 0.86 l
M
MP8–Fe(III) or 3A3–MP8
complex [obtained by preincubation of 0.86 l
M
MP8–
Fe(III) with 2 l
M
antibody 3A3 for 1 h at room tempera-
ture in 0.1
M
NaCl/P
i
(PBS), pH 7.4]. The evolution of
the UV-visible spectrum of the solution was monitored, as
Scheme 1. Mechanisms of oxygen transfer reactions catalyzed by per-
oxidases.

1278 R. Ricoux et al. (Eur. J. Biochem. 271) Ó FEBS 2004
a function of time, between 350 and 550 nm. Kinetic data
were then obtained by measuring the absorbance at the
maximum of absorption of the MP8–Fe(II)–RNO complex,
as a function of time.
S-oxidation of thioanisole by various oxidants catalyzed
by MP8 or the antibody–MP8 complex
Optimization of reaction conditions. Thioanisole, 84 l
M
in 0.1
M
Tris buffer, pH 7.5, was oxidized at room
temperature using MP8 (0.2 l
M
), as a catalyst, and various
oxidants (30 l
M
), such as H
2
O
2
, meta-chloroperbenzoic
acid (mCPBA) or tert-butyl hydroperoxide (t-BuOOH), in
the presence of 10% of various organic solvents [methanol,
CH
3
CN, tert-butyl alcohol (t-BuOH)]. The reactions were
initiated by adding the oxidant, and the rate of S-oxidation
was observed by monitoring the decrease in the absorbance
at 254 nm for 10 min. The concentrations of product

formed after 10 min were calculated using a m
M
absorption
coefficient (e ¼ 7.87 m
M
)1
Æcm
)1
at 254 nm) in the differ-
ence spectrum between the sulfide and the corresponding
sulfoxide.
Stereoselective oxidation of thioanisole. Standard incu-
bations (total volume, 0.5 mL) were performed at room
temperature in Tris buffer (0.1
M
, pH 7.5) containing
thioanisole (100 l
M
) and the catalyst, either 0.3 l
M
MP8
alone or 3A3–MP8 prepared by preincubation of 0.3 l
M
MP8 with 0.87 l
M
antibody for 1 h at room temperature.
An oxidant – H
2
O
2

, t-BuOOH or mCPBA (final
concentration, 50 l
M
) – was then added dropwise to the
solution at a rate of 20 · 5 lL drops over a period of
1.5 h. The reaction was quenched by the addition of
excess of Na
2
SO
3
. The organic products were then
extracted with ethyl-acetate (CH
3
COOEt) and analyzed
by GC (to determine the degree of conversion of the
sulfide) and by HPLC on a Chiracel OD-H column (iso-
hexane/propan-2-ol; 95 : 5; v/v) to determine the enantio-
meric excess of the sulfoxide thus obtained.
Results and discussion
Reaction of MP8 and 3A3–MP8 complexes with
N-monosubstituted hydroxylamines
The addition of the N-monosubstitued hydroxylamines 1
or 2 (350 l
M
) to a solution of MP8–Fe(III) (0.86 l
M
),
preincubated for 1 h at room temperature in 0.1
M
NaCl/P

i
,
pH 7.4, with antibody 3A3 (2 l
M
),ledtonewcomplexes,3a
and 3b, respectively, characterized by an absorption spec-
trum similar to those observed for the MP8–Fe(II)–RNO
complexes, with aborption maxima at approximately 413
and 530 nm (Table 1) [20]. The reactivity of these new
complexes was very similar to that of the MP8–Fe(II)–RNO
complexes and that of other already reported hemoprotein–
Fe(II)–RNO complexes [20], in that (a) they were stable
for 2 h in 0.1
M
NaCl/P
i
, pH 7.4, in the presence of 1 m
M
sodium dithionite, and (b) conversely, they were rapidly
destroyed upon the addition of 100 l
M
potassium ferri-
cyanide [Fe(CN)
6
K
3
], with regeneration of the 3A3–MP8–
Fe(III) complex. This strongly suggested a 3A3–MP8–
Fe(II)–RNO structure for these new complexes 3a and 3b.
Such a structure was confirmed by the following result, that

the addition of 2 l
M
3A3 to a solution of MP8–Fe(II)–
(CH
3
)
2
NO, prepared previously by reaction of hydroxyl-
amine 1 (350 l
M
)with1 l
M
MP8 in 0.1
M
NaCl/P
i
, pH 7.4,
led to a spectrum that was almost identical to that of 3a
(data not shown). Consequently, the above results show
that the oxydation of N-monosubstituted hydroxylamines
in the presence of the hemoabzyme 3A3–MP8–Fe(III) leads
to the formation of 3A3–MP8–Fe(II)–RNO complexes.
This constitutes a new reaction of hemoabzymes, and is also
the first example of an iron(II)–metabolite complex among
the familly of porphyrin–antibody complexes. Such com-
plexes constitute good models for those formed not only
in vitro, but also in vivo, during the oxidative metabolism of
drugs containing an amine function, such as amphetamine
or macrolids [20], and which lead to an inhibition of the
catalytic functions of cytochrome P450. In addition, the

Table 1. UV-visible characteristics of microperoxidase 8 (MP8) and 3A3–MP8–RNO complexes and kinetic constants for their formation by reaction
of MP8 and 3A3–MP8 with N-substituted hydroxylamines.
Fe(II)–RNO complex. R ¼
UV-visible
MP8
b
3A3–MP8
c
MP8
b
3A3-MP8
b
k
max
(nm), e (m
M
)1
Æcm
)1
)
a
k(min
)1
)
d
C
50
(l
M
)

e
k(min
)1
)C
50
(l
M
)
414 (96), 532 413 (80), 530 0.32 ± 0.03 285 ± 5 0.19 ± 0.02 565 ± 5
413 (77), 530 413 (50), 530 0.77 ± 0.04 300 ± 5 0.72 ± 0.02 285 ± 5
a
Calculated from the absorbance at 413 nm after reaction of 400 equivalents of RNHOH with 0.86 l
M
MP8–Fe(III) and 2 l
M
antibody
3A3 in 0.1
M
NaCl/P
i
(PBS) buffer, pH 7.4.
b
Ricoux et al. 2000.
c
This work.
d
k-values were calculated from the curves in Fig. 1 which were
fitted to pseudo first-order kinetics according to the equation: C ¼ C
max
(1-e

–kt
), using Kaleidagraph.
e
The RNHOH concentration which
leads to 50% conversion of MP8 or 3A3–MP8 into the corresponding Fe(II)–RNO complex.
Ó FEBS 2004 N- and S-oxidations catalyzed by a hemoabzyme (Eur. J. Biochem. 271) 1279
above results validate the use of hemoabzymes as a
convenient model for hemoproteins used in toxicology
and pharmacology, such as cytochrome P450, peroxidases
and nitric oxide synthase (NOS). It is probable that the
mechanism of formation of these complexes is similar to
that described for the formation of the MP8–Fe(II)–RNO
complexes and for the Fe(II)–RNO complexes of hemo-
proteins (Scheme 2). It should involve, first, a one-electron
reduction of the Fe(III) into Fe(II) by the monosubstituted
hydroxylamine to give the RNHOH
•+
radical cation. A
second, one-electron oxidation could then be achieved using
O
2
which, after losing two protons, should produce the
nitrosoalkane RNO that binds to MP8–Fe(II).
The values of the molar extinction coefficients at
413 nm for the 3A3–MP8–Fe(II)–RNO complexes have
been calculated according to Ricoux et al.[20](Table1).
From Table 1, it is clear that (a) the e-values depend on
the nature of the R substituent of the hydroxylamine and
(b) for both hydroxylamines, the e-valuesarelowerforthe
3A3–MP8–Fe(II)–RNO complexes than for their MP8–

Fe(II)–RNO counterparts. Indeed, when R ¼ (CH
3
)
2
CH-, calculated e-values are 77 m
M
)1
Æcm
)1
for the
MP8–Fe(II)–RNO complex and 50 m
M
)1
Æcm
)1
for the
3A3–MP8–Fe(II)–RNO complex. Similarly, when R ¼
(Cl-Ph-CH
2
)(CH
3
)CH-, a larger e-value is found for the
MP8–Fe(II)–RNO complex (96 m
M
)1
Æcm
)1
)thanforthe
3A3–MP8–Fe(II)–RNO complex (80 m
M

)1
Æcm
)1
). Overall,
the minor changes observed when comparing the spectral
characteristics of the 3A3–MP8–Fe(II)–RNO complexes
with those of the MP8–Fe(II)–RNO complexes (i.e.
almost no shift and a slightly lower absorbance of the
soret band) have already been observed when inserting
MP8 into 3A3 [2]. They are consistent with the insertion
of the MP8–Fe(II)–RNO complex into a hydrophobic
pocket with no change of the Fe(II) spin state and no
replacement of any of the two axial ligands of the iron,
His18 or RNO, by an amino acid side-chain of the
antibody protein.
Binding site topology of antibody 3A3
Figure 1 shows the changes in the concentration of the
Fe(II)–RNO complex, formed upon addition of RNHOH
to MP8–Fe(III) or 3A3–MP8–Fe(III), as a function of time.
From this figure, it appears that the formation of the Fe(II)–
RNO complexes follows pseudo first-order kinetics, and
that the formation rate of MP8–Fe(II) or 3A3–MP8–
Fe(II)–RNO complexes depends on the hydroxylamine
structure (Fig. 1, Table 1). Interestingly, in both instances,
Fe(II)–RNO complexes derived from the smaller aliphatic
hydroxylamine (1), formed more rapidly than those derived
from the more bulky aromatic hydroxylamine (2). Indeed,
rate constants of 0.77 ± 0.04 min
)1
and 0.72 ± 0.02

min
)1
, and of 0.32 ± 0.03 min
)1
and 0.19 ± 0.02 min
)1
could be calculated, respectively, for hydroxylamines 1 and
2 in the case of MP8 and 3A3–MP8. In addition, it is
clear from these values that, for both hydroxylamines, the
rate of complex formation is lower in the presence of the
antibody, with a decrease in the rate constant of  7% being
observed with hydroxylamine 1 andof>40%withthe
more bulky hydroxylamine 2.
The amount of Fe(II)–RNO complex formed after
adding increasing concentrations of hydroxylamine 1 or 2
to a solution of either 0.86 l
M
MP8 or 0.86 l
M
3A3–MP8
in 0.1
M
NaCl/P
i
buffer, pH 7.4, was determined using
UV-visible spectroscopy. With both hydroxylamines 1 and
2, the amount of Fe(II)–RNO complex increased with the
RNHOH concentration, MP8 and 3A3–MP8 being totally
converted into the corresponding Fe(II)–RNO complex at
concentrations higher than 3 m

M
. However, the concentra-
tion necessary to convert 50% of MP8 or 3A3–MP8
(0.86 l
M
) into the Fe(II)–RNO complex (C
50
)alsodepen-
ded on the hydroxylamine structure (Table 1). Indeed,
whereas very similar concentrations of hydroxylamine 1
and 2 were needed to convert 50% of 0.86 l
M
MP8 into
the corresponding Fe(II)–RNO complex (300 ± 5 l
M
and
285 ± 5 l
M
, respectively) (Table 1), a much higher con-
centration of hydroxylamine 2 (C
50
¼ 565 ± 5 l
M
)than
of hydroxylamine 1 (C
50
¼ 360 ± 5 l
M
) was needed to
convert 50% of 3A3–MP8 (0.86 l

M
) into the corresponding
Fe(II)–(pCl-Ph)NO complex.
From the results presented above, it first appears that the
N-substituted hydroxylamine carrying a ramified donating
Fig. 1. Time dependence of the formation of MP8- or 3A3–MP8–RNO
complexes for the reaction of 0.86 l
M
MP8 or 0.86 l
M
MP8, associated
with 2 l
M
antibody 3A3, with 333 l
M
RNHOH in 0.1
M
NaCl/P
i
(PBS)
buffer, pH 7.4. The concentration of Fe(II)–RNO complex, as a
function of time, is shown. Besides the points corresponding to
experimental values, the indicated curves represent fit of the data to
pseudo first-order kinetics, calculated from C ¼ C
max
(1-e
–kt
), using
KALEIDAGRAPH
3.0.2, where C is the concentration of Fe(II)–RNO

complex formed at a given time and C
max
is the maximum concen-
tration of Fe(II)–RNO complex formed. MP8, microperoxidase 8;
RNO, Fe(II)–nitrosoalkane complex.
Scheme 2. Mechanism of the formation of 3A3–MP8Fe(II)–RNO
complexes and oxidation of these complexes by potassium ferricyanide.
1280 R. Ricoux et al. (Eur. J. Biochem. 271) Ó FEBS 2004
alkyl group, N-isopropyl-hydroxylamine-1, is more reactive
than hydroxylamine 2,whichissubstitutedbyanelectro-
attractive p-chlorophenyl group. Indeed, it leads to the
highest rate constant and the lowest concentration necessary
to convert them into an Fe(II)–RNO complex (C
50
), with
either MP8 or 3A3-MP8 (Fig. 1, Table 1). Second, with
both hydroxylamines, a decrease in the reaction rate, as well
as an increase in the C
50
value, are observed with the 3A3–
MP8 complex, when compared with MP8 alone. These
phenomena are particularly important in the case of the
more bulky and hydrophobic N-substituted hydroxylamine
2, as the reaction rate decreases by a factor of 1.7 while the
C
50
value increases by a factor of 2 (Table 1). This suggests
that, although the antibody 3A3 does not prevent the
binding of a ligand, such as nitrosoalcane, to the iron of
MP8, it brings a partial steric hindrance on the distal face of

MP8 and thus controls access of the nitrosoalcane ligand
to the iron atom of MP8. Such a phenomenon has already
been observed in the case of hemoproteins having a narrow
active site, like hemoglobin, myoglobin and catalase, which
are unable to form Fe(II)–nitrosoamphetamine complexes,
and whose active site only enables the access of small
molecules, such as methyl and propylhydroxylamine, to the
heme iron [21]. This is also consistent with our previously
reported results, which showed that the antibody protein
induced a regioselectivity of the nitration of phenol by
NO
2
–
/H
2
O
2
catalyzed by 3A3–MP8 in favor of the forma-
tion of 2-nitrophenol [3]. Considering these findings, it was
reasonable to envision that the hemoabzyme, 3A3–MP8,
could catalyze the selective oxidation of other substrates,
such as compounds containing sulfur, which are known to
play an important role in medicine and agriculture.
Sulfoxidation of thioanisole
In a first experiment, H
2
O
2
(final concentration, 30 l
M

)was
added to a solution of 84 l
M
thioanisole and 0.2 l
M
MP8 in
0.1
M
Tris buffer, pH 7.5, at room temperature. The
concentration of product formed after 10 min was calcula-
ted as described in the Materials and methods. The reaction
was quenched by the addition of excess Na
2
SO
3
, and the
organic products were then extracted with CH
3
COOEt and
analyzed by GC. The only product formed, with a 2.5%
yield, was the corresponding sulfoxide that was identified by
comparison with an authentic sample (Fig. 2, Table 2).
The involvement of the iron atom of MP8 in the catalysis
was indicated by the 100% inhibition of the reactions
performed in the presence of 100 m
M
CN
–
(data not
shown). Indeed, CN

–
anions are known to bind strongly to
the Fe(III) of MP8 [22], replacing the labile H
2
O ligand
in the sixth coordination position of the iron, to produce
a very stable and catalytically inactive hexacoordinate
MP8Fe–CN complex. The effect of radical scavengers was
also investigated. The reaction was performed under the
conditions described above, but in the presence of 200 l
M
ascorbic acid that quenches free radicals (data not shown).
Under those conditions, a 100% inhibition of the sulfoxi-
dation was observed, which means that, in this instance, a
peroxidase-like mechanism was involved (Scheme 1).
Optimization of reaction conditions
Before the 3A3–MP8 complex was assayed as a catalyst for
the S-oxidation of thioanisole, the reaction conditions were
optimized with MP8 alone acting as a catalyst. For this
purpose, thioanisole, 84 l
M
in 0.1
M
Tris buffer, pH 7.5,
was oxidized at room temperature with the use of MP8
(0.2 l
M
) as a catalyst, and various oxidants (30 l
M
), such

as H
2
O
2
, mCPBA or t-BuOOH, in the presence of 10%
various organic solvents (methanol, CH
3
CN, t-BuOH). The
reactions were initiated by adding the oxidant, and the
concentrations of product formed after 10 min were calcu-
lated as described in the Materials and methods. The values
thus obtained are compared in Table 2. It first appeared
that H
2
O
2
was the best oxidant for the sulfoxidation of
thioanisole, as it produced the best yield in sulfoxide,
regardless of the solvent used. When tBuOOH was used in
the buffer alone, no oxidation was observed. However, in
the presence of organic solvents, sulfoxide was produced,
but in a lower yield than when using H
2
O
2
as an oxidant.
Finally, whatever the conditions, no sulfoxide was formed
when mCPBA was used as an oxidant, which confirmed
that the reaction occurred through a peroxidase Ôtwo-step
oxygen-transfer mechanismÕ, involving a radical–cation

intermediate, and not by a one-step oxygen-transfer mech-
anism (Scheme 1). With both H
2
O
2
and tBuOOH, the
S-oxidation of thioanisole was more efficient in the presence
of an organic solvent, the best of which was t-BuOH. As
it has been reported previously that (a) the addition of
alcohols to the reaction buffer increased the rate of
peroxidase-catalyzed asymmetric sulfoxidation of thioani-
sole, owing to a better solubilization of the thioanisole
substrate [23], and (b) the addition of 20–50% of organic
solvent, such as methanol (v/v), to solutions of MP8 in
water decreased the formation of MP8 dimers and aggre-
gates [12], the increased concentration of sulfoxide could
arise from the combination of two effects resulting from the
addition of t-butyl alcohol to the reaction medium, namely,
a better solubilization of thioanisole and an increase in the
catalytically active monomeric form of MP8.
Fig. 2. Activators and inhibitors of the sulfoxidation catalyzed by 3A3–
MP8. MP8, microperoxidase 8.
Table 2. Concentration of product for the S-oxidation of thioanisole by
H
2
O
2
, t-BuOOH or meta-chloroperbenzoic acid (mCPBA) in the pres-
ence of various organic solvents, with 0.2 l
M

microperoxidase 8 (MP8)
as the catalyst.
Oxidant
PhSOCH
3
(%)
Buffer
alone
+ 10%
methanol
+ 10%
CH
3
CN
+ 10%
t-BuOH
H
2
O
2
2.5 4.7 7.2 10.0
t-BuOOH – 2.1 6.2 2.8
mCBPA – – – –
Ó FEBS 2004 N- and S-oxidations catalyzed by a hemoabzyme (Eur. J. Biochem. 271) 1281
Stereoselective S-oxidation of thioanisole
As the above results showed that the best system for the
S-oxidation of thioanisole associated H
2
O
2

as an oxidant
with MP8 as a catalyst in the presence of tBuOH as an
organic co-solvent (Table 2), the stereoselective S-oxidation
of thioanisole (100 l
M
) was performed at room tempera-
ture, in 0.1
M
Tris buffer, pH 7.5, containing 5% t-butyl
alcohol, in the presence of either 0.3 l
M
MP8 alone or
0.3 l
M
antibody–MP8 complex acting as a catalyst. More-
over, H
2
O
2
(final concentration of 50 l
M
) was added
dropwise to this solution, at a rate of 20 · 5 lL drops over a
period of 1.5 h, in order to avoid a too high concentration
of oxidant in the reaction medium [6]. This was implemen-
ted not only to limit the degradation of the catalyst, but also
to avoid a direct reaction of the sulfide with H
2
O
2

that could
lead to racemic sulfoxide. The reaction was then quenched
by the addition of excess Na
2
SO
3
, and the organic products
were then extracted with CH
3
COOEt and analyzed by GC
and HPLC, as described in the Materials and methods. The
results shown in Table 3 show that the antibody–MP8
complex is a more efficient catalyst than MP8 alone, either
with or without 5% tBuOH, and generates sulfoxide yields
of 30% and 49%, respectively, under these conditions,
whereas MP8 alone generates yields of 10% and 23%,
respectively, under the same conditions. The yields did not
exceed 49%, even in the best case, because an oxidative
degradation of the catalyst occurred. This was shown by a
progressive disappearance, in its absorption spectrum, of
the soret band at 396 nm that is characteristic of the heme
moiety. This degradation was less important in the case of
the antibody–MP8 catalyst, which showed that the antibody
protected the heme against oxidative degradation and led to
higher yields in sulfoxide. In addition, whereas almost no
enantiomeric excess is observed in the presence of MP8
alone, an important enantiomeric excess is observed with
3A3–MP8 used as a catalyst, with the best value of 45%
obtained in favor of the R enantiomer in the presence of 5%
tBuOH. These results confirm the important role of the

antibody, previously observed [2,3]: a protection of MP8
against oxidative degradation, which leads to a higher
sulfoxide yield, and a steric hindrance on the distal face of
the heme, which significantly increases the enantioselectivity
of thioanisole’s S-oxidation.
Table 3 also compares the yields and enantiomeric excess
obtained for S-oxidation of thioanisole with various hemo-
proteins used as catalysts. With the exception of CcP [10],
which does not catalyze this reaction, all other peroxidases
led to yields ranging from 80 to 100%, higher than that
achieved with 3A3–MP8. CPO was the best catalyst and
produced the (R)-sulfoxide with a 100% yield and a 98%
enantiomeric excess [6]. Most other fungal and plant
peroxidases, such as HRP [9] and CiP [7], for which the
crystal structures and the protein sequence are known
to be quite homologous, produced the (S)-sulfoxide
with respective enantiomeric excess of 46 and 73%. The
mammalian peroxidases, MPO and LPO, which are also
quite homologous in protein sequence [24], both produced
the (R)-sulfoxide with respective enantiomeric excesses of 8
and 80%, like 3A3–MP8. Thus, although not as efficient as
peroxidases themselves, 3A3–MP8 constitutes an interesting
model system for hemoproteins, especially for mammalian
peroxidases, because it also leads to the oxidation of
thioanisole into the (R)-sulfoxide, like these enzymes. In
addition, the enantiomeric excess (45%) represents the
highest percentage reported, to date, for the oxidation of
sulfides catalyzed by porphyrin–antibody complexes: the
only other example is the stereoselective sulfoxidation of
thioanisole by iodosylbenzene, catalyzed by a Ru(II)–

porphyrin–antibody (SN 37.4), which produced the
S-enantiomer sulfoxide with a 43% enantiomeric excess
[25]. Our results thus validate the use of the hemoabzyme
3A3–MP8 as a catalyst for the selective oxidation of
interesting substrates such as alkanes and alkenes.
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Ó FEBS 2004 N- and S-oxidations catalyzed by a hemoabzyme (Eur. J. Biochem. 271) 1283