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: jpmahy@icmo.u-psud.fr 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]. 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Ó FEBS 2004 N- and S-oxidations catalyzed by a hemoabzyme (Eur. J. Biochem. 271) 1283 . New activities of a catalytic antibody with a peroxidase activity Formation of Fe(II)–RNO complexes and stereoselective oxidation of sulfides Re ´ my. nitrosoalcanes; microperoxidase 8; S-oxidation. Catalytic antibodies with a metalloporphyrin cofactor, or ÔhemoabzymesÕ, are not as efficient a category of catalysts as their