Báo cáo khoa học: Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N-hydroxyguanidines by hydrogen peroxide ppt

Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N -hydroxyguanidines by hydrogen peroxide Re ´ my Ricoux 1 , Jean-Luc Boucher 2 , Dominique Mandon 3 , Yves-Michel Frapart 2 , Yann Henry 4 , Daniel Mansuy 2 and Jean-Pierre Mahy 1 1 Laboratoire de Chimie Bioorganique et Bioinorganique, Institut de Chimie Mole ´ culaire d’Orsay, Universite ´ Paris-Sud XI, Orsay, France; 2 Universite ´ R. Descartes, Paris, France; 3 Institut LeBel, Strasbourg, France; 4 Institut Curie-Recherche, Universite ´ Paris-Sud XI, Orsay, France Nitric oxide (NO) is a potent intra- and intercellular mes- senger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of L -arginine catalysed by heme- proteins called NO-synthases with intermediate formation of N x -hydroxy- L -arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model com- pound, N-(4-chlorophenyl)-N¢-hydroxyguanidine, were tes- ted for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydro- gen peroxide. Nitrite and nitrate production along with selective formation of 4-chlorophenylcyanamide was observed from incubations of N-(4-chlorophenyl)-N¢- hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyana- mide, N d -cyano-L-ornithine, was too unstable under the conditions used and L -citrulline was the only product identified. A NO-specific conversion of 2-(4-carboxyphe- nyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4- carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and formation of MP8–Fe–NO complexes were observed by EPR spectroscopy and low-temperature UV/visible spectro- scopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxi- dation of exogenous hydroxyguanidines by hydrogen per- oxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N-hydroxyguanidines by peroxidases of physio- logical interest remains to be established in vivo. Keywords: N-hydroxyguanidines; nitric oxide synthase; iron nitrosyl complexes; microperoxidase 8; nitric oxide. Nitric oxide, NO, is an intra- and intercellular messenger with important functions in the physiology of mammalian cardiovascular, immune and nervous systems [1,2]. A major action of NO is to activate guanylate cyclase and to increase cyclic GMP levels in target cells. NO is also involved as an effector of the antiproliferative function exerted by cytotoxic macrophages against tumour cells or parasites [3,4]. Bio- synthesis of NO involves the oxidation of L -arg by haem- containing monooxygenases known as NO synthases (NOSs) that require haem, FAD, FMN and tetrahydro- biopterine (BH 4 ) as cofactors, and NADPH and O 2 as cosubstrates [1,2,5]. The reaction catalysed by NOSs occurs in two steps with generation of N x -hydroxy- L -arginine (NOHA) as an intermediate [6]. Mechanisms of NO formation from L -arg and NOHA catalysed by NOSs are the subjects of active studies. NOSs fully saturated with BH 4 specifically catalyse the oxidation of NOHA to L -citrulline and NO, whereas BH 4 -free NOSs oxidize NOHA to a mixture of L -citrulline and N d -cyano- L -ornithine (CN-orn), with formation of NO and nitrous oxide (N 2 O) as nitrogen oxides, N 2 O originating from the initial release of nitroxyl anion, NO – [7,8]. Strong differences in the reactivity of NOSs’ are also observed with some analogues of NOHA: mixtures of N-arylureas and N-arylcyanamides are formed when N-aryl-N¢-hydroxyguanidines are incubated in the presence of BH 4 -free NOS II, whereas BH 4 -saturated NOS II selectively forms N-aryl-ureas [9]. In addition to NOSs, some cytochrome P450s oxidize simple guanidines to the corresponding N-hydroxyguanidines [10] while distinct P450 isoforms catalyse the oxidation of N-hydroxyguani- dines to mixtures of ureas and cyanamides with formation of nitrogen oxides [10–12]. This reaction of P450s involves the generation of superoxide anion and is highly sensitive to the addition of low amounts of superoxide dismutase (SOD) [11]. In fact, various enzymatic and chemical systems forming superoxide can generate nitrogen oxides from Correspondence to J P. Mahy, Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, Institut de Chimie Mole ´ culaire d’Orsay, Universite ´ Paris-Sud XI, Baˆ timent 420, 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: NO, nitric oxide; MP8, microperoxidase 8; MP8-I, MP8 compound I; MP8-II, MP8 compound II; N-AcMP8, N-acetylmicroperoxidase 8; NOS, nitric oxide synthase; NOHA, N x -hydroxy-L-arginine; CN-orn, N d -cyano-L-ornithine; BH 4 , (6R)-5,6,7,8-tetrahydrobiopterin; P450, cytochrome P-450; HRP, horseradish peroxidase; NDA, 2,3-naphthalene- dicarboxaldehyde; CPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl 3-oxide; CPTI, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl. (Received 24 July 2002, revised 23 October 2002, accepted 7 November 2002) Eur. J. Biochem. 270, 47–55 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03358.x NOHA and N-alkyl- (or N-aryl-) N¢-hydroxyguanidines. For instance, NAD(P)H oxidases are involved in the formation of nitrogen oxides from NOHA during oxidative burst [13]. Chemical systems generating superoxide anion from KO 2 similarly oxidize N-hydroxyguanidines with cyanamides as main products with minor amounts of ureas [14]. Cyanamides and N 2 O are also the main products identified after the addition of oxidants such as K 3 Fe(CN) 6 , Ag 2 CO 3 or PbO 2 to N-hydroxyguanidines [15] whereas mixtures of ureas, cyanamides, NO and N 2 O were identified after the addition of Pb(OAc) 4 ,H 2 O 2 , peracids, hydroper- oxides or NO itself, to N-hydroxyguanidines [15–17]. It was previously observed that horseradish peroxidase (HRP) reacts with NOHA in the presence of H 2 O 2 with formation of L -citrulline and nitrogen oxides [18]. However, the putative cyanamide derived from NOHA, N d -cyano- L - ornithine, is a relatively unstable product [7] and the exact nature of the organic end-product(s) formed in this HRP- catalysed oxidation of NOHA remains to be determined. Furthermore, in addition to the previously identified ureas and cyanamides, it has been recently reported that a mutant of cytochrome c peroxidase is able to bind NOHA and to catalyse its oxidation by H 2 O 2 to a new compound identified as N x -nitroso- L -arginine without any significant accumulation of NO or L -citrulline [19]. N-aryl-N¢-hydroxy- guanidines, N-aryl ureas and N-aryl cyanamides are more stable compounds than their alkyl-counterparts [9] and we therefore reinvestigated the reactivity of N-aryl-N¢-hydroxy- guanidines in the presence of various oxidative systems. Haem-based mini-enzymes, called microperoxidases, form a new generation of biocatalysts obtained by hydro- lytic digestion of cytochrome c. Microperoxidase 8 (MP8) consists of an iron protoporphyrin IX cofactor covalently bound to an oligopeptide (amino acid residues 14–21 of horse cytochrome c) which contains His18 acting as the axial ligand to the haem iron [20–22]. These microperoxi- dases have been reported to catalyse peroxidase-like reac- tions involving one-electron oxidations of several typical peroxidase cosubstrates [21–23]. MP8 can also display cytochrome P450-type oxygen transfer reactions and cata- lyses the para-hydroxylation of aniline, the S-oxidation of thioethers and the N- and O-dealkylation of aromatic amines and ethers [24,25]. We recently demonstrated that some N-aryl-N¢-hydroxyguanidines bind to Fe III -MP8 with a high affinity (K d in the 10–100 l M range) and form new low-spin complexes [26]. In this paper, we describe the reactivity of a simple N-hydroxyguanidine, N-(4-chlorophe- nyl)-N¢-hydroxyguanidine 1 (Scheme 1) and of NOHA itself, in the presence of MP8 and H 2 O 2 . Our results show that MP8 selectively catalyses the oxidation of 1 to the corresponding cyanamide 2 with formation of only minor amounts of urea 3. Reaction of 1 and NOHA in the presence of MP8 and H 2 O 2 leads to NO formation as evidenced by EPR spectroscopy using a specific spin trap for NO and by low temperature UV/Visible detection of the MP8–Fe III –NO complex. Moreover, MP8 compound II (MP8-II) reacts with exogenous NOHA to yield the same MP8–Fe III –NO complex. We thus provide experimental evidence that a peroxidase-like mechanism explains such a selective cyanamide formation with generation of NO and NO – . Materials and methods Chemicals MP8 was prepared by sequential peptic and tryptic diges- tion of horse heart cytochrome c (Sigma) essentially as previously described [20]. The haem content was determined spectrophotometrically using e 397 ¼ 157 m M )1 Æcm )1 [20]. The purity of the sample was > 97% based on MALDI- TOF MS analysis. The ferrous form of MP8 was prepared by reacting MP8 with sodium dithionite in phosphate buffer under anaerobic conditions. Metal-free MP8 was prepared by reductive demetallation of Fe III –MP8 in the presence of Fe II chloride in glacial acetic acid containing concentrated HCl [27]. N-Acetylmicroperoxidase 8 (N-AcMP8) was obtained by treating MP8 with excess of acetic anhydride in sodium carbonate buffer, as described previously [28]. N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 was prepared in 65% yield by reaction of cyanamide 2 with hydroxylam- ine hydrochloride in anhydrous ethanol [29]. 4-chlorophe- nylurea 3 and 4-chlorophenylcyanamide 2 were obtained, respectively, by reacting 4-chloroaniline with potassium cyanate in 0.5 M HCl and 4-chloroaniline with cyanogen bromide [11,29]. All compounds were fully identified by 1 Hand 13 C NMR and mass spectra which were identical to those described previously. N d -Cyano- L -ornithine was synthesized from L -ornithine following the protocol des- cribed by Clague et al. [7]. Hydrogen peroxide (30% v/v) and bovine liver catalase were purchased from Sigma. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (CPTIO) and N x -hydroxy- L -arginine came from Alexis. Gaseous NO was from L’Air Liquide. Anaerobic solutions of NO were prepared by bubbling NO in 0.1 M phosphate buffer pH 7.4 carefully deoxygenated with argon. NO concentrations were measured using the con- version of oxyhemoglobin to methemoglobin following an Scheme 1. Structure of the studied compounds. 48 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003 established protocol [30]. The concentration of the solution of hydrogen peroxide was checked spectrophotometrically using e 240 ¼ 39.4 M )1 Æcm )1 [21]. All other reagents and solvents were of the highest commercially available purity. Incubation conditions and analyses of end-products Typical incubations contained 1 m M N-(4-chlorophenyl)- N¢-hydroxyguanidine 1 and 1 l M MP8 in 250 lL(final volume) 0.1 M phosphate buffer pH 7.4. Reactions were startedbytheadditionof2m M H 2 O 2 . Incubations were shaken at room temperature for the indicated times and stopped by the addition of 100 lL phosphate buffer containing 1000 UÆmL )1 catalase and 400 l M 4-chloro- benzamide (internal standard for RP-HPLC analysis). After 10 min at room temperature, aliquots (50 lL) were mixed with the same volumes of sulfanilamide 1% in HCl 0.5 M and N-(1-naphthyl)ethylenediamine 0.1% in HCl 0.5 M for measurements of NO 2 – (Griess reaction) [31]. Reduction of nitrate to nitrite was performed in the presence of nitrate reductase and a NADPH-regenerating system according to a previously described protocol [32] and the nitrite formed was quantified using the Griess reaction. The amounts of nitrate were thus obtained by difference between the two measurements. Calibration curves were made under iden- tical conditions in the presence of known amounts of NaNO 2 and KNO 3 . Separations of organic products were routinely carried out on a Thermo-Finnigan system equipped with a AS3000 auto-sampler, a Focus multiwave- length detector and a 250 · 4.6 mm Nucleosil ODS column (5 lm particle size). Flow rate was 1 mLÆmin )1 and the mobile phase was a gradient between solvent A (5 m M phosphoric acid in water, pH 2.6) and solvent B (acetonit- rile) using the following program: 0 min, 5% B; 5 min, linear gradient to 40% B in 10 min; 15 min, linear gradient to 100% B in 10 min; 30 min, linear gradient to 5% B in 5 min followed by 15 min re-equilibration. The absorbance was monitored between 200 and 400 nm. The retention times for 1, 2, 3 and 4-chlorobenzamide (internal standard) were 7.0, 18.3, 15.2 and 14.2 min, respectively. Calibration curves were made from identical mixtures containing various concentrations of 2, 3, and internal standard, but without MP8. Some incubations were also analysed on a Thermo-Finnigan LCQ Advantage system (RP-HPLC system coupled to a mass spectrometer). Separations were performed on a 150 · 2 mm Chromasil C 18 column (3.5 lm particle size). Flow rate was 250 lLÆmin )1 and the mobile phase was a gradient between solvent A (10 m M ammonium acetate) and solvent B (acetonitrile) using the following program: 0 min, 10% B; 5 min, linear gradient to 40% B in 10 min; 15 min, linear gradient to 100% B in 10 min; 28 min, linear gradient to 10% B in 5 min followed by 10 min re-equilibration. The absorbance was monitored between 200 and 400 nm and mass spectra were recorded in the 100–1000 mass range. Incubations of 1 m M NOHA in the presence of 1 l M MP8 and 2 m M H 2 O 2 were performed as previously described for N-hydroxyguanidine 1, except that stop buffer contained 1000 UÆmL )1 catalase only. Amino acid products were derivatized and separated by RP-HPLC according to the procedure described by Clague et al.[7].Thus,50lLof the reaction mixtures were mixed in HPLC vials containing 20 l M phenylalanine (internal standard), 100 lL0.1 M potassium borate (pH 9.5), 10 lL0.5 M NaCN in the same borate buffer and 50 lL10m M 2,3-naphthalenedicarbox- aldehyde (NDA) in methanol. The mixtures were allowed to stay at room temperature for 15 min and applied to a Nova- Pak C 18 column (150 · 3.9 mm, 4 lm particle size, Waters) which was maintained at 40 °C and equilibrated with 60% solvent A (5 m M ammonium acetate buffer, pH 6.0) and 40% methanol (solvent B). Elution conditions were as follows: 2 min at 40% solvent B, a linear increase from 40 to 60% solvent B over 20 min, and from 60 to 100% solvent B over the next 5 min, followed by 3 min of 100% solvent B, and a return to initial conditions in 3 min. The flow rate was 0.5 mLÆmin )1 and detection was performed at 420 nm. Retention time for NDA derivatives of NOHA, CN-orn, L -citrulline and phenylalanine were 10.0, 6.2, 5.0 and 18.5 min, respectively. EPR Spectroscopy Room temperature EPR measurements were performed on mixtures containing 1 l M MP8, 1 m M N-(4-chlorophenyl)- N¢-hydroxyguanidine 1 or NOHA, 200 l M H 2 O 2 and 20 l M 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (CPTIO) in 0.1 M phosphate buffer pH 7.4. After 1 min, the reaction mixture was transferred via a Teflon capillary tube to a Bruker Aqua-X cell inserted in a shq 0011 cavity (Bruker). EPR spectra were recorded on a Elexsys E 500 EPR spectrometer using the following instrument settings: microwave frequency, 9.82 GHz; field modulation frequency, 100 kHz; sampling time, 0.04 s; field sweep, 6 mTÆmin )1 , 0.05 mT modulation amplitude and microwave power, 10 mW. UV/visible spectroscopy UV/visible spectra were recorded on a Varian Cary 05E spectrophotometer equipped with a variable temperature Dewar using a home-made quartz cuvette with a 0.1-cm path length. Solutions of MP8 (2 l M in phosphate buffer containing 50% methanol as antifreeze adduct) were introduced into the quartz cuvette which was set up in the beam and used as reaction vessel. After equilibration at )15 °C, 2 m M N-hydroxyguanidine 1 or NOHA, and 10 m M H 2 O 2 , all in phosphate buffer containing 50% methanol, were introduced directly in the cuvette via a steel needle and the medium was homogenized by cold nitrogen bubbling prior to data collection. Results Reaction of N -(4-chlorophenyl)- N ¢-hydroxyguanidine 1 in the presence of H 2 O 2 and MP8 In preliminary experiments, mixtures containing 1 m M NOHA, 1 l M MP8 and 2 m M H 2 O 2 were incubated for 2 min at room temperature before the addition of catalase to stop the reaction. Amino acids were derivatized in the presence of NDA and NaCN [7], and analysis of the reaction mixtures by RP-HPLC showed the disappearance of NOHA with formation of citrulline as the only identified product (data not shown). Incubation of authentic CN-orn Ó FEBS 2003 Microperoxidase 8 and nitrogen oxides formation (Eur. J. Biochem. 270)49 followed by derivatization under the same conditions also resulted in the detection of citrulline (data not shown). These results indicated that the low stability of CN-orn under those conditions prevents any clear indentification of the end products of the reaction. Similar incubations containing 1 m M N-(4-chlorophe- nyl)-N¢-hydroxyguanidine 1,1l M MP8 and 2 m M H 2 O 2 were thus performed and stopped after 2 min by the addition of catalase. Analysis of the reaction mixtures by RP-HPLC using UV-detection revealed a significant decrease of the amount of starting N-hydroxyguanidine 1 and the formation of three new organic compounds (Fig. 1). The very predominant one was identified to be 4-chloro- phenylcyanamide 2 by comparison of its retention time and UV spectrum to those of the authentic compound and by coinjection with the authentic compound. A second, minor, metabolite was similarly found to be 4-chlorophenylurea 3. The amounts of N-hydroxyguanidine that had disappeared were almost identical to the amounts of cyanamide 2 and urea 3 formed. An unknown product exhibiting a retention time of 13.2 min and a UV peak at 240 nm was also detected by RP-HPLC (Fig. 1). Further studies of the reaction mixtures using RP-HPLC coupled to MS analysis completely confirmed the identification of 4-chlorophenyl- cyanamide 2 as the main end product of the reaction and of 4-chlorophenylurea 3 in minor amounts (data not shown). However, we were unable to identify unambiguously the unknown product using our RP-HPLC system coupled to a mass spectrometer, operating either in the positive or in the negative mode in the 100–1000 mass range (data not shown). Aliquots of the reaction mixtures were simulta- neously studied for NO 2 – and NO 3 – formation using a classical colorimetric assay. Nitrate ion was the predomin- ant nitrogen product with only low amounts of NO 2 – (Table 1). Total amounts of measured nitrogen oxides (NO 2 – +NO 3 – ) were about one-third of the sum of the organic products (2 + 3,Table1).Incontrolexperiments, 4-chlorophenylurea 3 remained unchanged when incubated 10 min at room temperature in the presence of 1 l M MP8 and 2 m M H 2 O 2 whereas 4-chlorophenylcyanamide 2 gave low amounts of unidentified products, but without any formation of urea 3 (data not shown). Formations of cyanamide 2,urea3 and (NO 2 – +NO 3 – ) upon MP8- and H 2 O 2 -dependent oxidation of 1 were linearly dependent upon the amounts of added MP8 (in the 0.1–2 l M range). Accumulation of 2 and 3 reached a maximum 3–4 min after the addition of MP8 at room temperature, indicating that degradation of MP8 had occured under those oxidative conditions (data not shown). The effect of changing the pH of the reaction medium on the efficiency and selectivity of the reaction of 1 in the presence of MP8 and H 2 O 2 was also investigated. Increasing the pH from 5.0 to 8.5 resulted in a fourfold increase in the rate of formation of cyanamide 2 and urea 3, but no change in the ratio 2/3 (35 ± 5) was measured (data not shown). This increase in activity is consistant with the fact that in this pH range, the substrate is HO 2 – rather than H 2 O 2 [21]. The reaction of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 in the presence of MP8 and H 2 O 2 was clearly dependent upon the presence of all components as formation of 4-chlorophenylcyanamide 2 and NO 3 – was not observed in incubations performed in the absence of either MP8 or H 2 O 2 . Complementary experiments performed under anaerobic conditions resulted in almost similar yields, without significant changes in the cyanamide/urea, and NO 2 – /NO 3 – ratios. This indicated that dioxygen was not involved in catalysis (Table 1). The reaction was strongly inhibited by the addition to the incubation mixture of ascorbic acid and N-acetyl- L -cysteine, two classical reductants (Table 1). Mannitol and dimethyl- sulfoxide, two scavengers of OH Æ , and EDTA, an iron chelator, were without effect on the reaction efficiency and selectivity, indicating that neither OH Æ nor free iron were directly involved in the formation of cyanamide. Further- more, implication of the iron atom of MP8 in the catalysis was indicated by the almost complete inhibition of the reaction performed in the presence of 1 l M metal-free MP8 (Table 1). Using N-AcMP8 in place of MP8 led to a significant increase in the efficiency of the reaction without significant modification of the cyanamide/urea ratio (Table 1). This higher activity could result from less degradation, or aggregation, of N-AcMP8 by comparison to MP8 [28]. Selective formation of cyanamide 2 was also observed when haemin was used instead of MP8 but the reaction with haemin was much less efficient (Table 1). As reported previously [15], a simple oxidant proceeding through monoelectronic transfers, Fe(CN) 6 K 3 ,alsoledtothe selective formation of cyanamide 2 (data not shown). This constitutes another argument in favour of a monoelectronic process in the reactions catalysed by MP8. Detection of nitric oxide by EPR spectroscopy To detect the possible generation of NO from the oxidation of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H 2 O 2 in the presence of MP8, we used a NO-specific reactant, the nitronyl-nitroxide CPTIO and EPR spectroscopy. Nitric oxide reacts with CPTIO to generate an imino- nitroxide, CPTI, and this NO-dependent conversion is easily Fig. 1. RP-HPLC profile obtained after reaction of 1 m M N-(4-chlo- rophenyl)-N¢-hydroxyguanidine 1 with 2 m M H 2 O 2 , in the presence of 1 l M MP8in0.1 M phosphate buffer pH 7.4. The reaction was started by the addition of H 2 O 2 at room temperature and was stopped after 2 min by addition of catalase and analysed by RP-HPLC as described in Materials and methods. IS: Internal standard. 50 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003 monitored by EPR at room temperature [33]. Fig. 2, trace a shows the EPR spectrum of 20 l M CPTIO in the presence of 1 m M N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 and 2m M H 2 O 2 . The spectrum consists of five lines with an intensityratioof1:2:3:2:1(Fig.2,tracea).Thesame kind of EPR spectrum was also obtained when N-hydroxy- guanidine 1,H 2 O 2 , or both reagents were omitted from the reaction mixture (data not shown). Moreover, the addition of 1 l M MP8 to this mixture led to a gradual disappearance ofthefive-lineEPRspectrumofCPTIOandtothe appearance of a seven-line spectrum (Fig. 2, trace b). After about 8 min, the only observable spectrum (Fig. 2, trace c) was identical to the spectrum of CPTI obtained after the addition of a solution of NO to CPTIO, without detection of any other transient radical (data not shown). The intensity of this spectrum slowly decreased and was barely detectable 20 min after the addition of MP8 to the mixture of CPTIO, 1 and H 2 O 2 . The simultaneous presence of all components of the reaction mixture was required for the formation of the CPTI spectrum. This disappearance of the EPR signal of CPTI could result from further oxidation of CPTI to EPR-silent products under those conditions or from the coupling of CPTI(O) to intermediate radicals derived from N-hydroxyguanidine 1.Incontrolexperi- ments: (a) CPTI formed from the addition of NO solutions to CPTIO was stable in the presence of 2 m M H 2 O 2 , but further addition of 1 l M MP8 led to the slow disappearance of any observable EPR spectra after 10 min; (b) the incubation of 20 l M CPTIO with 1 l M MP8 and 2 m M H 2 O 2 but without N-hydroxyguanidine 1 did not lead to any change in the five-line signal of CPTIO for at least 15 min. Very similar results were obtained using NOHA in place of N-hydroxyguanidine 1 (data not shown). These results show that oxidation of N-hydroxyguanidine 1 (or NOHA) in the presence of H 2 O 2 and MP8 leads to the transient formation of NO and that further reaction of NO either with O 2 inthepresenceofFe III –MP8, or with MP8-I/ MP8-II leads to its stable end-products, NO 2 – and NO 3 – .In order to get more information on the nature of nitrogen oxides involved during these oxidations, low temperature UV/visible studies were performed. Low temperature UV/visible spectroscopy In the absence of substrate, the reaction of MP8 with H 2 O 2 yields the oxo-ferryl derivative MP8 compound II (MP8-II) [34]. Monitoring the spectroscopic changes following the addition of H 2 O 2 to MP8 at )15 °C in phosphate buffer containing 50% methanol, showed absorption shifts from 395 to 408 nm for the Soret band and from 535 nm to 522 and 551 nm for the Q bands (Fig. 3A, traces a and b). MP8- II is known to be relatively unstable and to undergo complete bleaching within minutes [34]. However, when 10 m M H 2 O 2 and 2 m M NOHA were simultaneously added Table 1. Effects of incubation conditions on the oxidation of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H 2 O 2 catalysed by MP8. Reactions were performed in 0.1 M phosphate buffer pH 7.4 containing 1 m M N-(4-chlorophenyl)-N¢-hydroxyguanidine 1, 2 m M H 2 O 2 and 1 l M MP8. After 2 min at room temperature, the reactions were stopped by the addition of catalase and aliquots were analysed as described in Materials and methods for measurements of cyanamide 2, urea 3, NO 2 – and NO 3 – . Results are expressed in l M of products formed under the conditions used and are means ± SD from four to eight experiments (three experiments in the cases of anaerobic conditions, metal-free MP8 and N-AcMP8). Products Conditions Cyanamide 2 Urea 3 NO 2 – NO 3 – Complete system 175 ± 45 6 ± 2 5 ± 2 60 ± 15 –MP8 2 ± 1 2 ± 1 1 ± 1 2 ± 1 –H 2 O 2 2±1 2±1 1±1 2±1 –O 2 130±12 3±1 8±3 40±8 +10m M ascorbic acid 2 ± 1 3 ± 1 1 ± 1 2 ± 1 +10m M N-acetylcysteine 12 ± 4 3 ± 1 3 ± 1 12 ± 3 + 100 m M mannitol 165 ± 35 8 ± 3 5 ± 2 80 ± 10 + 100 m M dimethylsulfoxide 166 ± 20 12 ± 3 5 ± 2 85 ± 20 + 0.1 m M EDTA 155 ± 25 15 ± 5 5 ± 1 90 ± 20 Metal-free MP8 14 ± 5 2 ± 1 5 ± 3 6 ± 3 N-AcMP8 200 ± 15 3 ± 1 15 ± 3 75 ± 10 1 l M Heme + 2 m M H 2 O 2 58±10 2±1 5±1 38±10 Fig. 2. EPR detection of NO formation. Spectra obtained at room temperature from 20 l M CPTIO in 0.1 M phosphate buffer containing 1m M N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 and 2 m M H 2 O 2 without MP8 (a). (b) Same as in (a) but 2 min after the addition of 1 l M MP8. (c) Same as (b) 10 min after the addition of MP8. Ó FEBS 2003 Microperoxidase 8 and nitrogen oxides formation (Eur. J. Biochem. 270)51 to a 2-l M solution of MP8 at )15 °C, MP8-II formed first and smoothly turned into a new intermediate with maxima at 412, 528 and 563 nm (Fig. 3A, trace c). Contrary to MP8-II, the new intermediate slowly turned back to ferric MP8 (Fig. 3A, trace d). This intermediate was obtained as a more stable species upon careful addition of NOHA to a previously prepared solution of MP8-II maintained at )15 °C (data not shown). Therefore, this new intermediate was a reaction product of MP8-II with NOHA and was tentatively identified by comparison to the UV/visible spectra of the MP8–Fe III –NO and MP8–Fe II –NO com- plexes formed, respectively, by the addition of a NO-solu- tion to MP8, either in the ferric or ferrous form. These two Fe–NO complexes showed characteristic spectra with peaks at 414, 526 and 560 nm (MP8–Fe III –NO, Fig. 3B, trace a) and 411, 537 and 563 nm (MP8–Fe II –NO, Fig. 3B, trace b). It appears that the spectrum of the intermediate formed during the reaction was very close to that of the MP8–Fe III – NO complex but significantly differed from that of the MP8–Fe II –NO complex (compare Fig. 3A, trace c, and Fig. 3B, trace a). These data suggest that MP8–Fe III –NO was the main species formed during the oxidation of NOHA by H 2 O 2 in the presence of MP8. Similar results were observed using N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 instead of NOHA (data not shown). Discussion Our results show that MP8 efficiently catalyses the oxida- tion of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 in the presence of H 2 O 2 with predominant formation of 4-chlo- rophenylcyanamide 2 and only minor amounts of urea 3 (5–10% of cyanamide 2, Eqn 1). An unidentified product was formed in minor amounts (Fig. 1) but could not be fully characterized under our RP-HPLC conditions with MS operating in the positive or negative modes. This product did not display the typical absorption at 400 nm described for nitrosoguanidines [19] but could be a dimeric com- pound. Stable end-products of NO, NO 2 – and NO 3 – ,were detected in lower amounts than the organic compounds (Table 1), indicating that nitrogen oxides distinct from NO are formed but were not detected by our methods. The most plausible candidate is nitroxyl anion, NO – , which upon dimerization, protonation and dehydration would release gaseous N 2 O [7,15]. Using NOHA instead of hydroxygu- anidine 1 led to the detection of citrulline as the only stable identified derivative, in agreement with the known instabi- lity of the corresponding cyanamide, CN-orn [7]. NO x ¼ NO À ; NO Á ; NO À 2 ; NO À 3 ð1Þ The oxidation of N-hydroxyguanidine 1 and of NOHA by H 2 O 2 catalysed by MP8 clearly involves an active species derived from the haem of MP8 as metal-free MP8 was inactive. It also involves high oxidation states of the haem of MP8 as it is strongly inhibited by reductants such as ascorbate and N-acetylcysteine. Such a selective oxidation of an N-hydroxyguanidine to the corresponding cyanamide has been reported previously in the case of monoelectronic oxidants such as K 3 Fe(CN) 6 and Ag 2 CO 3 [15]. This new MP8-catalysed reaction thus displays strong analogies with chemical systems known to proceed by mono-electronic transfers and with peroxidase-type reactions catalysed by MP8. It probably involves initial oxidation of MP8- Fe III by H 2 O 2 to MP8 compound I (MP8 + –Fe IV ¼ O, MP8-I). Successive monoelectronic reductions of MP8-I by N-hydroxyguanidine could give MP8-compound-II (MP8-II) and restore MP8–Fe III (Scheme 2). One-electron oxidations of a N-hydroxyguanidine should result in the formation of the iminoxy-radical 4 and one can propose the mechanism shown in Scheme 2 for the oxidation of N-hydroxyguanidine 1 by H 2 O 2 in the presence of MP8. Further one-electron oxidations of the intermediate imin- oxy-radical 4 (or its mesomeric form, the nitroso-C-centered radical 5), by MP8-I (or MP8-II) can lead to the interme- diate nitroso-imine 6 and cyanamide 2 (Scheme 2, paths a or b). Such mechanisms can generate nitric oxide NO and nitroxyl anion, NO – . Formation of N 2 O could explain the marked differences observed between the amounts of nitrogenous compounds derived from NO (NO 2 – +NO 3 – ) Fig. 3. Low temperature UV/visible spectroscopy. (A) To a mixture of 2m M NOHA and 2 l M MP8 in 0.1 M phosphate buffer containing 50% methanol was added 10 m M H 2 O 2 at )15 °C and spectra were recorded at various time intervals: 0 min (a), 1 min (b), 2 min (c) and 5 min (d). (B) Spectra of authentic MP8–Fe III –NO (a) and MP8– Fe II -NO (b) obtained after the addition of a solution of NO to 2 l M MP8 followed by the addition of sodium dithionite. 52 R. Ricoux et al. (Eur. J. Biochem. 270) Ó FEBS 2003 and organic products (2 and 3) in our experiments (Table 1). It seems likely that, in the presence of H 2 O 2 and Fe III ,NO – could be oxidized to NO with formation of MP8–Fe III –NO [35,36]. Alternatively, NO – could add to MP8–Fe III ,form- ing MP8–Fe II –NO that can be reoxidized to MP8–Fe III – NO under those conditions (Scheme 2, path c). MP8-I and MP8-II could also react with NO and oxidize it either into NO + or into NO 2 . , leading after hydrolysis to NO 2 – and NO 3 – (Scheme 2). As control experiments showed that cyanamide 2 did not lead to any formation of urea and that dioxygen is probably not involved in the reaction, the formation of urea 3 could result from the hydrolysis of one of the intermediates indicated on Scheme 2, the most plausible candidate being nitroso-imine 6. These mechanisms are in complete agreement with those previously indicated for the oxidation of N-hydroxyguan- idines by one-electron oxidants and for the oxidation of benzamidoximes by H 2 O 2 in the presence of HRP [15,37,38]. In addition, the formation of intermediate nitroso-imines has been clearly established in the oxidation of disubstituted amidoximes by Pb(OAc) 4 [39]. This new reaction of MP8 is in full agreement with a peroxidase-like activity of MP8 as it involves high-valent compounds I and II of MP8 and proceeds through mono- electronic transfers that lead to the selective formation of a cyanamide and a mixture of nitrogen oxides, NO and N 2 O. It clearly differs from the oxidation of NOHA and N-hydroxy- guanidine 1 catalysed by hepatic cytochromes P450 and BH 4 -free NOS II that give 1 : 1 mixtures of the correspond- ing cyanamide and urea [9,11]. This is easily understandable as the active species in the later reactions has been shown to be O 2 •) derived from the oxidase function of these haem proteins [9,13]. This MP8-catalysed reaction also differs from the oxidations of NOHA and 1 by BH 4 -containing NOS II, Scheme 2. Possible mechanisms for the oxidation of N-(4-chlorophenyl)-N¢-hydroxyguanidine 1 by H 2 O 2 in the presence of MP8. Ó FEBS 2003 Microperoxidase 8 and nitrogen oxides formation (Eur. J. Biochem. 270)53 which led almost exclusively to citrulline and the corres- ponding urea 3 [6,9,40]. It has been proposed that the active species responsible for these NOS-catalysed reactions is either a Fe II –O 2 or a Fe III –O–O – complex [1,2]. The very different behaviour of the MP8–H 2 O 2 system, that should involve a high-valent iron-oxo species for cyanamide forma- tion (see above), seems to confirm that the active species responsible for urea formation in NOS-catalysed oxidations is not a high-valent iron-oxo complex which could have derived from the NOS–Fe III –O–O – intermediate. According to the proposed mechanism, many other one-electron oxidants could generate NO from N-hydroxy- guanidines through reactions that do not require the participation of BH 4 -containing NOS. The formation of NO in a model system for peroxidase may rationalize the observations that some N-hydroxyguanidines are potent vasodilators in endothelium-denuded vasculature [41,42]. 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Everett, S.A., Smith, K.A., Patel, K.B., Dennis, M.F., Stratford, M.R.L. & Wardman, P. (1996) The involvement of nitric oxide in the toxicity of hydroxyguanidine in leukaemia HL60 cells. Br. J. Cancer 74, S172–S176. Ó FEBS 2003 Microperoxidase 8 and nitrogen oxides formation (Eur. J. Biochem. 270)55 . Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N -hydroxyguanidines by hydrogen peroxide Re ´ my Ricoux 1 ,. NO À 3 ð1Þ The oxidation of N-hydroxyguanidine 1 and of NOHA by H 2 O 2 catalysed by MP8 clearly involves an active species derived from the haem of MP8 as metal-free