HYDROGEN PEROXIDE-SUPPORTED ACTIVITIES OF SEMISYNTHETIC FLAVOCYTOCHROME 2B4.

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1 Vol. 45, No. 1, June 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages HYDROGEN PEROXIDE-SUPPORTED ACTIVITIES OF SEMISYNTHETIC FLAVOCYTOCHROME 2B4. Victoria V. Shumyantseva, Yuliya L. Avdeenko, Tatyana L. Moskvitina, Tatyana V. Bulko and Alexander I. Archakov Institute of Biomedical Chemistry, Pogodinskaya St., 10, Moscow , Russia Received January 23, 1998 SUMMARY Semisynthetic flavocytochromes, obtained by covalent binding of riboflavin with cytochromes P450 2B4, were able to catalyze the H202 -mediated reactions of aniline p-hydroxylation, aminopyrine N-demethylation and p-nitroanizole O- dealkylation. The rates of the flavocytochrome-catalyzed, H202 -supported reactions far exceeded those of the appropriate NADH-dependent reactions and were comparable with the cytochrome P450 2B4-catalyzed, peroxide-mediated reaction rates. The kinetic parameters (kcat, Km) for the peroxide-dependent flavocytochrome P450 2B4 reactions were obtained. Sodium cyanide and SKF- 525A, a specific P450 inhibitor, were both shown to inhibit these reactions. The generation of active oxygen species by flavocytochrome 2B4 was registered by chemiluminescence intensity. Key words: cytochrome P450 2B4, flavocytochrome 2B4, hydrogen peroxide, luminol, chemiluminescence. INTRODUCTION Naturally occurring flavocytochromes are self-sufficient monooxygenases. They consists of a single polypeptide containing both a catalytic heme domain and FAD/FMN reductase domain which utilizes NAD(P)H as an electron donor [1]. Flavins are able to generate peroxides in the course of oxygen activation, or to interact with peroxides with formation of active oxygen species [2,3]. In view of this, was of interest to study peroxide-dependent oxidation of substrates by semisynthetic flavocytochrome and, particularly, the role of flavins in this process. Flavocytochromes, obtained by means of covalent binding of riboflavin with cytochrome P450 2B4, were able to catalyze the NAD(P)H-dependent aniline p- hydroxylation reactions as well as the aminopyrine and N,N-dimethylaniline N- demethylation reactions [4]. It is known that cytochrome P450, in the presence of hydrogen peroxide or organic peroxides, catalyzes the oxidation of many organic /98/ ~) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL substrates [5]. In the present study the flavocytochrome P450 2B4-catalyzed, H202-supported reactions of aniline p-hydroxylation, aminopyrine N- demethylation and p-nitroanisole O-dealkylation were explored. Used were the flavocytochromes that contain 8-10 riboflavin molecules per 1 cytochrome P450 2B4 molecule - as being most active in NAD(P)H-dependent reactions, catalyzed by semisynthetic flavocytochrome 2B4 [4]. MATERIALS AND METHODS The following reagents were used: NAD(P)H and riboflavin (REANAL, Hungary), Sephadex G25 (PHARMACIA, Sweden), luminol (MERCK, Germany), catalase (specific activity units/mg, SERVA, Germany). Spectral studies were carried out using a Beckman DU-65 (USA) and Hitachi 557 (Japan) spectrophotometers and an LKB-1205 chemiluminometer (Sweden). Cytochrome P450 2B4 was isolated and purified as described in [6]. Flavocytochrome 2B4 was obtained according to [4]. The rates of NADH- and H202-mediated aniline p- hydroxylation, aminopyrine N-demethylation and p-nitroanisole O-dealkylation were determined as in [7]. Initiation of free-radical oxidation of luminol was attained by H202 introduction (32 ram) into the cytochrome P450- or flavocytochrome 2B4-containing systems. Hydrogen peroxide concentration was determined spectrophotometrically with the use of ~240 =43.6 M -1 cm -1 [8] or by the potassium permanganate method [9]. RESULTS AND DISCUSSION Kinetic parameters of H202-dependent reactions, catalyzed by semisynthetic flavocytochrome 2B4. Cytochrome P450 in the presence of organic peroxides or hydrogen peroxide is able to catalyze substrates oxidation. The present study was aimed at investigating the role of the heme iron as well as the role of flavin residues, covalently bound to cytochrome P450, in the H202 -dependent reactions. The reactions of aniline p- hydroxylation, aminopyrine N-demethylation and p-nitroanisole O-dealkylation were carried out. The reactions were initiated by hydrogen peroxide addition to flavocytochrome in the presence of substrates. Michaelis constant (Km) was determined both for the substrates (aniline, aminopyrine) and for the co-substrate (H202). The hydrogen peroxide concentration was varied from 5 to 1200 ram; the aminopyrine and aniline concentrations, from 0.4 to 9.6 mm and from 0.75 to 24 mm, respectively. The data obtained are presented in Table 1. The K m values for the semisynthetic flavocytochrome are close to those obtained for microsomes, oligomeric cytochrome P450 2B4 and monomeric cytochrome P450 2B4 [7]. It was suggested 172

3 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Table 1 Kinetic parameters of H202-supported reactions, catalyzed by semisynthetic flavocytochrome 2B4. Reactions Kin, mm kcat, min -1 kca t/km, min -1 i202 substrate H202 substrate Aniline p-hydroxylation Aminopyrine _ I N-demethylation Note. The incubation mixture contained I00 mm K-phosphate buffer, ph 7.4, l~tivf flavocytochrome 2B4. When evaluating K m for H202 the concentration of aminopyrine or aniline were 8 mm and 3 ram, respectively. When evaluating K m and kca t for substrates, the concentration of H202 was 0.5M, The excess of H202 was degradated by addition of catalase ( U/ml). therefore that the cytochrome P450-catalyzed peroxidase reactions and the semisynthetic flavocytochrome-catalyzed reactions are probably governed by similar mechanisms. Semisynthetic flavocytochrome is able to effectively metabolize Type I and II substrates (aminopyrine and aniline) in the II202-supported reactions. The catalytic constants (kcat) for the aniline p-hydroxylation and aminopyrine N- demethylation reactions are close to the appropriate catalytic constants for the analogous systems - of microsomes and P450 2B4 oligomers (Table 2). The kca t value for the H202 -dependent aniline p-hydroxylation reaction was about half less than the kca t for the analogous reactions with microsomes and cytochrome P450 2B4 (oligomer). The possible reason for this difference is the "nonproductive" activation of hydrogen peroxides on flavin residues and their consequent incapability to enhance the rate of substrate metabolism. The use of hydrogen peroxide at concentration >600 mm in semisynthetic flavocytochromecatalyzed reactions leads to flavocytochrome inactivation. This process is probably connected with the lesser stability of flavocytochrome or with the generation of the enzyme-degrading active oxygen species on flavin residues [2,3]. Earlier it was shown that semisynthetic flavocytochromes 2B4, containing various amounts of covalently bound riboflavin, did not catalyze NADH-dependent reactions of p-nitroanisole and 7-ethoxycoumarine O-dealkylation [4]. Using I-I202 as donor of active oxygen species, we accomplished the flavocytochrome-catalyzed 173

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 2. The comparison of catalytic constants for I-I202 -supported reactions, catalyzed by different systems. Reactions kcat, min -I microsomes cytochrome P450 flavocytochrome 2B4, oligomers 2B4 Aniline 38 [6] 24 [6] 11 p-hydroxylation Aminopyrine - 20 [6] 16 N-demethylation p-nitroanisole O-dealkylation. The catalytic constant, kcat, for the peroxidedependent O-dealkylation was estimated to be min -1. Close kca t values (kcat=6.9 min -1 ) were earlier reported for the p-nitroanisole O-dealkylation reaction, catalyzed by cytochrome P450 2B4 with cumene hydroperoxide [10]. Effect of sodium cyanide on the H202-dependent reactions of flavoeytochrome 2B4. Cyanide-anion is a hemoprotein inhibitor and binds to ferric heme (Fe +3 ) [11]. Since the P450 heine iron is present in the peroxide-mediated reactions in the oxidized state, cyanides are effective inhibitors of these reactions [12]. In the course of the flavocytoehrome 2B4-catalyzed, H202-dependent reactions in the presence of 10 mm NaCN, there occurred the inhibition of the aminopyrine N-demethylation and aniline p-hydroxylation reactions. As controls, the analogous cytoehrome P450 2B4-catalyzed, peroxide-dependent reactions were conducted (Table 3). As seen from the Table, the flavocytochrome-eatalyzed, H202 - dependent reactions of aniline p-hydroxylation were inhibited by cyanide to 55-65%, while the analogous P450 2B4-catalyzed reactions, to more than 75%. In the case of the H202-dependent aminopyrine N-demethylation reaction, the sodium cyanide inhibition levels were 30-40% and 85-95% for the flavocytochromecatalyzed and the cytochrome P450 2B4-catalyzed reactions, respectively. Maerophagal nitric oxide synthase is catalytically self-sufficient monooxygenase that contains within one polypeptide chain both the heme and flavins. Experiments with potassium cyanide (10 mm) and maerophage nitric oxide synthase have shown 55% inhibition of N G -hydroxy-l-arginine/h202 activity [12]. 174

5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 3. The influence of sodium cyanide on H202 -supported reactions (% of inhibition). Reaction System cytochrome P450 2B4 flavocytochrome 2B4 cyanide SKF-525A cyanide SKF-525A Aniline p-hydroxylation Aminopyrine N-demethylation Note. The incubation mixture contained 100 mm K-phosphate buffer, ph 7.4, 1 pm cytochrome P450 2B4 or flavocytochrome 2B4, 0.01 mm SKF-525A or 10 mm NaCN, 3 mm aniline or 8 mm aminopyrine. The reaction was initiated by the addition of 0.5M K202 Influence of SKF-525A on H202-dependent reactions, catalyzed by flavocytochrome 2B4. SKF-525A (~,[3-diethylaminoethyldiphenilacetate) is a specific P450 inhibitor [5]. SKF-525A (0.01 ram) inhibited the peroxide-supported reactions of aniline p- hydroxylation and aminopyrine N-demethylation, as catalyzed by flavocytochrome 2B4. For comparison, the otherwise identical P450 2B4-catalyzed reactions were conducted. Addition of SKF-525A to the reaction mixture lowered product formation in the H202 dependent, aniline p-hydroxylation reactions by 85-95% and 65-75% for the cytochrome- and the flavocytochrome-catalyzed reactions, respectively. The reactions of H2Oa-dependent aminopyrine N-demethylation were inhibited by SKF-525A to 90-95% for the cytochrome P450-catalyzed reactions and to 70-75%, for the flavocytochrome-catalyzed ones (Table 4). In the case of the aniline p-hydroxylation and the aminopyrine N-demethylation reactions, both flavocytochrome-catalyzed, the inhibition by SKF-525A was, on the average, 20% less than for the analogous, cytochrome P450-catalyzed reactions. Why are the flavocytochrome 2B4-catalyzed peroxide-mediated reactions are less effectively inhibited by sodium cyanide and SKF-525A than the cytochrome P450 2B4-catalyzed peroxide-dependent reactions? Two explanations for this phenomenon may be offered: One is that, owing to the covalent binding of riboflavin molecules, flavocytochrome may undergo some conformational changes; 175

6 - which Vol. 45, No. 1, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL as a result of these, the access to the enzyme's catalytic center may be hampered in turn may diminish the stability of the cyanide ions - heine iron binding. Riboflavin molecules could also block the access to the heme. Another possible explanation is that the active oxygen species generation from H202 could possibly occur not only on the heme iron but also on the isoalloxasine residues of riboflavins [2,3]. At the same time, the possibility of flavins' participation in peroxidase reactions cannon be excluded either. Luminol chemiluminescence of the flavocytochrome 2B4 - hydrogen peroxide system. For the registration of active oxygen species formation, the chemiluminescence intensity of three systems (cytochrome P450 2B4 - ~I202, flavocytochrome - NADP, flavocytochrome H202. ) in the presence of luminol was estimated. Luminol interacts with hydroxyl radical (OH.) and, to a lesser extent, with superoxide anion (02-.) with formation of an active luminol radical whose luminescence was recorded [13, 14]. In the absence of one of a system's constituent components, no chemiluminescence was recorded. Experiments with the active oxygen species generation using the chemiluminescence detection failed to reveal any luminol-based chemiluminescence, indicating the lack of OH- or particles in the reaction volume. At the same time in the cytochrome P450 2B4 - H202 and flavocytochrome 2B4 - H202 systems the active oxygen species generation, as registed by the luminol chemiluminescence method, was observed. Chemiluminescence was registed immediately after H202 addition (final concentration in the sample, 32 mm), with the maximum chemiluminescence being observed 2-3 seconds after H202 addition. Then the gradual lowering of the chemiluminescence intensity was seen. The kinetic curves of chemiluminescence are presented in Fig. I. The lesser intensity of flavocytochrome chemiluminescence, as compared to cytochrome P450 2B4, was probably due to the chemiluminescence quenching by flavins, or to the lesser activity of flavocytochrome as an active oxygen species generator on H202-dependent reactions. The H202 -catalyzed generation of active oxygen species in the presence of sodium cyanide and the cytochrome P450 inhibitor, SKF-525A, was also studied. The percent inhibition of active oxygen species, generated during interaction of hydrogen peroxide (32 mm) with cytochrome P450 or flavocytochrome 2B4 (0.i pm), was calculated. The results of inhibition, calculated 176

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 8000 r-, E 4000 i.) 2000 I ' I ' I 20 4O 6O Time, s Fig. 1. H202-mediated chemiluminescence of cytochrome P450 2B4 and flavocytochrome 2B4 in the presence of luminol. The incubation mixture contained 100 ram K-phosphate buffer, ph 7.4, 200 pm luminol, 1 ~tm cytochrome P450 2B4 or flavocytochrome 2B4, 32 mm H202, 10 NaCN mm o - cytochrome P450 2B4 - cytochrome P450 2B4 + CN- 0 - flavocytochrome 2B4 0 - flavocytochrome 2B4 +CN- 177

8 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Table 4. The inhibition of chemiluminescence intensity by sodium cyanide or SKF- 525A System % of inhibition, sodium cyanide % of inhibition SKF-525A maximum of lightsum of maximum of lightsum Of chemi- chemi- chemi- chemiluminescence luminescence luminescence luminescence cytochrome P450 2B4 flavocytochrome B4 with the use of the chemiluminescence maximum data, are presented in Table 4. As expected, the results of inhibition of luminol-dependent chemiluminescence (Table 4) provide support for the inhibition of active oxygen species generation in the presence of NaCN and SKF-525A. The data obtained are in accord with those on inhibition, by sodium cyanide and SKF-525A, of H202 -dependent reactions of aniline p-hydroxylation and aminopyrine N-demethylation (Tables 2 and 3). Thus, the H202 -dependent reactions of aniline p-hydroxylation, aminopyrine N- demethylation and p-nitroanisole O-dealkylation, catalyzed by semisynthetic flavocytochrome, were investigated and kinetically charactarized. REFERENCES I. Shumyantseva V. V., Avdeenko Yu. L., Moskvitina T. L. & Archakov A. I. (1997) Voprosi reed. kchimii (in Russian), 43, Bruice T. C. (1980) Ace. Chem. Res. 13, Massey V. (1994) J. Biol. Chem. 269, Shumyantseva V. V., Uvarov V. Yu., Byakova O. E. & Archakov A. I. (1996) Biochem Mol. Biol. Int. 38, Archakov A. I. & Bachmanova G. I. (1990) Cytochrome P450 and active oxygen. London, New York, Philadelphia: Taylor and Francies, Kanaeva I. P., Skotselyas E. D., Kuznetsova G. P., Antonova G. I., Bachmanova G. I. & Archakov A. I. (1985) Biokchimia (in Russian) 50, Kanaeva I. P., Dedinskii I. R., Skotselyas E. D., Krainev A. G., Guleva I. V., Sevryukova I. F., Koen Ya. M., Kuznetsova G. P. & Archakov A. I. (1992) Arch. Biochem. Biophys. 298, Teselkin Yu. O., Babenkova I.V., Lyubitsky O.V., Klebanov Yu. A. & Vladimirov Yu. A. (1997) Voprosi reed. chimii (in Russian) 43,

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