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H2O2 and -NO Scavenging by Mycobacterium Leprae Truncated

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H2O2 and -NO Scavenging by Mycobacterium Leprae Truncated Biochemical and Biophysical Research Communications 373 (2008) 197–201 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Å H2O2 and NO scavenging by Mycobacterium leprae truncated hemoglobin O Paolo Ascenzi a,b,*, Elisabetta De Marinis a, Massimo Coletta c, Paolo Visca a,b a Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy b National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italy c Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘Tor Vergata’, Via Montpellier 1, I-00133 Roma, Italy article info abstract Article history: Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOAFe(III)) oxidation by H2O2 and of Received 28 May 2008 Å À trHbOAFe(IV)@O reduction by NO and NO2 are reported. The value of the second-order rate constant for Available online 9 June 2008 3 À1 À1 H2O2-mediated oxidation of trHbOAFe(III) is 2.4  10 M s . The value of the second-order rate con- Å stant for NO-mediated reduction of trHbOAFe(IV)@O is 7.8  106 MÀ1 sÀ1. The value of the first-order rate constant for trHbOAFe(III)AONO decay to the resting form trHbOAFe(III) is 2.1  101 sÀ1. The value Keywords: À 3 À1 À1 of the second-order rate constant for NO2 -mediated reduction of trHbOAFe(IV)@O is 3.1  10 M s . Mycobacterium leprae truncated Å À As a whole, trHbOAFe(IV)@O, generated upon reaction with H2O2, catalyzes NO reduction to NO2 .In hemoglobin O Å À Å turn, NO and NO2 act as antioxidants of trHbOAFe(IV)@O, which could be responsible for the oxidative H2O2 and NO scavenging Nitrosative and oxidative stress damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H2O2 Å Kinetics and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration. Ó 2008 Elsevier Inc. All rights reserved. During infection, Mycobacterium leprae is faced with the host antioxidants of the highly oxidizing heme-Fe(IV)@O derivative of macrophagic environment, where low pH, low pO2, high CO2 levels, heme-proteins. combined with the toxic activity of reactive nitrogen and oxygen Here, we report kinetics of M. leprae trHbOAFe(III) oxidation by Å ÅÀ Å À species, including nitrogen monoxide ( NO), superoxide (O2 ), H2O2 and of NO- and NO2 -mediated reduction of M. leprae and hydrogen peroxide (H2O2), contribute to limit the growth of trHbOAFe(IV)@O. As a whole, M. leprae trHbOAFe(IV)@O, obtained Å Å the bacilli. Remarkably, reactive nitrogen and oxygen species pro- by treatment with H2O2, catalyzes NO detoxification. In turn, NO À duced in vivo during the respiratory burst by monocytic/macro- and NO2 act as antioxidants of M. leprae trHbOAFe(IV)@O. There- phagic cells are an important cause of host tissue toxicity (i.e., fore, M. leprae trHbO can undertake within the same cycle not only Å nerve damage) [1–12]. NO and peroxynitrite scavenging [9,10,16–19] but also H2O2 The ability of M. leprae to persist in vivo in the presence of detoxification (present study). reactive nitrogen and oxygen species implies the presence in this elusive mycobacterium of (pseudo-)enzymatic detoxification sys- Materials and methods tems, including truncated hemoglobin O (trHbO) [8–10,13–18]. Å M. leprae trHbO has been reported to facilitate NO and peroxyni- Mycobacterium leprae trHbOAFe(III) was prepared as previously Å trite scavenging using O2 and NO as cofactors [10,16–19].As reported [37]. The M. leprae trHbOAFe(III) concentration was reported for some heme-proteins (e.g., hemoglobin (Hb) and myo- determined by measuring the optical absorbance at 409 nm 5 À1 À1 globin (Mb)) [20–28], M. leprae trHbO may undergo oxidation by (e409nm = 1.15  10 M cm ) [16]. M. leprae trHbOAFe(IV)@O H2O2, leading to the formation of the highly oxidizing ferryl was prepared by adding 10–25 equivalents of H2O2 to a buffered derivative (trHbOAFe(IV)@O), which could be responsible for the M. leprae trHbOAFe(III) solution. After a reaction time of 10– oxidative damage of the mycobacterium. Remarkably, recent 20 min, the trHbOAFe(IV)@O solution was stored on ice and used Å À Å À studies [29–36] suggest that NO and nitrite (NO2 ) can serve as within 1 h [33–35]. The H2O2, NO, and NO2 solutions were pre- pared as previously reported [10,16,34]. Kinetics of H2O2-mediated oxidation of trHbOAFe(III) was determined by mixing the trHbOAFe(III) (final concentration, @ À6 Abbreviations: Fe(III), ferric heme-protein; Fe(IV) O, ferryl heme-protein; 2.3  10 M) solution with the H2O2 (final concentration, A Fe(III) ONO, O-nitrito ferric heme-protein; Hb, hemoglobin; Lb, leghemoglobin; 1.0  10À5 to 5.0  10À5 M) solution [23–25]. Mb, myoglobin; TrHbO, truncated Hb O. A * Corresponding author. Fax: +39 06 5733 6321. The time course of H2O2-mediated oxidation of trHbO Fe(III) E-mail address: [email protected] (P. Ascenzi). was fitted to a single-exponential process (Scheme 1) [23–25]. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.168 198 P. Ascenzi et al. / Biochemical and Biophysical Research Communications 373 (2008) 197–201 Values of k were determined according to Eq. (1) [23–25]: Table 1 Values of kinetic parameters for H2O2-mediated oxidation of heme-Fe(III) A A ÀkÂt ½trHbO FeðIIIÞt ¼½trHbO FeðIIIÞi  e ð1Þ À1 À1 Heme-protein kon (M s ) The value of kon was determined according to Eq. (2) [23–25]: Mycobacterium leprae trHbOa 2.4  103 Horse heart Mbb 5.4  102 k ¼ k ½H O ð2Þ c 2 on 2 2 Sperm-whale Mb 6.6  10 Å A @ Human Mbd 3.4  104 Kinetics for NO-mediated reduction of trHbO Fe(IV) O was e 7 A @ Horseradish peroxidase 1.7  10 determined by mixing the trHbO Fe(IV) O (final concentration, Human myeloperoxidasef 1.9  107 À6 Å 1.2Â10 M) solution with the NO (final concentration, Human eosinophil peroxidaseg 4.3  107 5.0  10À6 to 2.0  10À5 M) solution [29–35]. Bovine lactoperoxidaseh 1.1  107 i 7 The time course of ÅNO-mediated reduction of trHbOAFe(IV)@O Catalase 1.7  10 was fitted to a two-exponential process (Scheme 2) [29–35]. a pH 7.2 and 20.0 °C. Present study. Values of h and l were determined according to Eqs. (3)–(5) [29– b pH 6.0 and 25.0 °C. From [24]. c 35,38]: pH 7.0 and 37.0 °C. From [25]. d pH 7.3 and 37.0 °C. From [23]. ½trHbOAFeðIVÞ@O ¼½trHbOAFeðIVÞ@O  eÀhÂt ð3Þ e pH 7.0 and 25.0 °C. From [21]. t i f A A A @ pH 7.0 and 15.0 °C. From [27]. ½trHbO FeðIIIÞ ONOt ¼½trHbO FeðIVÞ Oi g pH 7.0 and 15.0 °C. From [26]. h ðh ððeÀhÂt=ðl À hÞÞþðeÀlÂt=ðh À lÞÞÞÞ ð4Þ pH 7.0 and 15.0 °C. From [28]. i A A @ pH 7.0 and 20.0 °C. From [20]. ½trHbO FeðIIIÞt ¼½trHbO FeðIVÞ Oi A @ A A ð½trHbO FeðIVÞ Ot þ½trHbO FeðIIIÞ ONOtÞð5Þ responds to a monophasic process between 360 and 460 nm (Scheme 1). Values of k are wavelength-independent at fixed The value of hon was determined according to Eq. (6) [29–35]: Å [H2O2]. The plot of k versus [H2O2] is linear; the slope corresponds h ¼ hon ½NOð6Þ 3 À1 À1 to kon = 2.4  10 M s (Table 1). Å À Mixing of the M. leprae trHbOAFe(IV)@O and NO solutions Kinetics for NO2 -mediated reduction of trHbOAFe(IV)@O was determined by mixing the trHbOAFe(IV)@O (final concentration, brings about a shift of the optical absorption maximum of the Soret A @ 2.9  10À6 M) solution with the NO À (final concentration, band from 419 nm (i.e., trHbO Fe(IV) O) to 411 nm (i.e., 2 A A 2.5  10À5 to 2.0  10À4 M) solution [33–35]. trHbO Fe(III) ONO) and a change of the extinction coefficient 5 À1 À1 À from e419nm = 1.06  10 M cm (i.e., trHbOAFe(IV)@O) to The time course of NO2 -mediated reduction of 5 À1 À1 A A trHbOAFe(IV)@O was fitted to a single exponential process e411nm = 1.41  10 M cm (i.e., trHbO Fe(III) ONO). Then, the A A (Scheme 3) [33–35]. M. leprae trHbO Fe(III) ONO solution undergoes a shift of the Values of b were determined according to Eq. (7) [33–35]: optical absorption maximum of the Soret band from 411 nm (i.e., trHlbOAFe(III)AONO) to 409 nm (i.e., trHbOAFe(III)) [19] and a A @ A @ ÀbÂt ½trHbO FeðIVÞ Ot ¼½trHbO FeðIVÞ Oi  e ð7Þ change of the extinction coefficient from e411nm = 1.41  5 À1 À1 10 M cm (i.e., trHbO(III)AONO) to e409nm = 1.15  The value of bon was determined according to Eq. (8) [33–35]: 105 MÀ1 cmÀ1 (i.e., trHbOAFe(III)) [19]. À Å b ¼ bon ½NO2 ð8Þ Over the whole NO concentration range explored, the time Å À2 course for NO-mediated reduction of M.
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