Reductive nitrosylation and peroxynitrite-mediated oxidation of heme–hemopexin Paolo Ascenzi 1,2 , Alessio Bocedi 2 , Giovanni Antonini 1 , Martino Bolognesi 3 and Mauro Fasano 4 1 Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Rome, Italy 2 National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy 3 Department of Biomolecular Sciences and Biotechnology and CNR-INFM, University of Milan, Italy 4 Department of Structural and Functional Biology and Center of Neuroscience, University of Insubria, Busto Arsizio, Italy Heme scavenging by high- and low-density lipopro- teins, serum albumin and hemopexin (HPX) provides protection against heme and iron oxidative damage, limits access by pathogens to heme, and contributes to iron homeostasis by recycling the heme iron. During the first seconds after the appearance of heme in the plasma, > 80% of this powerful oxidizer binds to high- and low-density lipoproteins, and only the remaining 20% binds to serum albumin and HPX. Serum albumin and HPX then remove the heme from high- and low-density lipoproteins. Afterwards, heme transits to HPX, which releases it into hepatic paren- chymal cells only after internalization of HPX–heme by specific receptor-mediated endocytosis. After deliv- ering the heme intracellularly, HPX is released intact into the bloodstream and the heme is degraded [1–15]. HPX–heme is formed by two four-bladed b-propel- ler domains, resembling two thick disks that lock Keywords nitric oxide; peroxynitrite; peroxynitrite- mediated oxidation; rabbit hemopexin; reductive nitrosylation Correspondence P. Ascenzi, Department of Biology, University ‘Roma Tre’, I-00146 Rome, Italy Fax: +39 06 5517 6321 Tel: +39 06 5517 3200 ⁄ 2 E-mail: ascenzi@bio.uniroma3.it (Received 14 September 2006, revised 14 November 2006, accepted 21 November 2006) doi:10.1111/j.1742-4658.2006.05609.x Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX–heme(II) reversibly binds NO and facilitates NO scaven- ging by O 2 . HPX–heme is formed by two four-bladed b-propeller domains. The heme is bound between the two b-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX–heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX–heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX–heme(II)–NO are reported. In the presence of excess NO, HPX–heme(III) is converted to HPX– heme(II)–NO by reductive nitrosylation. The second-order rate constant for HPX–heme(III) reductive nitrosylation is (1.3 ± 0.1) · 10 1 1 m )1 Æs )1 ,at pH 7.0 and 10.0 °C. NO binding to HPX–heme(III) is rate limiting. In the absence and presence of CO 2 (1.2 · 10 )3 m), excess peroxynitrite reacts with HPX–heme(II)–NO (2.6 · 10 )6 m) leading to HPX–heme(III) and NO, via the transient HPX–heme(III)–NO species. Values of the second- order rate constant for HPX–heme(III)–NO formation are (8.6 ± 0.8) · 10 4 and (1.2 ± 0.2) · 10 6 m )1 Æs )1 in the absence and pres- ence of CO 2 , respectively, at pH 7.0 and 10.0 °C. The CO 2 -independent value of the first-order rate constant for HPX–heme(III)–NO denitrosyla- tion is (4.3 ± 0.4) · 10 )1 s )1 , at pH 7.0 and 10.0 °C. HPX–heme(III)–NO denitrosylation is rate limiting. HPX–heme(II)–NO appears to act as an effi- cient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO 2 (e.g. ONOO- C(O)O – ,CO 3 – , and NO 2 ). Abbreviations Hb, hemoglobin; HbI, haemoglobin I; HPX, hemopexin; Lb, leghemoglobin; Mb, myoglobin; Ngb, neuroglobin. FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 551 together at a 90° angle; the face of the N-terminal b-propeller domain packs against one edge of the C-terminal domain. Each propeller blade comprises a four-stranded antiparallel b-sheet, with the first and the fourth blades joined by disulfide bridges. The heme is bound between the two four-bladed b-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex (Fig. 1). Heme binding and release results from the opening and closing of the heme-binding pocket, via movement of the two b-propeller domains and ⁄ or the interdomain linker peptide [16]. Evidence suggests that heme-bound plasma proteins may display ligand binding (kinetic and thermody- namic) capacity and pseudo-enzymatic properties. Fer- rous high- and low-density heme–lipoproteins bind NO [17]. Ferrous heme–serum albumin has been reported to bind NO, O 2 and CO [14,18–21], and to exhibit weak catalase and peroxidase activity [22]. HPX– heme(II) binds CO and NO, however, O 2 induces HPX–heme(II) oxidation [17,23–27]. Furthermore, HPX–heme(III) binds cyanide [16]. NO appears to modulate heme binding to HPX–heme and, in turn, HPX–heme may play a key role in NO homeostasis [17,25–27]. Indeed, O 2 has been reported to react with HPX–heme(II)–NO yielding HPX–heme(III) and NO 3 – , by way of the ferric heme-bound peroxynitrite intermediate HPX–heme(III)–N(O)OO. Afterwards, peroxynitrite dissociates from HPX–heme(III)– N(O)OO and isomerizes to nitrate. HPX–heme(III) may reduce back to HPX–heme(II) and bind heme lig- ands [27]. The recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate; for peroxynitrous acid, it is hydrogen oxoperoxonitrate. The term per- oxynitrite is used in the text to refer generically to both ONOO ) and its conjugate acid HOONO [28]. Here, the kinetics of reductive nitrosylation of HPX–heme(III) and peroxynitrite-mediated oxidation of HPX–heme(II)–NO are reported. HPX–heme(II)– NO appears to act as an efficient scavenger of peroxy- nitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO 2 [e.g. ONOOC(O)O – ,CO 3 and NO 2 ). Our results have been analyzed in parallel with those of related heme–protein systems. Results and Discussion Reductive nitrosylation of HPX–heme(III) Addition of NO (either gaseous or dissolved in the buffer solution) to the HPX–heme(III) solution causes a shift in the maximum of the optical absorption spec- trum in the Soret band from 414 nm, i.e. HPX– heme(III), to 419 nm, i.e. HPX–heme(II)–NO, and a corresponding change in the extinction coefficient from e 414 ¼ 1.16 · 10 5 m )1 Æcm )1 to e 419 ¼ 1.45 · 10 5 m )1 Æcm )1 (Fig. 2A). The optical absorption spectrum of the reac- tion product HPX–heme(II)–NO (Fig. 2A) is identical to that obtained by adding NO to HPX–heme(II) [26,27]. Accordingly, optical absorption spectroscopic changes for the reaction of HPX–heme(III) with NO were not reversible. Pumping off gaseous NO causes a shift in the maximum of the optical absorption spec- trum in the Soret band from 419 nm, i.e. HPX– heme(II)–NO, to 428 nm, i.e. HPX–heme(II), and a corresponding change in the extinction coefficient from e 419 ¼ 1.45 · 10 5 m )1 Æcm )1 to e 428 ¼ 1.47 · 10 5 m )1 Æcm )1 . The optical absorption spectra of HPX–heme(III), HPX–heme(II)–NO and HPX–heme(II) here deter- mined correspond to those reported in the literature Fig. 1. Rabbit ferric HPX–heme structure, including the coordinating heme-iron residues His213 and His266 (PDB entry: 1QJS) [16]. The N-terminal domain (residues 1–208) is shown at the top. The C-ter- minal domain (residues 228–435) is shown at the bottom. The arrow indicates the interdomain linker peptide (residues 209–227). Heme group and His213 and His266 residues are shown in black. The figure was drawn with SWISS-PDB-VIEWER [75]. For details, see text. Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al. 552 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS [24,26,27,29]. As already reported for Glycine max leg- hemoglobin (Lb) [30], sperm whale myoglobin (Mb) [31], horse cytochrome c [31], human neuroglobin (Ngb) [32], Scapharca inaequivalvis hemoglobin I (HbI) [33] and human hemoglobin (Hb) [31], our data indi- cate that the reaction of HPX–heme(III) with an excess NO leads to reduction of the heme–Fe(III) atom and generation of the HPX–heme(II)–NO species (Table 1). Over the whole NO concentration range explored, the time course of reductive nitrosylation of HPX– heme(III) (2.6 · 10 )6 m) conforms to a single-exponen- tial decay for > 90% of its course between 360 and 460 nm (Fig. 2B). The pseudo-first-order rate constant for HPX–heme(III) reductive nitrosylation (i.e. k)is wavelength independent. The plot of k versus [NO] is linear (Eqn 2) with a y-intercept at 0, indicating that the reverse reaction rate is negligible (k off <1· 10 )4 s )1 ); the slope of the plot of k versus [NO] corres- ponds to k on ¼ (1.3 ± 0.1) · 10 1 m )1 Æs )1 (Fig. 2C, Table 1). The first-order rate constant for HPX– heme(II)–NO + conversion to HPX–heme(II)* (i.e. h) must exceed by at least one order of magnitude the value of k (6.5 · 10 )3 s )1 ) obtained at the highest NO concentration investigated (5.0 · 10 )4 m), i.e. h >7· 10 )2 s )1 (Scheme 1, Fig. 2C, Table 1), other- wise a hyperbolic plot of k versus [NO] would be observed [31,34,35]. Values of k on for reductive nitrosylation of HPX– heme(III), horse cytochrome c(III) [31,36] and human Ngb(III) [32] are lower than those reported for G. max 2 Lb(III) [30], sperm whale Mb(III) [31,36], S. inaequi- valvis HbI(III) [33] and human Hb(III) [31], possibly reflecting heme–Fe(III) atom hexa-coordination [16,37, 38] (Table 1). Values of k off for NO dissociation from the heme(III)–NO proteins considered range between <1 · 10 )4 and 1.4 · 10 1 s )1 (Table 1), reflecting the different stability of the heme–Fe(III)–NO complexes [30–33,36]. Values of h for NO + dissociation from HPX–heme(II)–NO + , human Ngb(II)–NO + [32] and S. inaequivalvis HbI(III) [33] are larger than those reported for reductive nitrosylation of G. max Lb(III) [30], sperm whale Mb(III) [31,36], horse cytochrome c [31,36] and human Hb(III) [31] (Table 1). Values of k for reductive nitrosylation of HPX– heme(III) (Fig. 2C), human Ngb(III) [32], G. max Lb(III) [30] and S. inaequivalvis HbI [33] depend line- arly on NO concentration over the whole range explored (i.e. between 5.0 · 10 )5 and 1.2 · 10 )3 m). In contrast, values of k for reductive nitrosylation of sperm whale Mb(III) [31], horse cytochrome c [31] and human Hb(III) [31] do not increase linearly with the NO concentration but tend to level off at [NO] > 2 · 10 )5 m. The transient heme(III)–NO spe- cies was observed during reductive nitrosylation of G. max Lb(III) [30], sperm whale Mb(III) [31], horse cytochrome c(III) [31] and human Hb(III) [31] Fig. 2. Kinetics of NO-mediated reductive nitrosylation of HPX– heme(III), at pH 7.0 and 10.0 °C. (A) Steady-state and kinetic differ- ence absorption spectra (line and circles, respectively) in the Soret region of HPX–heme(III) minus HPX–heme(II)–NO. (B) Time course of reductive nitrosylation of HPX–heme(III), k ¼ 420 nm. The NO concentration was 1.0 · 10 )4 M (trace a), 2.0 · 10 )4 M (trace b), and 5.0 · 10 )4 M (trace c). The time course analysis according to Eqn (1) allowed us to determine the following values of k: 1.2 · 10 )3 s )1 (trace a), 2.8 · 10 )3 s )1 (trace b), and 6.7 · 10 )3 s )1 (trace c). (C) Dependence of the pseudo-first-order rate constant for reductive nitrosylation of HPX–heme(III) (i.e. k) on the NO concen- tration. The continuous line was generated from Eqn (2) with k on ¼ (1.3 ± 0.1) · 10 1 M )1 Æs )1 . The HPX–heme(III) concentration was 2.6 · 10 )6 M. P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 553 only. Furthermore, the intermediate species HPX– heme(III)*, HPX–heme(III)–NO, HPX–heme(II)– NO + , HPX–heme(II)* and HPX–heme(II) (Scheme 1) were not detected. This suggests the following consid- erations. (a) Formation of HPX–heme(III)* does not appear to be rate limiting, as observed for reductive nitrosylation of Ngb(III) [32] and horse cytochrome c [31]. (b) HPX–heme(III)–NO hydrolyzes very rapidly under neutral conditions, similarly to human Ngb(III)– NO [32] and S. inaequivalvis HbI [33] and human Hb(III)–NO [31]. By contrast, G. max Lb(III)–NO [30], sperm whale Mb(III)–NO [31] and horse cyto- chrome c(III)–NO [31] are rather stable under neutral conditions and are hydrolyzed at a significant extent under alkaline conditions. (c) HPX–heme(II) nitrosyla- tion [27] is faster than the NO-induced reduction of the heme–Fe(III) atom, as observed for G. max Lb(II) [30,39], sperm whale Mb(II) [31,40], horse cyto- chrome c(II) [31,36], mouse Ngb(II) [32,41] (highly homologous to human Ngb [38,42,43]), S. inaequivalvis HbI(II) [33,44] and human Hb(II) [31,39,40,45]. How- ever, nitrosylation of hexa-coordinate HPX–heme(II), horse cytochrome c(II) [36] and mouse Ngb(II) [41] (highly homologous to human Ngb [38,42,43]) is slower than NO binding to penta-coordinate G. max Lb(II) [39], sperm whale Mb(II) [40], S. inaequivalvis HbI(II) [44] and human Hb(II) [39,40,45,46] (Table 1). As a whole, NO binding to HPX–heme(III), human Ngb(III) [32] and S. inaequivalvis HbI(II) [33] appears to be rate limiting (i.e. k < h; Scheme 1), whereas the conversion of heme(II)–NO + to heme(II) is rate limit- ing for reductive nitrosylation of G. max Lb(III) [30], sperm whale Mb(III) [31], horse cytochrome c [31] and human Hb(III) [31] (i.e. k > h, Scheme 1). Effect of CO 2 on peroxynitrite-mediated oxidation of HPX–heme(II)–NO In the absence and presence of CO 2 , mixing of the HPX–heme(II)–NO and peroxynitrite solutions causes a shift in the optical absorption maximum of the Soret band from 419 nm, i.e. HPX–heme(II)–NO, to 420 nm, i.e. HPX–heme(III)–NO, and a corresponding change in the extinction coefficient from e 419 ¼ 1.45 · 10 5 to e 420 ¼ 1.59 · 10 5 m )1 Æcm )1 (Fig. 3A). The HPX– heme(III)–NO solution undergoes a shift in the optical absorption maximum of the Soret band from 420 nm, i.e. HPX–heme(III)–NO, to 414 nm, i.e. HPX– heme(III), and a corresponding change in the extinction coefficient from e 420 ¼ 1.59 · 10 5 m )1 Æcm )1 to e 414 ¼ 1.16 · 10 5 m )1 Æcm )1 (Fig. 3A). The optical absorption spectrum of HPX–heme(III) obtained by mixing the HPX–heme(II)–NO and peroxynitrite solutions (e 414 ¼ 1.16 · 10 5 m )1 Æcm )1 ) (Fig. 3A) corresponds to that reported in the literature [24,26,27]. Values for the opti- cal absorption maximum and extinction coefficient of HPX–heme(II)–NO, HPX–heme(III)–NO and HPX– heme(III) are unaffected by CO 2 . Analogous to G. max Lb [30], horse Mb [47], human Ngb [32] and human Hb [28,47], our data indicate that the reaction of HPX– heme(II)–NO with an excess peroxynitrite leads to oxi- dation of the heme–Fe(II) atom and generation of the HPX–heme(III) species. Over the whole peroxynitrite concentration range explored (1.5 · 10 )5 )2.5 · 10 )4 m), the time course for the peroxynitrite-mediated oxidation of HPX– heme(II)–NO (2.6 · 10 )6 m) corresponds to a bipha- sic process, in the absence and presence of CO 2 (1.2 · 10 )3 m) (Fig. 3B). Values of the pseudo-first-order Table 1. Kinetic parameters for reductive nitrosylation of ferric heme-proteins (for details, see Scheme 1). ND, not determined. 5 Heme-protein k on (M )1 Æs )1 ) k off (s )1 ) h (s )1 ) l on (M )1 Æs )1 ) l off (s )1 ) Rabbit HPX–heme(III) 1.3 · 10 1a <1· 10 )4a >7· 10 )2a 6.3 · 10 3b 9.1 · 10 )4b Human Ngb(III) Fast reacting form c 2.1 · 10 1 2.5 · 10 )3 >2· 10 )1 ND ND Slow reacting form c 2.9 2.5 · 10 )3 >5· 10 )2 ND ND Mouse Ngb(III) d ND ND ND 2 · 10 5 2.0 · 10 )4 S. inaequivalvis HbI 3.2 · 10 1e <1· 10 )3e >6· 10 )1e 1.6 · 10 7f ND Horse cytochrome c 7.2 · 10 2g 4.4 · 10 )2g < 4.0 · 10 )3h 8.3 g 2.9 · 10 )5g Human Hb(III) a subunits 1.7 · 10 3i 6.5 · 10 )1i 1.3 · 10 )3j 2.6 · 10 7k 4.6 · 10 )5l b subunits 6.4 · 10 3i 1.5 i 1.3 · 10 )3j 2.6 · 10 7k 2.2 · 10 )5l G. max Lb(III) 1.4 · 10 5m 3.0 m 4.8 · 10 )4m 1.2 · 10 8l 2.4 · 10 )5l Sperm whale Mb(III) 1.9 · 10 5g 1.4 · 10 1g < 8.8 · 10 )4h 1.7 · 10 7n 1.2 · 10 )4n a pH 7.0 and 10.0 °C; this study. b pH 7.0 and 10.0 °C [27]. c pH 7.0 and room temperature [32]. d pH 7.0 and 25.0 °C [41]. e pH 7.5 and 20.0 °C [33]. f pH 7.0 and 20.0 °C [44]. g pH 6.5 and 20.0 °C [36]. h pH < 8.3 and 20.0 °C [31]. i pH 7.0 and 20.0 °C [74]. j pH 7.0 and 20.0 °C [31]. k pH 7.0 and 20.0 °C [45]. l pH 7.0 and 20.0 °C [39]. m pH 7.0 and 20.0 °C [30]. n pH 7.0 and 20.0 °C [40]. Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al. 554 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS rate constant for the formation of and the first-order decay of the transient HPX–heme(III)–NO species (i.e. b and d, respectively) are wavelength independent. As shown in Fig. 3C, the first step of kinetics for per- oxynitrite-mediated oxidation of HPX–heme(II)–NO (b on in Scheme 2) is a bimolecular process as observed under pseudo-first-order conditions. The plot of b versus [peroxynitrite] is linear (Eqn 6) with a y-intercept at 0, indicating that the reverse reaction rate is negli- gible; the slope of the plot of b versus [peroxynitrite] corresponds to b on ¼ (8.6 ± 0.8) · 10 4 m )1 Æs )1 and (1.2 ± 0.2) · 10 6 m )1 Æs )1 in the absence and presence of CO 2 , respectively (Table 2). By contrast, the second step (d in Scheme 2) follows a peroxynitrite-independ- ent monomolecular behavior (Fig. 3D), the average value of d is (4.3 ± 0.4) · 10 )1 s )1 (Table 2). Fig. 3. Kinetics of peroxynitrite-mediated oxidation of HPX–heme(II)–NO in the absence and presence of CO 2 , at pH 7.0 and 10.0 °C. (A) Steady-state and kinetic difference absorption spectra (line and symbols, respectively) in the Soret region of HPX–heme(II)–NO minus HPX– heme(III) (line and triangles) and fully populated HPX–heme(III)–NO minus HPX–heme(III) (squares). Steady-state and kinetic difference absorption spectra were independent of CO 2 . (B) Time course of the peroxynitrite-induced conversion of HPX–heme(II)–NO to HPX–heme(III) by way transient HPX–heme(III)–NO formation, in the absence (trace a) and presence (trace b) of CO 2 , k ¼ 425 nm. The time course analysis according to Eqns (3–5) allowed us to determine the following parameters: b ¼ 4.5 s )1 and d ¼ 4.3 · 10 )1 s )1 (trace a), and b ¼ 5.9 · 10 1 s )1 and d ¼ 4.5 · 10 )1 s )1 (trace b). The peroxynitrite concentration was 5.0 · 10 )5 M. (C) Dependence of the pseudo-first order rate constant for the peroxynitrite-induced conversion of HPX–heme(II)–NO to HPX–heme(III)–NO (i.e. b) on the peroxynitrite concen- tration, in the absence (diamonds) and presence (squares) of CO 2 . The continuous line was calculated according to Eqn (6) with b on ¼ (8.6 ± 0.9) · 10 4 M )1 Æs )1 in the absence of CO 2 (diamonds), and b on ¼ (1.2 ± 0.2) · 10 6 M )1 Æs )1 in the presence of CO 2 (squares). (D) Dependence of the first order rate constant for NO dissociation from HPX–heme(III)–NO (i.e. d) on the peroxynitrite concentration, in the absence (diamonds) and presence (squares) of CO 2 . The average value of d is (4.3 ± 0.4) · 10 )1 s )1 . The HPX–heme(II)–NO concentration was 2.6 · 10 )6 M. The CO 2 concentration was 1.2 · 10 )3 M. Table 2. Kinetic parameters for peroxynitrite-mediated oxidation of ferrous nitrosylated heme-proteins (for details, see Scheme 2). Heme–protein [CO 2 ](M) b on (M )1 Æs )1 ) d (s )1 ) Rabbit HPX–heme(II)–NO a – 8.6 · 10 4 4.3 · 10 )1 1.2 · 10 )3 1.2 · 10 6 4.3 · 10 )1 Human Hb(II)–NO b – 6.1 · 10 3  1 1.2 · 10 )3 5.3 · 10 4  1 G. max Lb(II)–NO c – 8.8 · 10 3 2.5 1.0 · 10 )3 1.2 · 10 5 2.5 Horse Mb(II)–NO – d 3.1 · 10 4d  1.2 · 10 1d 1.2 · 10 )3e 1.7 · 10 5e 1.1 · 10 1e Human Ngb(II)–NO f – 1.3 · 10 5 1.2 · 10 )1 a pH 7.0 and 10.0 °C; this study. b pH 7.2 and 20.0 °C [48]. c pH 7.3 and 20.0 °C [30]. d pH 7.5 and 20.0 °C [47]. e pH 7.0 and 20.0 °C [47]. f pH 7.2 and 20.0 °C [32]. P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 555 As observed for HPX–heme(II)–NO (Fig. 3C), val- ues of the pseudo-first-order rate constant b (Scheme 2) for peroxynitrite-mediated oxidation of G. max Lb(II)– NO [30], horse Mb [47], human Ngb(II)–NO [32] and human Hb(II)–NO [28,47] depend linearly over the whole peroxynitrite concentration range explored, in the absence and presence of CO 2 . Values of b on for per- oxynitrite-mediated oxidation of HPX–heme(II)–NO and human Ngb(II)–NO [32] exceed those reported for G. max Lb(II)–NO [30], horse Mb [47] and human Hb(II)–NO [28,47] (Table 2). CO 2 facilitates peroxy- nitrite-mediated oxidation of HPX–heme(II)–NO (Fig. 3C), G. max Lb(II)–NO [30], horse Mb [47] and human Hb(II)–NO [28,47] increasing values of b on (Table 2). A similar observation was made for the reac- tion of G. max Lb(II)–O 2 [48], sperm whale Mb(II)–O 2 [49] and human Hb(II)–O 2 [50] with peroxynitrite in the absence and presence of CO 2 . In the presence of CO 2 , peroxynitrite changes from a two- to a one-electron oxidant. In fact, CO 2 reacts rapidly with peroxynitrite leading to ONOOC(O)O – (second-order rate constant is  3 · 10 4 m )1 Æs )1 ), which in turn decays very rapidly to CO 3 – and NO 2 (first-order rate constant is  5 · 10 5 s )1 ). CO 3 – and NO 2 are stronger oxidant and nitrating agents than peroxynitrite; NO 2 nitrates with preference Tyr and Trp residues [51,52]. Although CO 2 facilitates the nitration of heme–protein aromatic residues by peroxy- nitrite [53], optical absorbance spectroscopy for HPX between 230 and 500 nm indicates that no appreciable aromatic nitration takes place (data not shown). This suggests that the CO 2 -induced increase in k on for per- oxynitrite-mediated oxidation of HPX ( 3 Table 2), reflects oxidation of the heme–Fe atom by CO 3 – rather than conformational transition(s) depending on the nitration of Tyr and Trp residues by NO 2 . As observed for HPX–heme(III)–NO (Fig. 3D), val- ues of the first-order rate constant d (Scheme 2) for NO dissociation from G. max Lb(III)–NO [30], horse Mb [47], human Ngb(III)–NO [32] and human Hb(III)–NO [28,47] are unaffected by CO 2 , ranging between 1 · 10 )1 and 1.2 · 10 1 s )1 (Table 2). The dis- sociation of heme(III)–NO adducts is facilitated by the consumption of NO via its reaction with peroxynitrite excess and⁄ or with the reactive species generated during peroxynitrite decomposition (e.g. NO 2 ). Under anaerobic conditions, the reaction of NO with peroxy- nitrite leads to N 2 O 3 and H 2 O, at pH < 7. At pH > 7, NO reacts with NO 2 leading to N 2 O 3 ,in turn, N 2 O 3 reacts with peroxynitrite leading to NO 2 – and NO 2 [28]. The transient species heme(III)–NO was observed during peroxynitrite-mediated oxidation of HPX– heme(II)–NO (Fig. 3B). This intermediate is also seen in G. max Lb(II)–NO [30], horse Mb [47], human Ngb(II)–NO [32] and human Hb(II)–NO [28,47]. By contrast, the transient penta-coordinate derivative of HPX–heme(III), i.e. HPX–heme(III)*, (Scheme 2) was not observed. This transient was also never observed in ligand-binding reaction(s) to human Ngb(III) [32]. This suggests the following: (a) NO dissociation from heme(III)–NO represents the rate-limiting step for per- oxynitrite-mediated oxidation of the heme(II)–NO proteins considered [28,30,32,47]; and (b) the HPX– heme(III)* fi HPX–heme(III) reaction (Scheme 2) does not appear to be rate limiting, as reported for peroxy- nitrite-mediated oxidation of human Ngb(II)–NO [32]. Under the experimental conditions, kinetic and spectroscopic properties of HPX–heme(II)–NO were unaffected by decomposed peroxynitrite. Conclusions Our data represent the first evidence for reductive nitrosylation of HPX–heme(III) and for peroxyni- trite-mediated oxidation of HPX–heme(II)–NO. As a general remark, the few data available from litera- ture concerning the reductive nitrosylation of ferric heme–proteins and the peroxynitrite-mediated oxida- tion of ferrous nitrosylated heme-proteins (in the absence and presence of CO 2 ) are reported for the purpose of a comparison with those of HPX–heme (Tables 1,2). Although HPX–heme(III) reduction and nitrosyla- tion occur physiologically and modulate HPX–heme complex (de-)stabilization [27,54–56], reductive nitrosy- lation of HPX–heme(III) appears too slow to occur in vivo despite NO concentrations > 10 )5 m under pathological conditions [57–61]. The same considera- tions hold also for reductive nitrosylation of human Ngb(III), sperm whale Mb(III), G. max Lb(III) and human Hb(III) [30–32]. However, as shown in Table 1, heme reduction kinetics are facilitated in bis-histidyl hexa-coordinate heme–proteins (i.e. HPX–heme and human Ngb) [62]. The reactivity of peroxynitrite with HPX–heme(II)– NO (Table 2) is high enough to protect against peroxynitrite-mediated damage, and to impair the for- mation of strong oxidants and nitrating agents (e.g. ONOOC(O)O – ,CO 3 – and NO 2 ), in the absence and presence of CO 2 [63]. In fact, values of the second- order rate constant b on for peroxynitrite-mediated oxi- dation of HPX–heme(II)–NO are larger than those for the reaction of peroxynitrite with (macro)molecular targets (e.g. cysteine residues;  4 · 10 3 m )1 Æs )1 ) and with CO 2 ( 3 · 10 4 m )1 Æs )1 ) [51,52] (Table 2). Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al. 556 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS The high value of the reaction rate of HPX– heme(II)–NO with peroxynitrite (i.e. b on ; Table 2) may reflect structural features reminiscent those of heme– proteins endowed with (pseudo-)enzymatic activities [64]. Indeed, the imidazole ring of the proximal His266 residue of HPX–heme is eclipsed with respect to the heme N–Fe–N coordination bonds [16], as observed for the proximal His residue in horseradish peroxidase [65], and in Alcaligenes eutrophus and Escherichia coli flavohemoglobins [66,67], although His266 is rotated by 90° with respect to heme propionates in HPX– heme. Furthermore, the negatively charged residue Glu226 occurs in the neighborhood of the proximal His266 residue of HPX–heme [16]. Similarly, horserad- ish peroxidase [65], and A. eutrophus and E. coli flavo- hemoglobins [66,67] all display Asp or Glu residues in the neighborhood of the proximal His. In horseradish peroxidase [65], and A. eutrophus and E. coli flavoh- emoglobins [66,67], the proximal Asp⁄ Glu residue is hydrogen-bonded to the proximal His ND1 atom, partly setting the orientation of the proximal imidazole [66,67]. In HPX–heme the orientation of the proximal imidazole is defined by a hydrogen bond connecting the carbonyl O atom of Ser267 to the His266 ND1 atom, whereas the carboxylate of Glu226 falls at  0.45 nm from the proximal imidazole ring [16]. Although the local stereochemistry makes unlikely the achievement of a hydrogen bond between His266 and Glu226 in HPX–heme(III), Glu226 may modulate heme binding to HPX and HPX–heme ligand binding capacity by affecting the protonation state of the prox- imal His266 residue via electrostatic control of the resi- due pK a [26]. In conclusion, our results describe a curious situ- ation in which heme binding to a nonheme-protein (i.e. HPX) confers (although transiently) functional properties (e.g. peroxynitrite scavenging) and may be predictive of (pseudo-enzymatic) function(s) of heme- carriers (e.g. heme–albumin as well as high and low density heme–lipoproteins). The system studied here may suggest that the effects arising from heme binding to HPX might have some role in the regulation of bio- logical functions. Because these effects involve tran- sient reactive functions, dependent on the interaction with specific molecules (i.e. the heme), they have been called ‘chronosteric’ effects [68]. Experimental procedures Chemicals Hemin [iron(III)–protoporphyrin(IX)] was obtained from Sigma Chemical Co. (St Louis, MO). Gaseous NO was purchased from Aldrich Chemical Co. (Milwaukee, WI). NO was purified by flowing through a NaOH column in order to remove acidic nitrogen oxides. The NO stock solution was prepared by keeping in a closed vessel the 1.0 · 10 )1 m phosphate buffer solution (pH 7.0) under purified NO, at 760.0 mmHg and 20.0 °C, anaerobically. The solubility of NO in the aqueous buffered solution is 2.05 · 10 )3 m, at 760.0 mmHg and 20.0 °C. The NO stock solution was diluted with degassed 1.0 · 10 )1 m phosphate buffer to reach the desired concentration [26,27,69]. The 1.0 · 10 )1 m phosphate buffer solution was kept under helium. Peroxynitrite was prepared from KO 2 and NO and from HNO 2 and H 2 O 2 , under anaer- obic conditions. Peroxynitrite was purified by freeze frac- tionation. The peroxynitrite concentration was determined by measuring the optical absorbance at 302 nm (e 302 ¼ 1.67 · 10 3 m )1 Æcm )1 ). The peroxynitrite stock solution was diluted with degassed 1.0 · 10 )2 m NaOH to reach the desired concentration. The 1.0 · 10 )2 m NaOH solution was kept under helium. Decomposed peroxynitrite was prepared by acidification of the peroxynitrite solution with HCl, then the solution was neutralized with 1.0 · 10 )1 m NaOH [53,70,71]. For the experiments car- ried out in the absence of CO 2 , the 1.0 · 10 )1 m phos- phate buffer and the 1.0 · 10 )2 m NaOH solutions were prepared fresh daily, thoroughly degassed, and kept under helium. Experiments in the presence of CO 2 (1.2 · 10 )3 m) were carried out by adding to the protein solution the required amount from a freshly prepared 5.0 · 10 )1 m sodium bicarbonate solution. The CO 2 con- centration is always expressed as the true concentration in equilibrium with HCO 3 – . The value of the constant of the hydration–dehydration equilibrium 4 CO 2 þ H 2 O $ H þ þ HCO  3 at pH 7.0 and 10.0 ° C is 6.38 · 10 )7 m. The bicarbonate concentration present during the reac- tions was 9.5 · 10 )3 m [28,30,32,47,53]. All the other chemicals were obtained from Merck AG (Darmstadt, Germany). All products were of analytical or reagent grade and used without purification unless stated. HPX Rabbit serum HPX was prepared as reported previously [25,72]. Protein contaminants were < 3% of the HPX sam- ple as judged by gel electrophoresis and N-terminal amino acid sequence determination. The HPX–heme(III) solution (2.0 · 10 )6 )1.5 · 10 )4 m) was prepared by adding 1.2- molar excess of the HPX solution to the heme(III) solution (1.0 · 10 )1 m phosphate buffer, pH 7.0), at 10.0 °C [26,27]. Under these conditions, no free heme is present in solution [17,25,26,72]. In fact, the value of the dissociation equilib- rium constant for heme binding to HPX is < 10 )9 m [25,72]. The HPX–heme(II)–NO solution (2.6 · 10 )6 m) was prepared by reductive nitrosylation of HPX– heme(III) under anaerobic conditions, i.e. by adding to P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 557 HPX–heme(III) either gaseous NO or the buffered NO solution (see below). Reductive nitrosylation of HPX–heme(III) The value of the second-order rate constant for reductive nitrosylation of HPX–heme(III) (k on ) was determined by mixing the HPX–heme(III) solution (final concentration 2.6 · 10 )6 m) with the NO solution (final concentration, 1.0 · 10 )4 )5.0 · 10 )4 m) under anaerobic conditions, at pH 7.0 (1.0 · 10 )1 m phosphate buffer) and 10.0 °C [30–33]. No gaseous phase was present. Kinetics was monitored between 360 and 460 nm. Under all experimental condi- tions, final pH measured after mixing ranged always between 6.9 and 7.1. For an homogeneous comparison with the available functional data [26,27], kinetics was obtained at 10.0 °C. Time courses were fitted to the minimum reaction mech- anism represented by Scheme 1 [30–33], where HPX–heme* indicates the transient penta-coordinate HPX–heme species. Values of the NO-dependent pseudo-first-order rate con- stant for reductive nitrosylation of HPX–heme(III) (i.e. k) have been determined from data analysis, according to Eqn (1) [30–33]: ½HPX–hemeðIIIÞ t ¼½HPX–hemeðIIIÞ i  e kt ð1Þ The value of the second-order rate constant for reductive nitrosylation of HPX–heme(III) (i.e. k on ) was obtained from the linear dependence of k on the NO concentration (i.e. [NO]) according to Eqn (2) [30–33]: k ¼ k on ½NOð2Þ The difference optical absorption spectrum in the Soret region of HPX–heme(III) minus HPX–heme(II)–NO was obtained under steady-state conditions by subtracting the absorbance change in HPX–heme(II)–NO from that of HPX–heme(III). The kinetic difference optical absorption spectrum in the Soret region of HPX–heme(III) minus HPX–heme(II)–NO was reconstructed from the difference optical absorption spectrum of HPX–heme(II)–NO minus HPX–heme(II)– NO (De ¼ 0.0 m )1 Æcm )1 ) obtained under steady-state conditions plus the total absorbance changes of the HPX– heme(III) reductive nitrosylation process. Peroxynitrite-mediated oxidation of HPX–heme(II)–NO Values of the second-order rate constant for peroxynitrite- mediated conversion of HPX–heme(II)–NO to HPX– heme(III)–NO (i.e. b on ) and of the first-order rate constant for NO dissociation from the HPX–heme(III)–NO complex (i.e. for the formation of HPX–heme(III); d) were determined by rapid mixing the HPX–heme(II)–NO solution (final concentration 2.6 · 10 )6 m) with the peroxy- nitrite solution (final concentration, 1.5 · 10 )5 m to 2.5 · 10 )4 m) under anaerobic conditions, at pH 7.0 (1.0 · 10 )1 m phosphate buffer) and 10.0 °C, in the absence and presence of CO 2 (1.2 · 10 )3 m) [28,30,32,47]. The dead time of the SX18MV-R rapid-mixing stopped-flow appar- atus (Applied Photophysiscs Ltd, Leatherhead, UK) was 1.6 ms. No gaseous phase was present. Kinetics was monit- ored between 360 and 460 nm. Under all the experimental conditions, the final pH value measured after mixing ranged always between 6.9 and 7.1. Kinetics was obtained at 10.0 °C in order to avoid loss of the initial part of the HPX–heme(II)–NO fi HPX–heme(III)–NO reaction especi- ally in the presence of CO 2 , and for an homogeneous com- parison with the available functional data [26,27]. The time courses were fitted to two consecutive mono- exponential processes according to the minimum reaction mechanism depicted in Scheme 2 [28,30,33,47]. Values of the (pseudo-)first-order rate constants for the formation of the HPX–heme(III)–NO complex (i.e. b) and for NO dissociation from the transient HPX–heme(III)–NO complex (i.e. for the formation of HPX–heme(III); d) have been determined from data analysis, according to Eqns (3– 5) [73]: ½HPX–heme(II)–NO t ¼½HPX–heme(II)–NO i  e bt ð3Þ Scheme 1. Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al. 558 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS ½HPX–heme(III)–NO t ¼½HPX-heme(II)–NO i ðb ððe bt=ðdbÞ Þ þðe dt=ðbdÞ ÞÞÞ ð4Þ ½HPX–heme(III) t ¼½HPX–heme(II)–NO i ð½HPX–heme(II)–NO t þ½HPX–heme(III)–NO t Þð5Þ The value of b on was obtained from the linear dependence of b on the peroxynitrite concentration (i.e. [peroxynitrite]) according to Eqn (6) [28,30,32,47]: b ¼ b on ½peroxynitriteð6Þ The difference optical absorption spectrum in the Soret region of HPX–heme(II)–NO minus HPX–heme(III) was obtained under steady-state conditions by subtracting the absorbance change of HPX–heme(III) from that of HPX– heme(II)–NO. The kinetic difference optical absorption spectra in the Soret region of HPX–heme(II)–NO minus HPX–heme(III) and of HPX–heme(III)–NO minus HPX–heme(III) were reconstructed from the difference optical absorption spec- trum of HPX–heme(III) minus HPX–heme(III) (De ¼ 0.0 m )1 Æcm )1 ) obtained under steady-state conditions plus the absorbance changes of the overall process HPX– heme(II)–NO + HOONO fi HPX–heme(III) + NO and of the partial reaction HPX–heme(III)–NO fi HPX– heme(III) + NO (Scheme 2). The absolute optical absorption spectrum of HPX– heme(III)–NO in the Soret region was reconstructed from the optical absorption spectrum of HPX–heme(III) obtained under steady-state conditions plus the absorbance changes of the partial reaction HPX–heme(III)– NO fi HPX–heme(III) + NO (Scheme 2). Nitration of HPX Tyr and Trp residues was investigated by optical absorbance spectroscopy [53]. Briefly, the heme was removed from HPX–heme(III), obtained by reacting HPX–heme(II)–NO (1.5 · 10 )4 m) with peroxynitrite (1.0 · 10 )3 m), by mixing the HPX–heme(III) solution (1.5 · 10 )4 m) with the cold ()20.0 °C) acid acetone solu- tion (2–3 mL of 2.0 m HCl per liter of acetone). One vol- ume of HPX–heme(III) in water was added slowly with vigorous stirring to about 30 vol of the cold acid acetone solution. The precipitated HPX was collected by centrifuga- tion and then dissolved in a minimum amount of water. The dissolved HPX was dialyzed in the cold against a dilute bicarbonate solution (1.0 · 10 )3 m) and subsequently against 1.0 · 10 )2 m phosphate buffer at pH 7.0. The pro- tein precipitate was removed by centrifugation [69]. Then, the optical absorbance spectrum of HPX ( 1 · 10 )4 m) was recorded between 230 and 500 nm. Data analysis All experiments were carried out at least in quadruplicate. The results are given as mean values plus or minus the standard deviation. 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