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Inhibition of Azotobacter vinelandii rhodanese by NO-donors
BBRC Biochemical and Biophysical Research Communications 306 (2003) 1002–1007 www.elsevier.com/locate/ybbrc
Inhibition of Azotobacter vinelandii rhodanese by NO-donorsq Andrea Spallarossa,a Fabio Forlani,b Silvia Pagani,b Luca Salvati,c Paolo Visca,c,d Paolo Ascenzi,c,e Martino Bolognesi,f,g and Domenico Bordog,* a Dipartimento di Scienze Farmaceutiche, Universit a di Genova, Via Benedetto XV 3, I-16132 Genoa, Italy Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, I-20133 Milan, Italy c Dipartimento di Biologia, Universit a ‘Roma Tre,’ Viale G. Marconi 446, I-00146 Rome, Italy Istituto Nazionale per le Malattie Infettive IRCCS ‘Lazzaro Spallanzani,’ Unit a di Microbiologia Molecolare, Via Portuense 292, I-00149 Rome, Italy e Laboratorio Interdipartimentale di Microscopia Elettronica, Universit a ‘Roma Tre,’ Via della Vasca Navale 79, I-00146 Rome, Italy f Dipartimento di Fisica, Istituto Nazionale di Fisica della Materia, Centro di Eccellenza per la Biologia e la Medicina, Universit a di Genova, Via Dodecaneso 33, I-16146 Genoa, Italy g Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, I-16132 Genoa, Italy b
d
Received 15 May 2003
Abstract Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), ()E -4-ethyl-2-[E -hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetyl-penicillamine (SNAP) on the catalytic activity of Azotobacter vinelandii rhodanese (RhdA) has been investigated. GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion. The absorption spectrum of the NOR-3-treated RhdA displays a maximum at 335 nm, indicating NO-mediated S-nitrosylation. RhdA inhibition by NO-donors correlates with S-nitrosothiol formation. The reducing agent dithiothreitol prevents RhdA inhibition by NO-donors, fully restores the catalytic activity, and reverts the NOR-3-induced RhdA absorption spectrum to that of the active enzyme. These results indicate that RhdA inhibition occurs via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Rhodanese; Sulfurtransferase; Phosphatase; NO-donors; Enzyme inhibition
Sulfurtransferases (EC 2.8.1.x) are enzymes involved in the formation, interconversion, and transport of compounds containing sulfane sulfur atoms. The best characterized sulfurtransferase is bovine rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), which in vitro catalyses transfer of a sulfane sulfur atom from thiosulfate to cyanide, with formation of sulfite and thiocyanate [1–3]. This reaction involves the tranq
Abbreviations: RhdA, Azotobacter vinelandii rhodanese; PTP, protein tyrosine phosphatase; human PTP1B, human protein tyrosine phosphatase 1B; NO, nitric oxide; GSNO, S-nitroso-glutathione; GSH, glutathione; NOR-3, ()-E -4-ethyl-2-[E -hydroxyimino]-5-nitro3-hexenamide; NOR-3*, NO-deprived NOR-3; SNAP, S-nitroso-Nacetyl-penicillamine; NAP, N-acetyl-penicillamine; DTT, dithiothreitol. * Corresponding author. Fax: +39-010-5737306. E-mail address: [email protected] (D. Bordo).
sient formation of a persulfide-containing intermediate (Rhd–S), hosting an extra sulfur atom as persulfide modification of the catalytic Cys residue: 2 S2 O2 3 þ Rhd ! SO3 þ Rhd–S Rhd–S þ CN ! Rhd þ SCN
ðScheme 1Þ
Given the abundance of rhodanese in bovine liver mitochondria, this enzyme is deemed to be primarily involved in the elimination of toxic cyanogenic compounds arising from vegetal food digestion in mammals [4,5]. Recently, the extensive genome sequencing has shown that genes coding for proteins endowed with significant sequence similarity to mammalian rhodaneses are widely distributed in the three major evolutionary phyla, and that often several rhodanese-like proteins are encoded by the same genome. The latter observation suggests functional and specificity diversification within
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01067-2
A. Spallarossa et al. / Biochemical and Biophysical Research Communications 306 (2003) 1002–1007
the rhodanese homology family (see, e.g., the COG database, http://www.ncbi.nlm.nih.gov/COG). This fact has brought about new hypotheses on the biological function of rhodanese-like enzymes, such as sulfur mobilization in sulfur–iron cluster biosynthesis [6], or involvement in sulfur metabolism [7]. The ability of rhodanese to bind selenium in the presence of glutathione (GSH) suggests that rhodanese-like enzymes may also play a role in selenophosphate synthesis [8]. In the nitrogen-fixing bacterium Azotobacter vinelandii, the detection of rhodanese activity has led to the identification of the associated rhdA gene and to the biochemical characterization of its protein product [9,10]. The 29.6 kDa protein RhdA displays moderate but significant sequence similarity to bovine rhodanese (22% identity) [9,10]. Bovine rhodanese and RhdA also share the same overall three-dimensional structure, consisting of two similarly folded domains, hosting the catalytic Cys in the C-terminal domain [11–13]. In native RhdA crystals, the catalytic Cys230 is found persulfurated; the extra sulfur atom could be removed by a number of nucleophilic acceptors such as cyanide and phosphate [14]. Given the high reactivity of nitric oxide (NO) versus thiols [15–19], the effect of NO-donors S-nitroso-glutathione (GSNO), ()-E -4-ethyl-2-[E -hydroxyimino]-5nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetylpenicillamine (SNAP) on RhdA activity has been investigated. Herein, we report that NO-donors inhibit RhdA via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue.
Materials and methods Chemicals. The NO-donors NOR-3 and SNAP as well as GSH, Nacetyl-penicillamine (NAP), and dithiothreitol (DTT) were purchased from Sigma Chemical (St. Louis, MO, USA). The NO-donor GSNO was prepared by mixing equimolar amounts of an aqueous solution of NaNO2 and a freshly prepared GSH solution in 2.5 101 M HCl and 1.0 104 M EDTA (pH 1.5). The resulting mixture was incubated at 25 °C for 5 min and then neutralized with NaOH. The GSNO solution was stored at )20 °C [20]. The NO-deprived NOR-3 (NOR-3*) was obtained by treating NOR-3 at alkaline pH and 25 °C for 3 days [21]. All other products were obtained from Merck AG (Darmstadt, Germany). All chemicals were of analytical grade and were used without further purification. RhdA characterization and enzyme assay. Recombinant RhdA was prepared in the active persulfurated form, as previously reported [13]. The RhdA sulfurtransferase activity (i.e., the enzyme-catalyzed formation of thiocyanate; see Scheme 1) was assayed through a discontinuous method that quantitates the product thiocyanate, based on the absorbance of Fe(SCN)3 at 460 nm [4]. The optimized reaction mixture (0.65 ml) contained 6.0 102 M KCN, 5.0 102 M KH2 PO4 , and 1.0 106 –5.0 106 M RhdA in 5.0 102 M Tris–HCl, pH 8.0. The reaction was initiated by the addition of Na2 S2 O3 (6.0 102 M, final concentration) and continued for 2 min at 37 °C. The reaction was stopped by adding 0.1 ml formaldehyde (38%, v/v). Then, the reaction product SCN was detected by adding 0.25 ml of the iron reagent solution (0.25 M Fe(NO3 )3 and 13%, v/v, HNO3 , in water) to yield the
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Fe(SCN)3 complex. The assay was calibrated using a standard curve obtained from known concentrations of Fe(SCN)3 . In each experiment, the baseline was always assessed by pre-incubating RhdA with formaldehyde. One unit of RhdA activity is defined as the amount of enzyme that produces 1.0 lmol thiocyanate min1 in the assay condition. Effect of NO-donors on RhdA activity. The effect of GSNO, GSH, NOR-3, NOR-3*, SNAP, and NAP on the RhdA catalytic activity was determined by incubation of the active enzyme (final concentration, 1.0 106 –5.0 106 M range) with NO-donors and NO-deprived compounds, at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 5 min to 60 min. The final concentration of GSNO, GSH, NOR-3, NOR-3*, SNAP, and NAP ranged between 1.0 104 and 2.0 103 M. The RhdA residual sulfurtransferase activity was then assayed [4]. The [NO]/[RhdA] (i.e., [NOR-3]/[RhdA]) stoichiometry was determined by incubation of the active enzyme (final concentration, 5.0 106 M) with NOR-3 (final concentration, 1.0 106 – 2.0 105 M range), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 180 min. The RhdA residual sulfurtransferase activity was then assayed [4]. The NO concentration was calculated taking into account the half-time for NO release from NOR-3 (54 3 min) and the RhdANOR-3 incubation time (180 min) [19]. Protective effect of DTT on RhdA activity. The protective effect of DTT on the NO-mediated inhibition of RhdA was investigated by the simultaneous incubation of the active enzyme (final concentration, 1.0 106 –5.0 106 M range) with DTT (final concentration, 1.0 103 M) and NOR-3 (final concentration, 1.0 104 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min. The RhdA residual sulfurtransferase activity was then assayed [4]. Recovery of RhdA activity by DTT. The effect of DTT on the NOmediated inhibition of RhdA was investigated by incubating the inhibited enzyme (final concentration, 1.0 106 – 5.0 106 M range) with DTT (final concentration, 1.0 103 M), at pH 8.0 (5.0 102 M Tris– HCl) and 37 °C, for 60 min. Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 1.0 104 M) for 60 min. The RhdA residual sulfurtransferase activity was then assayed [4]. Effect of NOR-3, NOR-3*, and DTT on RhdA spectroscopic properties. The effect of NOR-3 and NOR-3* on RhdA absorbance spectroscopic properties between 300 and 400 nm was investigated by incubation of the active enzyme (final concentration, 1.0 104 M) with the NO-donor and the NO-deprived compound (final concentration, 2.0 104 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min. Reversibility of the absorption spectrum of the inhibited Snitrosylated RhdA was achieved by incubation of the inhibited enzyme (final concentration, 1.0 104 M) with DTT (final concentration, 1.0 103 M), KH2 PO4 (final concentration, 5.0 102 M), and Na2 S2 O3 (final concentration, 6.0 102 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min.
Results and discussion NO-donors GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion (Fig. 1 and Table 1). Such inhibition obeys apparent first-order kinetics (Fig. 1, panel A). The double-reciprocal plots of the relative apparent first-order inhibition constant for RhdA inhibition by NO-donors (ki =k) as a function of the GSNO, NOR-3, and SNAP concentration ([NO-donor]1 ) yield straight lines (Fig. 1, panel B), indicating that enzyme inhibition is a relatively rapid pre-equilibrium followed by a limiting first-order process, as previously reported for NO inhibition of protein tyrosine phosphatases (PTP’s) and
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A. Spallarossa et al. / Biochemical and Biophysical Research Communications 306 (2003) 1002–1007 Table 1 Effect of GSNO, GSH, NOR-3, NOR-3*, SNAP, NAP, and DTT on the catalytic activity of RhdA, at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C
Fig. 1. RhdA inhibition by GSNO, NOR-3, and SNAP. (A) Time course of RhdA inhibition by SNAP. The enzyme activity was assayed after treatment with 2.0 104 M (circles), 5.0 104 M (squares), and 1.0 103 M (triangles) SNAP. The effect of 1.0 103 M NAP (diamonds) on the RhdA activity is shown for comparison. The RhdA concentration was 5.0 106 M. The continuous lines were calculated according to Eq. (1) [22,23]: ½RhdA ¼ ½RhdA ð1 ekt Þ;
ð1Þ
where [RhdA*] is the inhibited enzyme concentration and [RhdA] is the total active enzyme concentration at t ¼ 0 min, with the following values of k: 0.050 min1 (circles), 0.10 min1 (squares), and 0.16 min1 (triangles). (B) Dependence of the pseudo first-order rate constant k for RhdA inhibition by GSNO (circles), NOR-3 (squares), and SNAP (triangles) on the NO-donor concentration. The continuous lines were calculated according to Eq. (2) [22,23]: ki =k ¼ Ki ½NO-donor 1 þ 1
ð2Þ
with values of ki , ki =Ki , and Ki given in Table 2. All data were obtained at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C. For further details, see text.
papain [22,23]. Values of the apparent first-order ratelimiting constant (ki ), of the apparent second-order rate constant (ki =Ki ), and of the apparent dissociation equilibrium constant (Ki ) for RhdA inhibition by GSNO, NOR-3, and SNAP are reported in Table 2. As shown in Fig. 2, one equivalent of NO, released from NOR-3, inhibits one equivalent of RhdA. Notably, enzyme activity is essentially unaffected by NO-deprived compounds (i.e., GSH, NAP, and NOR-3*) (Fig. 1, panel A, and Table 1). To test whether RhdA inhibition occurred via NOmediated S-nitrosylation, the effect of DTT on RhdA inhibition by NOR-3 and on the enzyme spectroscopic properties was investigated. As expected [19,22,24–26], the simultaneous incubation of DTT and NOR-3 with RhdA prevents the enzyme inhibition (Table 1). More-
Inhibitor/activator
Enzyme activity (%)
None GSNOa GSHa;b NOR-3a NOR-3*a; b NOR-3 + DTT (time ¼ 0 min)c NOR-3 + DTT (time ¼ 60 min)d SNAPa NAPa;b
100 5 73 93 6 63 94 7 91 7 92 8 42 95 6
a The concentration of NO-donors and NO-deprived compounds (final concentration, 1.0 104 M) exceeded that of RhdA (final concentration, 5.0 106 M). The incubation time was 60 min. For further experimental details, see text. b As expected [19], GSH, NO-deprived NOR-3 (NOR-3*), and NAP do not affect RhdA activity. c The simultaneous addition of DTT (final concentration, 1.0 103 M) and NOR-3 (final concentration, 1.0 104 M) to active RhdA (final concentration, 5.0 106 M) prevents the enzyme inhibition (time ¼ 0 min). For further experimental details, see text. d Addition of DTT (final concentration, 1.0 103 M) to inhibited RhdA (final concentration, 5.0 106 M) restores the enzyme activity (time ¼ 60 min). Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 1.0 104 M) for 60 min. For further experimental details, see text.
over, the incubation of inhibited, NOR-3-treated, RhdA with DTT restores enzyme activity (Table 1). The absorption spectrum of the NOR-3 treated RhdA displays a maximum at 335 nm (Fig. 3), suggesting the NO-mediated nitrosylation of the thiol group [19,22,24,25,27] of the unique Cys230 catalytic residue [10]. Coherently [19,22,24,25,27], DTT reverses the absorption spectrum of the NOR-3 inhibited RhdA (Fig. 3). Notably, NOR-3* does not affect the RhdA spectroscopic properties (data not shown). The catalytic activity of several unrelated enzymes has been shown to be modulated by NO through chemical modification(s) of reactive residues (e.g., Cys and Tyr), as well as by binding to metal centers [18,19,24–26]. In particular, the NO-mediated chemical modification of Cys residues can be promoted by the protein structural environment, such as neighboring residues or metals, implicated in cysteinate stabilization [17–19,26]. Such a situation may occur also in RhdA, where a strongly positive active site electrostatic field and specific hydrogen bonds to the catalytic Cys230 residue (Fig. 4) could stabilize the side chain anionic species [13]. In keeping with such structural considerations, the results presented here show that RhdA is inhibited by the NO-donors GSNO, NOR-3, and SNAP, via NO-mediated S-nitrosylation of the Cys230 catalytic residue. The catalytic domain of RhdA (Fig. 4) displays the same three-dimensional fold of Cdc25A and Cdc25B
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Table 2 Values of kinetic and equilibrium parameters for the inhibition of RhdA, human PTPB1, and Yersinia PTP by GSNO, NOR-3, and SNAP Enzyme
NO-donor
ki (min1 )
ki =Ki (M1 min1 )
Ki (M)
RhdAa
GSNO NOR-3 SNAP
5.1 102 1.3 3.6 101
8.1 101 5.0 103 3.0 102
6.3 104 2.6 104 1.2 103
Human PTPB1b
GSNO SNAP
4.6 102 1.1 101
9.9 101 1.1 102
4.7 104 9.7 104
Yersinia PTPb
GSNO SNAP
3.8 102 2.1
6.6 101 6.1 102
5.7 104 3.4 103
a b
pH 8.0 and 37 °C. Present study. pH 7.4 and 25 °C. From [23].
Fig. 2. Dependence of the RhdA activity on the [NO]/[RhdA] molar ratio. The intersect of the straight lines (arrow) indicates that one equivalent of NO, released from NOR-3, reacts with one equivalent of RhdA. The open symbol on the ordinate indicates the relative RhdA activity (100%) in the absence of NOR-3 (i.e., NO). RhdA concentration was 5.0 106 M. The NOR-3 concentration ranged between 1.0 106 M and 2.0 105 M. The NO concentration was calculated taking into account the half-time for NO release from NOR-3 ( ¼ 54 3 min) and the RhdA-NOR-3 incubation time (180 min) [19]. All data were obtained at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C. For further details, see text.
human cell cycle-control phosphatase catalytic domains, hinting at a common evolutionary origin for the two enzyme families [13,28–30]. Furthermore, the RhdA and
Fig. 3. Difference absorption spectrum of the active enzyme minus the inhibited S-nitrosylated RhdA. Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 2.0 104 M) for 60 min. Incubation of inhibited S-nitrosylated RhdA with DTT (final concentration, 1.0 103 M), KH2 PO4 (final concentration, 5.0 102 M), and Na2 S2 O3 (final concentration, 6.0 102 M), for 60 min, induced the disappearance of the difference absorption spectrum, restoring the RhdA activity (see Table 1). The RhdA concentration was 1.0 104 M. All data were obtained at pH 8.0 (5.0 102 M Tris– HCl) and 37 °C. For further details, see text.
Cdc25 phosphatase active sites display striking conformational similarity with the active site loop present in PTP’s and in low molecular weight phosphatases (for a review, see [31]). In this respect, active site and overall structural similarities have led to propose that Cdc25
Fig. 4. Stereoview of the RhdA active site. In active persulfurated RhdA an extra sulfur atom (Sd) is bound to the Cys230 Sc atom. N, C, O, and S atoms are represented in blue, black, red, and yellow, respectively. The picture was prepared with the programs MOLSCRIPT [36] and BOBSCRIPT [37] and rendered with the program RASTER3D [38].
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phosphatases, PTP’s, and low molecular weight phosphatases share a common evolutionary ancestor, from which each protein family may have diverged after substantial rearrangement of the respective three-dimensional fold [28,31–33]. As observed in RhdA (Fig. 4), the active site loop of these phosphatases forms a cradlelike structure defining the catalytic pocket, whose center is occupied by the reactive Cys Sc atom. The active site loop of RhdA is based on a –Cys–X4 –Arg– sequence motif (Fig. 4), while phosphatases display an active site loop composed of seven amino acids, based on a –Cys– X5 –Arg– motif [13,14,28–31,34]. Despite the similar values of the kinetic and equilibrium parameters for the NO-dependent inhibition of RhdA (present study), of the low molecular weight human PTP1B [23], of bovine and rat liver PTP [35], and of Yersinia PTP [23,35] (Table 2), enzyme inhibition occurs via different mechanisms in RhdA and in PTP’s. In RhdA, the NO-mediated S-nitrosylation of the unique Cys230 catalytic residue provides a molecular basis for enzyme inhibition by GSNO, NOR-3, and SNAP. On the other hand, NO inhibits bovine liver PTP by catalyzing the formation of a disulfide bond between the active site Cys12 residue and the neighboring Cys17 side chain, possibly linked to transient Cys12 S-nitrosylation [35]. In the context of a general consensus on NO reactivity for protein substrates, the present results show that the reaction mechanisms observed with distinct proteins, albeit structurally similar, are controlled by the protein fold and chemical properties of selected amino acid residues in the near surroundings of the NO reactive site [15–19]. Such concepts appear evident in the comparison of the structural and functional properties of RhdA and PTP’s, which bear similarly structured active site loops, are inhibited by NO-donors, and yet display different inhibition mechanisms.
Acknowledgments The authors wish to thank Prof. Angelo Azzi (University of Bern, Switzerland) and Prof. Maurizio Paci (University of Rome ‘Tor Vergata,’ Italy) for helpful discussions. The present study was partly supported by grants from the Ministry of Education, University, and Research of Italy (MIUR; ‘Fondi per lo Sviluppo, 2001’ and ‘FIRB’ to P.A. and ‘Solfotransferasi Procariotiche, 2001–2003’ to M.B.), from the National Research Council of Italy (CNR; ‘Agenzia 2000’ to P.A.), and from the Italian Space Agency (ASI; grant IR/294/02). M.B. is grateful to CINRO and Istituto G. Gaslini (Genova) for continuous support.
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[31] E.B. Fauman, M.A. Saper, Structure and function of the protein tyrosine phosphatases, Trends Biochem. Sci. 21 (1996) 413–417. [32] N.V. Grishin, Fold change in evolution of protein structures, J. Struct. Biol. 134 (2001) 167–185. [33] I. Zegers, J.C. Martins, R. Willem, L. Wyns, J. Messens, Arsenater reductase from S. aureus plasmid pI258 is a phosphatase drafted for redox duty, Nat. Struct. Biol. 8 (2001) 843– 847. [34] J.M. Denu, J.E. Dixon, Protein tyrosine phosphatases: mechanisms of catalysis and regulation, Curr. Opin. Chem. Biol. 2 (1998) 633–641. [35] A. Caselli, G. Camici, G. Manao, G. Moneti, L. Pazzagli, G. Cappugi, G. Ramponi, Nitric oxide causes inactivation of the low molecular weight phosphotyrosine protein phosphatase, J. Biol. Chem. 269 (1994) 24878–24882. [36] P.J. Kraulis, MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures, J. Appl. Crystallogr. 24 (1991) 946–950. [37] B.M. Esnouf, An extensively modified version of MOLSCRIPT that includes greatly enhanced coloring capabilities, J. Mol. Graph. 15 (1997) 112–113, see also 132–134. [38] E.A. Merrit, M.E.P. Murphy, Raster3d, a program for photorealistic molecular graphics, Acta Crystallogr. D 50 (1994) 869– 873.
Inhibition of Azotobacter vinelandii rhodanese by NO-donorsq Andrea Spallarossa,a Fabio Forlani,b Silvia Pagani,b Luca Salvati,c Paolo Visca,c,d Paolo Ascenzi,c,e Martino Bolognesi,f,g and Domenico Bordog,* a Dipartimento di Scienze Farmaceutiche, Universit a di Genova, Via Benedetto XV 3, I-16132 Genoa, Italy Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, I-20133 Milan, Italy c Dipartimento di Biologia, Universit a ‘Roma Tre,’ Viale G. Marconi 446, I-00146 Rome, Italy Istituto Nazionale per le Malattie Infettive IRCCS ‘Lazzaro Spallanzani,’ Unit a di Microbiologia Molecolare, Via Portuense 292, I-00149 Rome, Italy e Laboratorio Interdipartimentale di Microscopia Elettronica, Universit a ‘Roma Tre,’ Via della Vasca Navale 79, I-00146 Rome, Italy f Dipartimento di Fisica, Istituto Nazionale di Fisica della Materia, Centro di Eccellenza per la Biologia e la Medicina, Universit a di Genova, Via Dodecaneso 33, I-16146 Genoa, Italy g Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, I-16132 Genoa, Italy b
d
Received 15 May 2003
Abstract Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), ()E -4-ethyl-2-[E -hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetyl-penicillamine (SNAP) on the catalytic activity of Azotobacter vinelandii rhodanese (RhdA) has been investigated. GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion. The absorption spectrum of the NOR-3-treated RhdA displays a maximum at 335 nm, indicating NO-mediated S-nitrosylation. RhdA inhibition by NO-donors correlates with S-nitrosothiol formation. The reducing agent dithiothreitol prevents RhdA inhibition by NO-donors, fully restores the catalytic activity, and reverts the NOR-3-induced RhdA absorption spectrum to that of the active enzyme. These results indicate that RhdA inhibition occurs via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Rhodanese; Sulfurtransferase; Phosphatase; NO-donors; Enzyme inhibition
Sulfurtransferases (EC 2.8.1.x) are enzymes involved in the formation, interconversion, and transport of compounds containing sulfane sulfur atoms. The best characterized sulfurtransferase is bovine rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), which in vitro catalyses transfer of a sulfane sulfur atom from thiosulfate to cyanide, with formation of sulfite and thiocyanate [1–3]. This reaction involves the tranq
Abbreviations: RhdA, Azotobacter vinelandii rhodanese; PTP, protein tyrosine phosphatase; human PTP1B, human protein tyrosine phosphatase 1B; NO, nitric oxide; GSNO, S-nitroso-glutathione; GSH, glutathione; NOR-3, ()-E -4-ethyl-2-[E -hydroxyimino]-5-nitro3-hexenamide; NOR-3*, NO-deprived NOR-3; SNAP, S-nitroso-Nacetyl-penicillamine; NAP, N-acetyl-penicillamine; DTT, dithiothreitol. * Corresponding author. Fax: +39-010-5737306. E-mail address: [email protected] (D. Bordo).
sient formation of a persulfide-containing intermediate (Rhd–S), hosting an extra sulfur atom as persulfide modification of the catalytic Cys residue: 2 S2 O2 3 þ Rhd ! SO3 þ Rhd–S Rhd–S þ CN ! Rhd þ SCN
ðScheme 1Þ
Given the abundance of rhodanese in bovine liver mitochondria, this enzyme is deemed to be primarily involved in the elimination of toxic cyanogenic compounds arising from vegetal food digestion in mammals [4,5]. Recently, the extensive genome sequencing has shown that genes coding for proteins endowed with significant sequence similarity to mammalian rhodaneses are widely distributed in the three major evolutionary phyla, and that often several rhodanese-like proteins are encoded by the same genome. The latter observation suggests functional and specificity diversification within
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01067-2
A. Spallarossa et al. / Biochemical and Biophysical Research Communications 306 (2003) 1002–1007
the rhodanese homology family (see, e.g., the COG database, http://www.ncbi.nlm.nih.gov/COG). This fact has brought about new hypotheses on the biological function of rhodanese-like enzymes, such as sulfur mobilization in sulfur–iron cluster biosynthesis [6], or involvement in sulfur metabolism [7]. The ability of rhodanese to bind selenium in the presence of glutathione (GSH) suggests that rhodanese-like enzymes may also play a role in selenophosphate synthesis [8]. In the nitrogen-fixing bacterium Azotobacter vinelandii, the detection of rhodanese activity has led to the identification of the associated rhdA gene and to the biochemical characterization of its protein product [9,10]. The 29.6 kDa protein RhdA displays moderate but significant sequence similarity to bovine rhodanese (22% identity) [9,10]. Bovine rhodanese and RhdA also share the same overall three-dimensional structure, consisting of two similarly folded domains, hosting the catalytic Cys in the C-terminal domain [11–13]. In native RhdA crystals, the catalytic Cys230 is found persulfurated; the extra sulfur atom could be removed by a number of nucleophilic acceptors such as cyanide and phosphate [14]. Given the high reactivity of nitric oxide (NO) versus thiols [15–19], the effect of NO-donors S-nitroso-glutathione (GSNO), ()-E -4-ethyl-2-[E -hydroxyimino]-5nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetylpenicillamine (SNAP) on RhdA activity has been investigated. Herein, we report that NO-donors inhibit RhdA via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue.
Materials and methods Chemicals. The NO-donors NOR-3 and SNAP as well as GSH, Nacetyl-penicillamine (NAP), and dithiothreitol (DTT) were purchased from Sigma Chemical (St. Louis, MO, USA). The NO-donor GSNO was prepared by mixing equimolar amounts of an aqueous solution of NaNO2 and a freshly prepared GSH solution in 2.5 101 M HCl and 1.0 104 M EDTA (pH 1.5). The resulting mixture was incubated at 25 °C for 5 min and then neutralized with NaOH. The GSNO solution was stored at )20 °C [20]. The NO-deprived NOR-3 (NOR-3*) was obtained by treating NOR-3 at alkaline pH and 25 °C for 3 days [21]. All other products were obtained from Merck AG (Darmstadt, Germany). All chemicals were of analytical grade and were used without further purification. RhdA characterization and enzyme assay. Recombinant RhdA was prepared in the active persulfurated form, as previously reported [13]. The RhdA sulfurtransferase activity (i.e., the enzyme-catalyzed formation of thiocyanate; see Scheme 1) was assayed through a discontinuous method that quantitates the product thiocyanate, based on the absorbance of Fe(SCN)3 at 460 nm [4]. The optimized reaction mixture (0.65 ml) contained 6.0 102 M KCN, 5.0 102 M KH2 PO4 , and 1.0 106 –5.0 106 M RhdA in 5.0 102 M Tris–HCl, pH 8.0. The reaction was initiated by the addition of Na2 S2 O3 (6.0 102 M, final concentration) and continued for 2 min at 37 °C. The reaction was stopped by adding 0.1 ml formaldehyde (38%, v/v). Then, the reaction product SCN was detected by adding 0.25 ml of the iron reagent solution (0.25 M Fe(NO3 )3 and 13%, v/v, HNO3 , in water) to yield the
1003
Fe(SCN)3 complex. The assay was calibrated using a standard curve obtained from known concentrations of Fe(SCN)3 . In each experiment, the baseline was always assessed by pre-incubating RhdA with formaldehyde. One unit of RhdA activity is defined as the amount of enzyme that produces 1.0 lmol thiocyanate min1 in the assay condition. Effect of NO-donors on RhdA activity. The effect of GSNO, GSH, NOR-3, NOR-3*, SNAP, and NAP on the RhdA catalytic activity was determined by incubation of the active enzyme (final concentration, 1.0 106 –5.0 106 M range) with NO-donors and NO-deprived compounds, at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 5 min to 60 min. The final concentration of GSNO, GSH, NOR-3, NOR-3*, SNAP, and NAP ranged between 1.0 104 and 2.0 103 M. The RhdA residual sulfurtransferase activity was then assayed [4]. The [NO]/[RhdA] (i.e., [NOR-3]/[RhdA]) stoichiometry was determined by incubation of the active enzyme (final concentration, 5.0 106 M) with NOR-3 (final concentration, 1.0 106 – 2.0 105 M range), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 180 min. The RhdA residual sulfurtransferase activity was then assayed [4]. The NO concentration was calculated taking into account the half-time for NO release from NOR-3 (54 3 min) and the RhdANOR-3 incubation time (180 min) [19]. Protective effect of DTT on RhdA activity. The protective effect of DTT on the NO-mediated inhibition of RhdA was investigated by the simultaneous incubation of the active enzyme (final concentration, 1.0 106 –5.0 106 M range) with DTT (final concentration, 1.0 103 M) and NOR-3 (final concentration, 1.0 104 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min. The RhdA residual sulfurtransferase activity was then assayed [4]. Recovery of RhdA activity by DTT. The effect of DTT on the NOmediated inhibition of RhdA was investigated by incubating the inhibited enzyme (final concentration, 1.0 106 – 5.0 106 M range) with DTT (final concentration, 1.0 103 M), at pH 8.0 (5.0 102 M Tris– HCl) and 37 °C, for 60 min. Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 1.0 104 M) for 60 min. The RhdA residual sulfurtransferase activity was then assayed [4]. Effect of NOR-3, NOR-3*, and DTT on RhdA spectroscopic properties. The effect of NOR-3 and NOR-3* on RhdA absorbance spectroscopic properties between 300 and 400 nm was investigated by incubation of the active enzyme (final concentration, 1.0 104 M) with the NO-donor and the NO-deprived compound (final concentration, 2.0 104 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min. Reversibility of the absorption spectrum of the inhibited Snitrosylated RhdA was achieved by incubation of the inhibited enzyme (final concentration, 1.0 104 M) with DTT (final concentration, 1.0 103 M), KH2 PO4 (final concentration, 5.0 102 M), and Na2 S2 O3 (final concentration, 6.0 102 M), at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C, for 60 min.
Results and discussion NO-donors GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion (Fig. 1 and Table 1). Such inhibition obeys apparent first-order kinetics (Fig. 1, panel A). The double-reciprocal plots of the relative apparent first-order inhibition constant for RhdA inhibition by NO-donors (ki =k) as a function of the GSNO, NOR-3, and SNAP concentration ([NO-donor]1 ) yield straight lines (Fig. 1, panel B), indicating that enzyme inhibition is a relatively rapid pre-equilibrium followed by a limiting first-order process, as previously reported for NO inhibition of protein tyrosine phosphatases (PTP’s) and
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A. Spallarossa et al. / Biochemical and Biophysical Research Communications 306 (2003) 1002–1007 Table 1 Effect of GSNO, GSH, NOR-3, NOR-3*, SNAP, NAP, and DTT on the catalytic activity of RhdA, at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C
Fig. 1. RhdA inhibition by GSNO, NOR-3, and SNAP. (A) Time course of RhdA inhibition by SNAP. The enzyme activity was assayed after treatment with 2.0 104 M (circles), 5.0 104 M (squares), and 1.0 103 M (triangles) SNAP. The effect of 1.0 103 M NAP (diamonds) on the RhdA activity is shown for comparison. The RhdA concentration was 5.0 106 M. The continuous lines were calculated according to Eq. (1) [22,23]: ½RhdA ¼ ½RhdA ð1 ekt Þ;
ð1Þ
where [RhdA*] is the inhibited enzyme concentration and [RhdA] is the total active enzyme concentration at t ¼ 0 min, with the following values of k: 0.050 min1 (circles), 0.10 min1 (squares), and 0.16 min1 (triangles). (B) Dependence of the pseudo first-order rate constant k for RhdA inhibition by GSNO (circles), NOR-3 (squares), and SNAP (triangles) on the NO-donor concentration. The continuous lines were calculated according to Eq. (2) [22,23]: ki =k ¼ Ki ½NO-donor 1 þ 1
ð2Þ
with values of ki , ki =Ki , and Ki given in Table 2. All data were obtained at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C. For further details, see text.
papain [22,23]. Values of the apparent first-order ratelimiting constant (ki ), of the apparent second-order rate constant (ki =Ki ), and of the apparent dissociation equilibrium constant (Ki ) for RhdA inhibition by GSNO, NOR-3, and SNAP are reported in Table 2. As shown in Fig. 2, one equivalent of NO, released from NOR-3, inhibits one equivalent of RhdA. Notably, enzyme activity is essentially unaffected by NO-deprived compounds (i.e., GSH, NAP, and NOR-3*) (Fig. 1, panel A, and Table 1). To test whether RhdA inhibition occurred via NOmediated S-nitrosylation, the effect of DTT on RhdA inhibition by NOR-3 and on the enzyme spectroscopic properties was investigated. As expected [19,22,24–26], the simultaneous incubation of DTT and NOR-3 with RhdA prevents the enzyme inhibition (Table 1). More-
Inhibitor/activator
Enzyme activity (%)
None GSNOa GSHa;b NOR-3a NOR-3*a; b NOR-3 + DTT (time ¼ 0 min)c NOR-3 + DTT (time ¼ 60 min)d SNAPa NAPa;b
100 5 73 93 6 63 94 7 91 7 92 8 42 95 6
a The concentration of NO-donors and NO-deprived compounds (final concentration, 1.0 104 M) exceeded that of RhdA (final concentration, 5.0 106 M). The incubation time was 60 min. For further experimental details, see text. b As expected [19], GSH, NO-deprived NOR-3 (NOR-3*), and NAP do not affect RhdA activity. c The simultaneous addition of DTT (final concentration, 1.0 103 M) and NOR-3 (final concentration, 1.0 104 M) to active RhdA (final concentration, 5.0 106 M) prevents the enzyme inhibition (time ¼ 0 min). For further experimental details, see text. d Addition of DTT (final concentration, 1.0 103 M) to inhibited RhdA (final concentration, 5.0 106 M) restores the enzyme activity (time ¼ 60 min). Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 1.0 104 M) for 60 min. For further experimental details, see text.
over, the incubation of inhibited, NOR-3-treated, RhdA with DTT restores enzyme activity (Table 1). The absorption spectrum of the NOR-3 treated RhdA displays a maximum at 335 nm (Fig. 3), suggesting the NO-mediated nitrosylation of the thiol group [19,22,24,25,27] of the unique Cys230 catalytic residue [10]. Coherently [19,22,24,25,27], DTT reverses the absorption spectrum of the NOR-3 inhibited RhdA (Fig. 3). Notably, NOR-3* does not affect the RhdA spectroscopic properties (data not shown). The catalytic activity of several unrelated enzymes has been shown to be modulated by NO through chemical modification(s) of reactive residues (e.g., Cys and Tyr), as well as by binding to metal centers [18,19,24–26]. In particular, the NO-mediated chemical modification of Cys residues can be promoted by the protein structural environment, such as neighboring residues or metals, implicated in cysteinate stabilization [17–19,26]. Such a situation may occur also in RhdA, where a strongly positive active site electrostatic field and specific hydrogen bonds to the catalytic Cys230 residue (Fig. 4) could stabilize the side chain anionic species [13]. In keeping with such structural considerations, the results presented here show that RhdA is inhibited by the NO-donors GSNO, NOR-3, and SNAP, via NO-mediated S-nitrosylation of the Cys230 catalytic residue. The catalytic domain of RhdA (Fig. 4) displays the same three-dimensional fold of Cdc25A and Cdc25B
A. Spallarossa et al. / Biochemical and Biophysical Research Communications 306 (2003) 1002–1007
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Table 2 Values of kinetic and equilibrium parameters for the inhibition of RhdA, human PTPB1, and Yersinia PTP by GSNO, NOR-3, and SNAP Enzyme
NO-donor
ki (min1 )
ki =Ki (M1 min1 )
Ki (M)
RhdAa
GSNO NOR-3 SNAP
5.1 102 1.3 3.6 101
8.1 101 5.0 103 3.0 102
6.3 104 2.6 104 1.2 103
Human PTPB1b
GSNO SNAP
4.6 102 1.1 101
9.9 101 1.1 102
4.7 104 9.7 104
Yersinia PTPb
GSNO SNAP
3.8 102 2.1
6.6 101 6.1 102
5.7 104 3.4 103
a b
pH 8.0 and 37 °C. Present study. pH 7.4 and 25 °C. From [23].
Fig. 2. Dependence of the RhdA activity on the [NO]/[RhdA] molar ratio. The intersect of the straight lines (arrow) indicates that one equivalent of NO, released from NOR-3, reacts with one equivalent of RhdA. The open symbol on the ordinate indicates the relative RhdA activity (100%) in the absence of NOR-3 (i.e., NO). RhdA concentration was 5.0 106 M. The NOR-3 concentration ranged between 1.0 106 M and 2.0 105 M. The NO concentration was calculated taking into account the half-time for NO release from NOR-3 ( ¼ 54 3 min) and the RhdA-NOR-3 incubation time (180 min) [19]. All data were obtained at pH 8.0 (5.0 102 M Tris–HCl) and 37 °C. For further details, see text.
human cell cycle-control phosphatase catalytic domains, hinting at a common evolutionary origin for the two enzyme families [13,28–30]. Furthermore, the RhdA and
Fig. 3. Difference absorption spectrum of the active enzyme minus the inhibited S-nitrosylated RhdA. Inhibited RhdA was obtained by pretreatment with NOR-3 (final concentration, 2.0 104 M) for 60 min. Incubation of inhibited S-nitrosylated RhdA with DTT (final concentration, 1.0 103 M), KH2 PO4 (final concentration, 5.0 102 M), and Na2 S2 O3 (final concentration, 6.0 102 M), for 60 min, induced the disappearance of the difference absorption spectrum, restoring the RhdA activity (see Table 1). The RhdA concentration was 1.0 104 M. All data were obtained at pH 8.0 (5.0 102 M Tris– HCl) and 37 °C. For further details, see text.
Cdc25 phosphatase active sites display striking conformational similarity with the active site loop present in PTP’s and in low molecular weight phosphatases (for a review, see [31]). In this respect, active site and overall structural similarities have led to propose that Cdc25
Fig. 4. Stereoview of the RhdA active site. In active persulfurated RhdA an extra sulfur atom (Sd) is bound to the Cys230 Sc atom. N, C, O, and S atoms are represented in blue, black, red, and yellow, respectively. The picture was prepared with the programs MOLSCRIPT [36] and BOBSCRIPT [37] and rendered with the program RASTER3D [38].
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phosphatases, PTP’s, and low molecular weight phosphatases share a common evolutionary ancestor, from which each protein family may have diverged after substantial rearrangement of the respective three-dimensional fold [28,31–33]. As observed in RhdA (Fig. 4), the active site loop of these phosphatases forms a cradlelike structure defining the catalytic pocket, whose center is occupied by the reactive Cys Sc atom. The active site loop of RhdA is based on a –Cys–X4 –Arg– sequence motif (Fig. 4), while phosphatases display an active site loop composed of seven amino acids, based on a –Cys– X5 –Arg– motif [13,14,28–31,34]. Despite the similar values of the kinetic and equilibrium parameters for the NO-dependent inhibition of RhdA (present study), of the low molecular weight human PTP1B [23], of bovine and rat liver PTP [35], and of Yersinia PTP [23,35] (Table 2), enzyme inhibition occurs via different mechanisms in RhdA and in PTP’s. In RhdA, the NO-mediated S-nitrosylation of the unique Cys230 catalytic residue provides a molecular basis for enzyme inhibition by GSNO, NOR-3, and SNAP. On the other hand, NO inhibits bovine liver PTP by catalyzing the formation of a disulfide bond between the active site Cys12 residue and the neighboring Cys17 side chain, possibly linked to transient Cys12 S-nitrosylation [35]. In the context of a general consensus on NO reactivity for protein substrates, the present results show that the reaction mechanisms observed with distinct proteins, albeit structurally similar, are controlled by the protein fold and chemical properties of selected amino acid residues in the near surroundings of the NO reactive site [15–19]. Such concepts appear evident in the comparison of the structural and functional properties of RhdA and PTP’s, which bear similarly structured active site loops, are inhibited by NO-donors, and yet display different inhibition mechanisms.
Acknowledgments The authors wish to thank Prof. Angelo Azzi (University of Bern, Switzerland) and Prof. Maurizio Paci (University of Rome ‘Tor Vergata,’ Italy) for helpful discussions. The present study was partly supported by grants from the Ministry of Education, University, and Research of Italy (MIUR; ‘Fondi per lo Sviluppo, 2001’ and ‘FIRB’ to P.A. and ‘Solfotransferasi Procariotiche, 2001–2003’ to M.B.), from the National Research Council of Italy (CNR; ‘Agenzia 2000’ to P.A.), and from the Italian Space Agency (ASI; grant IR/294/02). M.B. is grateful to CINRO and Istituto G. Gaslini (Genova) for continuous support.
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