Contrasting Effects of Metal Ions on S-Nitrosoglutathione, Related to

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Chem. Res. Toxicol. 2004, 17, 392-403

Contrasting Effects of Metal Ions on S-Nitrosoglutathione, Related to Coordination Equilibria: GSNO Decomposition Assisted by Ni(II) vs Stability Increase in the Presence of Zn(II) and Cd(II) Artur Kre¸ z˘ el† and Wojciech Bal*,‡ Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland, and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland Received September 24, 2003

Complex formation between nitrosoglutathione (GSNO) and Zn(II), Cd(II), and Ni(II) ions was studied by potentiometry and spectroscopic techniques. GSNO forms simple ML and ML2 type complexes (L ) GSNO) with these ions. The stability of GSNO in HEPES buffer solution, pH 7.4, increased in the presence of both Zn(II) and Cd(II), due to an indirect mechanism. A concentration-dependent destabilization of GSNO by Ni(II) ions was found to be linearly dependent on the NiL complex concentration. NiL forms ternary complexes readily. The NiLAstoichiometry was found for L-His, and NiHLB3- and NiLB4- complexes were detected for GSSG as the second ligand. The formation of these complexes was found to inhibit GSNO decay, by limiting the concentration of the NiL complex. The mechanism of Ni(II)-assisted GSNO decomposition contains several steps, with a hypothetical ternary complex with GSH as a likely active form. These results provide experimental evidence for the stabilization of GSNO in solution by metal ions, which may provide an additional level of control and/or impairment of cellular redox signaling. The Ni(II)-dependent destabilization of GSNO may constitute a novel epigenetic mechanism in nickel carcinogenesis.

Introduction Nitric oxide (NO) has emerged as a key regulator of many physiological functions, such as vasodilation, oxygen transport, cardiac activity, immune response, neurotransmission, and apoptosis (1, 2). The NO molecule is a free radical gas and is thus readily reactive toward many biochemical targets. The chemical processes involved include nitration of aromatic rings and nitrosation/ nitrosylation of amines, transition metal ions, such as Fe(II), and thiols (2, 3). The latter of these processes yields nitrosothiols, also named thionitrites. The nitroso derivatives of protein cysteines are considered to be key forms in the process called generically protein nitrosylation and are regarded as a major pathway of intracellular signaling, in parallel with phosphorylation (1, 4). Two molecular mechanisms of protein nitrosylation have been proposed. One, precedent historically, involves direct interaction of a protein with NO gas or a more reactive product of its oxidation with oxygen, such as, e.g., N2O3 or NO2-, dubbed collectively reactive nitrogen species (RNS) (4-6). It is currently considered to occur predominantly in cellular membranes and other hydrophobic compartments, including hydrophobic protein interiors. The latter option has recently been indicated as a likely mechanism of albumin-mediated formation of nitrosothiols in blood serum (7). The other mechanism is a transnitrosation reaction, where a donor molecule * To whom correspondence should be addressed. Tel: +48-226597072 ext 2353. Fax: +48-22-6584636. E-mail: [email protected]. † University of Wroclaw. ‡ Polish Academy of Sciences.

Scheme 1. Molecule of S-Nitrosoglutatione in Its Fully Protonated Form (GSNO)+

exchanges its NO moiety with the target site. A Fe(II) complex with NO and glutathione (GSH) has been identified as one such donor (8). However, it is the thiolto-thiol transnitrosation that emerges as a major signaling pathway (9, 10). The donor nitrosothiol (RSNO,1 also called thionitrite) can be preformed in a direct reaction with a RNS, which may proceed by a radical mechanism (5, 11). GSH is the most abundant cellular thiol, and thus, it is no surprise that its NO derivative, nitrosoglutathione (GSNO) (Scheme 1), is considered to be an important 1 Abbreviations: RSNO, any nitrosothiol; GSNO, S-nitrosoglutathione; L, GSNO molecule in its fully deprotonated form; A, L-His molecule in its fully deprotonated form; B, GSSG molecule in its fully deprotonated form; D, any fully deprotonated ligand molecule; M, metal ion; kobs, apparent 0th order kinetic constant; k1, computed 1st order kinetic constant.

10.1021/tx034194i CCC: $27.50 © 2004 American Chemical Society Published on Web 02/07/2004

Decomposition of GSNO by Metals

intracellular NO carrier (12). The unique relevance of GSNO has been confirmed by a discovery of an evolutionarily conserved enzyme, decomposing GSNO to GSSG and ammonia (13). Once formed, GSNO can exert site specific protein modifications, at two known kinds of consensus sites (8, 14), which include glutathionylation and sulfenic acid formation, in addition to transnitrosation. The discovery of this combination of versatility and site specificity led Stamler et al. to propose the existence of a general intracellular signaling pathway, based on redox modification of protein Cys residues (15, 16). GSNO may also have a distinct physiological function, which does not involve protein transnitrosation or thiolation (17). The formation of the S-NO bond is reversible, and its stability is modulated by the structure of the specific nitrosothiol and by external factors. Much attention has been recently devoted to mechanisms of nitrosothiol decomposition in aqueous solution. The picture emerging is a complicated one. NO and disulfide are major decomposition products. Three classes of reaction mechanisms have been proposed, as reviewed recently (18). These include (i) the unimolecular homolytic scission of the S-N bond, (ii) the metal ion-catalyzed reductive decomposition, and (iii) various higher order reactions. The major variant of mechanism i includes the two step reaction, propagated by thyil radicals. The discrepancies in determinations of reaction rates, rate laws, and enthalpies of the homolytic S-N bond dissociation among various studies led to a proposal that mechanism ii, enabled by the adventitious presence of traces of redox active transition metals in reaction buffers, contributes significantly, or even decisively, to the overall process of nitrosothiol decomposition. The metal ions identified as capable of RSNO decomposition in a redox process are Cu(I), Cu(II), and also Fe(II) (19-22). A recent study suggested that iron, rather than copper, may be the crucial impurity in phosphate buffers (23). Other redox capable metal ions, which might in principle be reactive toward GSNO, are Ni(II), Co(II), Mn(II), Cr(III), and Fe(III). They were, however, declared as exerting no effect on RSNO stability, along with such nonredox ions as Zn(II), Ca(II), and Mg(II) (19). Ag(I) and Hg(II) were found to decompose RSNO in a different manner, via a stoichiometric hydrolysis reaction, which yielded nitrite and a metal thiolate complex (24). Studies specific to GSNO indicated that Cu(I), Cu(II), and Fe(II) catalyze its decay to GSSG and NO (22, 25, 26). These studies demonstrated the complexity of the overall process, inhibited by the accumulation of GSSG, which served as an inhibitory chelator for the catalytic metal. We have been studying potential molecular mechanisms of nickel carcinogenesis, as reviewed recently (2729). Our recent work indicated a competition for a zinc finger binding as a potential link between Zn(II) physiology and Ni(II) toxicity (30). We have also studied Zn(II) and Ni(II) complexes with GSH, which may be intracellular low molecular weight carriers for these metal ions under certain conditions (31, 32). Here, we present an extension of these studies over GSNO, demonstrating that Zn(II) and Cd(II) ions can stabilize GSNO, while Ni(II) accelerates its decomposition. To obtain a deeper understanding of the latter phenomenon, we have included studies of Ni(II) complexes with histidine and GSSG.

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Experimental Section Materials. Reduced GSH, oxidized glutathione (GSSG), Cd(NO3)2‚4H2O, NaOD (40% w/v in D2O), sodium (3-trimethylsilyl)2,2,3,3-tetradeuteriopropionate (TSP), L-His, L-Ala, Chelex-100 resin, phosphoric acid and sodium phosphates, NaH2PO4‚H2O, Na2HPO4‚7H2O, and Na3PO4‚12H2O were purchased from Sigma Chemical Co. (St. Louis, MO). NaOH, NaClO4‚H2O, Ni(ClO4)2‚ 6H2O, ZnSO4‚7H2O, and KNO3 were purchased from Merck (Darmstadt, Germany). GSNO was synthesized from GSH, according to the published procedure (33), using NaNO2 and HCl from POCH (Gliwice, Poland). D2O (99.9%) and DCl (35% solution in D2O) were from Cambridge Isotope Laboratories. Potentiometry. Potentiometric titrations of GSNO, its Zn(II), Cd(II), and Ni(II) complexes, as well as L-His and GSSG, their binary Ni(II) complexes, and the ternary systems with GSNO in the presence of 0.1 M KNO3 were performed at 25 °C using pH metric titrations over the pH range 2.5 to 11.0 (Molspin automatic titrator, Molspin, Newcastle, U.K.) with 0.1 M NaOH as the titrant. Changes in pH were monitored with a combined glass-Ag/AgCl electrode (InLab 422, Mettler Toledo AG, Greifensee, Switzerland), calibrated daily in hydrogen ions concentrations by HNO3 titrations (34). Sample volumes of 1.5 mL, GSNO concentrations of 2 mM, and M:L molar ratios of 1:0 (ligand titrations), 1:1, 1:2, and 1:4 were used for studies of binary systems. For ternary systems, the Ni(II) and ligand concentrations were kept at ca. 2 mM. These data were analyzed using the SUPERQUAD program (35). Standard deviations computed by SUPERQUAD refer to random errors only. Electronic Absorption (UV-vis). The spectra of 10 mM samples of GSNO, GSSG, L-His, and their binary and ternary complexes were recorded at 25 °C in the wavelength range of 200-1100 nm on a Cary 50 Bio (Varian Inc., Palo Alto, CA) using 1 cm cuvettes. The solutions contained 50 mM HEPES, pH 7.4. The spectra of analogous samples, containing 10 mM L-Ala and 5 and 10 mM Ni(II), were also recorded as reference. Only the bands below 900 nm could be quantified reliably; the long wavelength tails present are therefore not reported in the Results section. The decay of GSNO was followed on a Cary 50 Bio spectrophotometer, in 1 cm cuvettes, using the maximum of GSNO absorption at 330 nm as the monitor wavelength. The whole spectra in the wavelength range of 200-1100 nm were also recorded in some experiments to ensure the absence of appearance of further chromophoric reaction products. The 1 mM samples of GSNO in 50 mM HEPES buffer, pH 7.4, were used, containing between 0 and 1 mM metal ion salts, between 0 and 0.5 mM GSSG, between 0 and 2 mM L-His, or between 0 and 50 µM GSH, where appropriate. The buffer solutions and the dissolved GSNO samples were purged with argon to remove oxygen. Sealed cuvettes were used to prohibit the leakage of oxygen into the reaction mixtures. Triplicate runs were measured for all experiments. NMR. One-dimensional (1D) 1H NMR spectra of 10 mM samples of GSNO in D2O, both metal-free and containing 5 mM Zn(II) or Cd(II), were recorded at 25 °C, on a AMX-300 spectrometer (Bruker, Karlsruhe, Germany) at 300 MHz. TSP was used as internal 1H standard. Capillary Electrophoresis. The analysis of decay of GSNO was performed on a P/ACE 5000 system (Beckman Coulter, Fullerton, CA) using a micellar electrokinetic method (36) with a slight modification. Shortly, the aliquots of GSNO samples identical to those studied by UV were diluted with HCl to the final concentrations of 0.1 mM, pressure injected for 3 s on the unmodified fused silica capillary (effective length 50 cm × 50 µm internal diameter), and ran at 15 kV (+263.2 V/cm) at 25 °C. The control runs with 0.25 mM GSSG were done in the same way. The micellar running buffer contained 25 mM NaH2PO4, 15 mM H3BO3 and 50 mM SDS, pH 8.0.

Results The acid-base properties of GSNO and the formation of GSNO complexes with Zn(II) and Cd(II) were charac-

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Table 1. Protonation Constants of GSNO, GSSG, and (Collectively D), Determined at 25 °C and I ) 0.1 M (KNO3)

L-His

GSNO logβb

species HD H2D H3D H4D H5D H6D

GSSG pKa

logβ

L-Hisa

pKa

9.084 (3) 9.084 9.695 (4) 12.617 (6) 3.785 18.583 (4) 15.07 (1) 2.530 22.524 (6) 25.690 (7) 28.10 (1) c

logβ

pKa

9.695 9.129 (1) 9.129 8.888 15.165 (2) 6.036 3.971 16.85 (5) 1.685 3.166 2.41

a Protonation constants of L-His from this determination were published previously in a study of Zn(II) complexes (31). b βjk ) [HjD]/([H+]j[D]), statistical errors on the last digits of stability constants are given in parentheses. c Not determined.

Table 2. Apparent Values of pKa, Sensed at Individual Nondissociable Protons of GSNO, Determined at 25 °C proton

acidic pKa

alkaline pKa

R-Glu β-Glu γ-Glu R-Cys β-Cys(NO)1b β-Cys(NO)2b Gly

2.34 (1)a 2.36 (2) 2.37 (2) 3.91 (3) 3.84 (3) 3.69 (2) 3.72 (1)

9.16 (2) 9.16 (2) 9.15 (2) 9.07 (2) 9.25 (1) 9.13 (1)

a Statistical errors on the last digits of stability constants are given in parentheses. b Assignments arbitrary.

Table 3. Stability Constants (log β)a of Zn(II), Cd(II), and Ni(II) (Collectively M) Complexes with GSNO (L) and of Ni(II) Complexes with L-His (A) and GSSG (B) Determined at 25 °C and I ) 0.1 M (KNO3) species

Ni(II)

Zn(II)

Cd(II)

ML ML2 MH-1L2 MH-2L2 MA MA2 MLA MHB MB M2B MHLB MLB

5.37 (2) 9.72 (2) -0.89 (6) -11.65 (8) 8.670 (4) 15.532 (5) 13.26 (2) 15.15 (1) 9.412 (2) 12.12 (5) 20.85 (3) 12.99 (1)

4.85 (1) 8.64 (2) -0.06 (1) -9.95 (1)

4.02 (2) 6.83 (3) -3.38 (5) -13.57 (2)

Figure 1. NMR spectra at pH* 7.5 and 25 °C. Top: GSNO, 10 mM; middle: GSNO, 10 mM + Zn(II), 5 mM; and bottom: GSNO, 10 mM + Cd(II), 5 mM. Signal assignment: 1, R-Cys(NO); 2, β-Cys(NO); 3, Gly; 4, R-Glu; 5, γ-Glu; and 6, β-Glu. The signal of R-Cys(NO) is partially obscured by the HDO resonance at this pH*.

aβ 2+ i + j k ijk ) [MiHjDk]/([M ] [H ] [D] ), D denotes any ligand; statistical errors on the last digits of stability constants are given in parentheses.

terized by potentiometry and 1H NMR. UV-vis was used as a spectroscopic tool for studying Ni(II) complexes, instead of NMR, due to their paramagnetism. The protonation constants of GSNO, obtained by potentiometry, are presented in Table 1. This table presents additionally the protonation constants of other ligands studied in this work, GSSG and L-His, also derived from potentiometry. Table 2 presents the values of apparent protonation constants, calculated from dependencies of chemical shifts on pH* for individual nondissociable protons of the GSNO molecule. Table 3 presents the stability constants of metal ion complexes studied as follows: binary GSNO complexes with Zn(II), Cd(II), and Ni(II), binary Ni(II) complexes with GSSG and L-His, as well as ternary Ni(II) complexes with GSNO and each of L-His and GSSG, calculated on the basis of potentiometric titrations. Figure 1 shows the examples of 1D NMR spectra (300 MHz) of GSNO and its Zn(II) and Cd(II) complexes at pH* 7.5. The large shifts of R-Glu and β-Glu resonances upon Zn(II) and Cd(II) binding can be seen

Figure 2. Comparison of dependence on pH* of chemical shifts of individual nondissociable protons in 10 mM GSNO in the absence (O) and presence (b) of 5 mM Zn(II).

clearly. The titration curves of nondissociable protons of GSNO complexed to Zn(II) or Cd(II) ions, derived from chemical shifts, are presented in Figures 2 and 3, respectively, overlapped on the titration curves of GSNO alone. The shifts of the titration curves for glutamic acid residues toward lower pH* values, as compared to that of the metal-free GSNO, support the interaction of Zn(II) and Cd(II) with the glutamic acid moiety of GSNO above pH* 6. However, a very subtle effect of metal ion binding can also be traced on Cys(NO) residue signals. Figure 4 shows the potentiometric species distribution


Figure 3. Comparison of dependence on pH* of chemical shifts of individual nondissociable protons in 10 mM GSNO in the absence (O) and presence (b) of 5 mM Cd(II).

Figure 4. Species distribution plots for GSNO complexes with Zn(II) (top) and Cd(II) (bottom), calculated using data from Tables 1 and 3, for 10 mM GSNO (L) and 5 mM Zn(II) or Cd(II) (M), as used in NMR experiments. 1, M2+; 2, ML; 3, ML22-; 4, MH-1L23-; and 5, MH-2L24-.

plots for GSNO and its complexes with Zn(II) and Cd(II). The comparison of Figures 2 and 3 with Figure 4 clearly confirms the glutamic acid moiety as the binding site for Zn(II) and Cd(II) in GSNO. The formation of complexes containing Ni(II) was studied by potentiometry, aided by UV-vis spectra. Potentiometric titrations indicated that in addition to binary Ni(II)-GSNO complexes, ternary species were also formed with L-His and GSSG. The protonation and Ni(II) stability constants of the latter two molecules were thus redetermined to ensure the coherence of conditions in the analysis of formation of ternary complexes. The protonation and Ni(II) complex formation data for L-His


Figure 5. Species distribution plots for binary and ternary Ni(II) complexes. (A) 10 mM GSNO + 5 mM Ni(II); (B) 1 mM GSNO + 0.5 mM Ni(II); (C) 10 mM GSSG + 10 mM Ni(II); (D) 10 mM L-His + 5 mM Ni(II); (E) 10 mM GSNO + 10 mM GSSG + 10 mM Ni(II); and (F) 10 mM GSNO + 10 mM L-His + 10 mM Ni(II). Labels of individual species are as follows: 1, Ni2+; 2, NiHD; 3, NiK; 4, NiD2; 5, NiH-1D2; 6, Ni2D; 7, NiHLE; and 8, NiLE, where D is any of GSNO, GSSG, or L-His, L is GSNO, and E is GSSG or L-His. Only the major binary species of GSSG and L-His are labeled in ternary distribution plots E and F. The ternary complexes in these plots are marked with thicker lines. Charges are omitted for simplicity.

and GSSG are presented in Tables 1 and 3, respectively. Figure 5 shows the species distribution plots for Ni(II) complexes with GSNO, GSSG, and L-His, as well as for ternary GSNO complexes, including Ni(II) and either L-His or GSSG. These plots were calculated for GSNO, L-His, and GSSG concentrations of 10 mM, as used to record the UV-vis spectra, except for Figure 5B, where the concentrations of 1 mM for GSNO and 0.5 mM for Ni(II) were used, similar to those used for kinetic studies (below). The UV-vis spectra of these Ni(II) complexes at pH 7.4 (controlled using 50 mM HEPES buffer) are provided in Figure 6. The literature data were used to assign absorption bands of GSNO (37). The spectra of Ni(II) complexes all have pseudo-octahedral character (38). HEPES has negligible affinity toward Ni(II) ions. The spectra of metal ion-free GSNO and Ni(II) complexes with L-Ala under the same conditions were recorded for the sake of comparison. The parameters characterizing these spectra are presented in Table 4, and the spectrum of metal-free GSNO is shown in Figure 6A. The spectra of L-Ala complexes are not shown, to maintain the clarity of Figure 6B. They are very similar to the spectrum of the GSSG complex, as evidenced by their parameters in Table 4. UV-vis spectroscopy was used as a tool for studying GSNO decay, monitored primarily at 330 nm. Figure 7 compares the stability of 1 mM solutions of metal-free GSNO in the 50 mM HEPES buffer, pH 7.4, with that of solutions, containing 0.5 mM Zn(II). A clear stabilizing effect of Zn(II) was seen, while the kinetics of the decay remained to be linear (pseudo-0th order type). An analogous addition of Cd(II) instead of Zn(II) also resulted in

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Figure 6. UV-vis spectra of Ni(II) complexes at pH 7.4 (50 mM HEPES) and 25 °C. (A, B) Systems containing GSNO: 1, 10 mM GSNO; 2, 10 mM GSNO + 10 mM Ni(II); 3, 10 mM GSNO + 5 mM Ni(II); and 4, 10 mM GSNO + 10 mM Ni(II) + 10 mM L-His. (C) Systems without GSNO: 5, 10 mM GSSG + 10 mM Ni(II); 6, 10 mM L-His + 10 mM Ni(II); and 7, 10 mM L-His + 5 mM Ni(II).

Figure 7. Kinetics of spontaneous decay of 1 mM GSNO in 50 mM HEPES (pH 7.4), at 25 °C, monitored at 330 nm (b), as compared with the decay of 1 mM GSNO in the presence of 0.5 mM Zn(II) under the same conditions (9). Triplicate runs are presented for each case.

Table 4. Parameters of UV-vis Spectra of GSNO and Its Binary and Ternary Complexes Determined at 25 °C in 50 mM HEPES Buffer, pH 7.4 components

λmaxa

b

10 mM GSNO

291 370c 515c 545

158d 100d 8.8e 14.8e

10 mM GSNO 10 mM Ni(II)

291 370c 515c 545 647 730c

158d 100d 8.8e 14.5e 2.8f 1.7f

10 mM GSNO 5 mM Ni(II)

291 370c 515c 545 638 730c

158d 100d 8.8e 14.5e 3.2f 1.4f

10 mM L-Ala 10 mM Ni(II)

384 646 720c

4.7f 2.1f 1.4f

10 mM L-Ala 5 mM Ni(II)

379 642 720c

5.5f 2.9f 1.4f

10 mM GSSG 10 mM Ni(II)

380 642

5.6f 2.3f

10 mM L-His 10 mM Ni(II)

371 606

6.3f 3.1f

10 mM L-His 5 mM Ni(II)

357 555

4.4f 2.9f

10 mM GSNO 10 mM L-His 10 mM Ni(II)

288 370c 515c 545 595

161d 100d 8.8e 16.0e 3.1f,g

a Units are nanometers. b Units are per molar per centimeter (M-1 cm-1); intensities were calculated per Ni(II), except for intraligand GSNO bands (d, e), which are calculated per GSNO. c Shoulder. d π f π* transition in the -SNO group (37). e n f π* transition in the -SNO group (37). f d-d transition in pseudooctahedral Ni(II) (38). g Estimated from a difference spectrum vs metal-free GSNO.

Figure 8. Kinetics of decay of 1 mM GSNO in 50 mM HEPES (pH 7.4), at 25 °C, monitored at 330 nm (0), as compared with the decay of 1 mM GSNO in the presence of 0.1 (9), 0.3 (2), 0.5 (b), and 1 ([) mM Ni(II). The decay in the presence of 0.5 mM Ni(II) and 0.5 mM GSSG is also presented (O). Single runs are shown for simplicity.

GSNO stabilization. The 0th order kinetic constants (kobs) for GSNO and its Zn(II) and Cd(II) complexes, averaged over triplicate runs, are presented in Table 5, along with the concentrations of major solution components. The difference of averages of k0 between free GSNO and its Zn(II) constant was statistically significant (p ) 0.001). In contrast, Ni(II) ions accelerated GSNO decay under the same conditions, in a concentration-dependent fashion, as presented in Figure 8. Single runs are presented in this figure for the sake of clarity of presentation. The inhibitory effect of GSSG on Ni(II)-dependent GSNO decomposition was demonstrated by its addition to the reaction mixture. A nonlinear kinetic behavior was seen for Ni(II) experiments with Ni(II)-to-GSNO ratios higher than 1:10. In contrast to this, the rates of GSNO decomposition in the presence of Ni(II) and L-His, presented in Figure 9, exhibited linear kinetics. Quantitative



Table 5. Momentary Velocities (Pseudo 0th Order Kinetic Constants) of GSNO Decay, Determined at 25 °C in 50 mM HEPES Buffer, pH 7.4 initial concentrations of componentsa GSNO, 1 GSNO, 1 + Zn(II), 0.5 GSNO, 1 + Cd(II), 0.5 GSNO, 1 + Ni(II), 0.1 GSNO, 1 + Ni(II), 0.3 GSNO, 1 + Ni(II), 0.5 GSNO, 1 + Ni(II), 1 GSNO, 1 + Ni(II), 0.5 + GSSG 0.5 GSNO, 1 + Ni(II), 0.5 + His 0.5 GSNO, 1 + Ni(II), 0.5 + His 1 GSNO, 1 + Ni(II), 0.5 + His 2 GSNO, 1 + GSH 0.02 GSNO, 1 + GSH 0.05 GSNO, 1 + Ni(II), 0.5 + GSH 0.02 GSNO, 1 + Ni(II), 0.5 + GSH 0.05

Rc

kobsb 10-10

6.0 (5) × 3.1 (4) × 10-10 5 (1) × 10-10 1.2 (1) × 10-9 6.4 (5) × 10-9 4.4 (7) × 10-9 9.7 (8) × 10-9 5.3 (9) × 10-9 1.8 (2) × 10-8 1.1 (1) × 10-8 4.2 (4) × 10-9 1.1 (1) × 10-9 3.1 (3) × 10-9 1.5 (2) × 10-9 6.0 (3) × 10-10 1.3 (1) × 10-9 2.0 (1) × 10-9 2.0 (7) × 10-8 1.4 (2) × 10-9 1.9 (3) × 10-8 2.0 (1) × 10-9

1.0d 1.0d 1.0d 1.0d 1.0 0.94 1.0 0.88 1.0 0.7 1.0 0.94 1.0d 1.0d 1.0d 1.0d 1.0d 1.0 0.90 1.0 0.90

species distributiona 1e

Ltot Ltot 0.715; ZnL 0.242; ZnL2 0.043 Ltot 0.916; CdL 0.082; CdL2 0.002 Ltot 0.89; NiL 0.061; NiL2 0.049 Ltot 0.698; NiL 0.186; NiL2 0.116 Ltot 0.839, NiL 0.168; NiL2 0.101; NiHLA, 0.006; NiLA 0.002 Ltot 0.556; NiL 0.307; NiL2 0.149 Ltot 0.502; NiL 0.266; NiL2 0.117, NiHLA, 0.005; NiLA 0.002 Ltot 0.378; NiL 0.547; NiL2 0.075 Ltot 0.219, NiL 0.4; NiL2 0.077; NiHLA, 0.003; NiLA 0.001 Ltot 0.839, NiL 0.045; NiL2 0.034; NiHLA, 0.061; NiLA 0.021 Ltot 0.799, NiL 0.036; NiL2 0.023; NiHLA, 0.061; NiLA 0.021 Ltot 0.816, NiL 0.043; NiL2 0.03, NiLA 0.141 Ltot 0.966, NiLA 0.034 Ltot 0.997, NiLA 0.003 f f f f f f

a Units are millimolar (mM). b Units are molar per second (M s-1). c Reaction progress variable, molar fraction of GSNO remaining in solution. d Pseudo 0th order reaction, kobs independent of R. e Ltot, total concentration of L, consists of HL-, 98.0%, and L2- 2.0%, at a constant ratio. f Not comparable at excess GSH (see text).

Figure 9. Kinetics of decay of 1 mM GSNO in 50 mM HEPES (pH 7.4), at 25 °C, monitored at 330 nm, in the presence of 0.5 mM Ni(II) and 0.5 (9), 1 (2), and 2 (b) mM L-His. Single runs are shown for simplicity.

inspection of the nonlinear kinetic curves indicated that deceleration of the reaction rates did not conform to any regular rate law. This evolving reaction kinetics suggests the presence of product inhibition. The effect of low concentrations of added GSH on GSNO stability in the absence and presence of Ni(II) was also studied, with the kinetic curves presented in Figure 10. In the absence of Ni(II), the reaction progressed linearly, although faster than without added GSH. In the presence of Ni(II), the initial fast phase was seen, followed by a linear phase, with the momentary rate very similar to that recorded in the absence of Ni(II). Table 5 presents the averages of pseudo-0th rate constants, which represent these momentary reaction rates, for all kinetic experiments. The values for the nonlinear curves were obtained asymptotically at the start of the reaction and near the end of its monitoring period. Figure 11 demonstrates capillary electropherograms of the substrate solution of 1 mM

Figure 10. Kinetics of decay of 1 mM GSNO in 50 mM HEPES (pH 7.4), at 25 °C, monitored at 330 nm, in the presence of 0 (9), 20 (2), and 50 (b) µM added GSH and 0.5 mM Ni(II), together with 0 (0), 20 (4), and 50 (O) µM added GSH. Single runs are shown for simplicity.

GSNO, of this solution with 1 mM Ni(II) added, immediately and 6 h after the addition of Ni(II), and of a solution of GSSG. These measurements confirmed GSSG as the sole major product of GSNO decay, with a continuous presence of low amounts of GSH.

Discussion Acid-Base Properties of GSNO. Protonation constants of GSNO, obtained by potentiometry (Table 1), can be assigned to individual protons by comparison with group constants, derived from chemical shifts of individual GSNO protons (Table 2). These comparisons clearly assign the lowest pKa value to the Glu carboxylate, the next one to the Gly carboxylate, and the alkaline constant to the amine, in full accord with expectations, based upon comparisons with GSH and GSSG. These

398


Figure 11. Capillary electropherograms of 1 mM GSNO in 50 mM HEPES (pH 7.4) before the addition of 0.5 mM Ni(II) (A), immediately after the addition of 0.5 mM Ni(II) (B), and following a 6 h incubation with 1 mM Ni(II) at 25 °C (C). Panel D presents the control of 0.25 mM GSSG in 50 mM HEPES (pH 7.4). Assignment of peaks: 1, GSNO; 2, GSH; 3, unidentified impurity in HEPES buffer; and 4, GSSG.

constants can also be compared quantitatively to those of GSH on one hand and its S-methyl derivative on another. The GSNO carboxyl groups are slightly more basic (0.3-0.4 pH units) than those of GSH, as measured recently in our laboratory under identical experimental conditions (31, 32). The analogous data for S-methylated or any other S-substituted GSH derivatives are not available in the literature. In contrast, the amino group in GSNO is more acidic than those in GSH, pKa of 9.66 (31, 32), and S-methyl-GSH, pKa of 9.38 at I ) 0.4 M (39). The quantitative comparisons with GSSG are less straightforward, because of the presence of statistical factors, which modulate the values of the macroconstants for two chemically equivalent sets of proton binding groups (40). All values determined by us (Table 1) reside within the ranges set by two previous determinations at a similar temperature and ionic strength (41, 42). The separation between the pairs of values for Gly carboxylates and Glu amines in GSSG is ca. 0.8 log units, which corresponds to the statistical separation factor of 0.6 log units plus weak negative cooperativity, expectable for charged residues. Therefore, the average values for comparisons are 3.57 for Gly carboxylate and 9.29 for Glu amine. The amine of GSNO is thus more acidic, and the Gly carboxylate more basic than those of GSSG, as well. The apparent protonation constants, exhibited by individual nondissociable protons of GSNO (Table 2), clearly demonstrate that Cys(NO) residue is sensitive to deprotonations of Gly carboxylate and Glu amine but not to that of Glu carboxylate. This fact, together with the effects on pKa values, indicates the presence of specific interactions within the GSNO molecule, which stabilize the uncharged forms of the Gly carboxyl and Glu amine and involve the SNO moiety. Because of a significant sequential separation of all of these moieties, excluding any through-bond effects (43), this must be a throughspace interaction between the SNO moiety and the


amine. We have recently discovered similar interactions in GSH, which are responsible for a specific susceptibility of the amine-protonated GSH to thiol oxidation (32, 44). These interactions have been interpreted in terms of a preferred solution conformation of GSH, resembling the shape of the letter E (44). A more in-depth study of structural effects in GSNO is required, but the results presented here tend to support a similar conformation for GSNO. GSNO Complexes with Zn(II) and Cd(II). As demonstrated by NMR (Figures 1-3), GSNO coordinates Zn(II) and Cd(II) predominantly with its glutamic acid {N,O} donor set. This fact is confirmed by the potentiometrically derived stoichiometries of ML and ML22- for the complexes present at the neutral pH range. The stability constants, presented in Table 3, together with the species distribution plots in Figure 4, demonstrate that the complexes formed are rather weak, and those of Cd(II) are weaker than those of Zn(II). A comparison of log βML and log βML2 values for GSNO with those for amino acids Ala, Ser, and Leu, which share the {N,O} donor set and noncoordinating side chains, confirms a close quantitative match among all of the respective log β values, with Zn(II) complexes significantly stronger than those of Cd(II) (45, 46). However, the NMR spectra indicate that R and β protons of the Cys(NO) residue also sense Zn(II) and Cd(II) coordination, in parallel with the Glu protons. The magnitude of this effect, in the terms of ∆δ values, is similar to that described above for metalfree GSNO (see Figures 2 and 3) and is thus likely to have the same nature. A formation of a direct bond between the SNO moiety and the Zn(II) or Cd(II) is rather out of question because there are no effects of Zn(II) or Cd(II) in absorption spectra of GSNO (data not shown). Weak interactions through, e.g., bridging water molecules, cannot be excluded, but there is no evidence for them in our results. Scheme 2 presents a ML22- complex. The ML22- complex is superseded by MH-1L23- and MH-2L24- species with pKa values of 8.7 and 9.9 for Zn(II) and 10.2 and 10.2 for Cd(II), respectively. The NMR titrations indicate that the formation of these new complexes is associated with the gradual shift of δ toward the values for metal-free GSNO. The above pKa values exhibit an excellent match with the published values for the formation of Zn(II) and Cd(II) hydroxides (47, 48) and for the formation of ternary hydroxocomplexes of Zn(II) (49). Therefore, the formation of MH-1L23- and MH-2L24species is due to alkaline hydrolysis. Binary Ni(II) Complexes. Binary Ni(II) complexes with GSNO, NiL, and NiL22- (Table 3) are somewhat more stable than the corresponding ones for Zn(II) and Cd(II). The stability constants of these complexes match the quantitative trend, set forth by previous studies of Ni(II) complexes with Ala, Ser, and Leu, very well (45, 46, 50). The pKa values of 10.6 and 10.8 indicate also that the NiH-1L23- and NiH-2L24- complexes are the products of partial or complete hydrolysis (51). The UV-vis spectra of Ni(II) complexes of GSNO at pH 7.4 (Table 4 and Figure 6), where NiL and NiL22- complexes prevail (Figure 5A), demonstrate that the coordination of Ni(II) has no effect on SNO electronic transitions, in particular those at 515-545 nm, which are very sensitive to the electronic density on the nitrogen of the SNO moiety (52). The same was the case for Zn(II) and Cd(II). This factpoints against an involvement of the SNO moiety in the Ni(II) binding. The spectra of all complexes studied



Scheme 2a

pH 7.4 occurs via one or two {N,O} donor sets in all nonhistidine complexes and via one or two {N,N,O} donor sets in L-His complexes. It should be noted that the same kind of coordination was proposed for the Cu(II) complex of GSNO on the basis of frozen solution EPR spectra (56). The binary systems Ni(II)/GSSG and Ni(II)/L-His were studied before on several occasions. We reproduced potentiometric determinations of these systems in order to obtain input data for the determination of ternary complexes, discussed below. The coordination models and stability constants determined by us are in a very good agreement with the literature values, including our own previous experiments (32, 41, 50). Ternary Ni(II) Complexes. The preliminary analysis of kinetic experiments indicated a presence of product inhibition in the GSNO decay process. To find its molecular basis, potentiometric titrations of a potential ternary systems, with GSNO and GSSG, a product of GSNO decay, were performed. These measurements indeed indicated the formation of ternary complexes of the formula NiHLB3- and NiLB4-, where according to the labeling scheme of Table 3, L is GSNO and B is GSSG. The species distribution plot for these complexes (Figure 5E) demonstrates that the NiHLB3- complex is a major component of the system at neutral pH. Its stoichiometry indicates that one of the amine groups in GSSG is protonated. The pKa of its release, to form NiLB4-, is 7.86, lower than those in free GSSG, average of ca. 9.3, but much higher than that accompanying the formation of the binary complex NiB2- from NiHB-, 5.74. By virtue of stoichiometry, each of GSNO and GSSG must contribute one {N,O} donor set to Ni(II) chelation in NiHLB3-. As expected, the UV-vis spectra did not discern between NiL22-, NiB2-, and NiHLB3- complexes (data not shown). The comparable stability of NiB2- and NiHLB3-, visualized in Figure 5E, suggests that the elevation of the amine pKa in NiHLB3-, as compared to NiHB-, is compensated by interligand interactions in the ternary complex. The formation of NiLB4- likely involves the addition of the second {N,O} donor set of GSSG to Ni(II). The study of the Ni(II)/GSNO/L-His ternary system was prompted by our recent results, indicating that L-His may be a major intracellular chelator for Ni(II) (32). Only one ternary complex, of NiLA3- stoichiometry, was detected. This stoichiometry was easily predictable, on assumption of the conservation of donor sets present in parent binary NiL and NiA+ complexes, {N,O} + {N,N,O}. NiLA- is a relevant complex at physiological pH, in equilibrium with binary L-His complexes (Figure 5F). Its stability can be expressed with the equilibrium constant Ke of the competition reaction:

a (A) Structure of a M(GSNO) 4- Complex Where M ) Zn(II), 2 Cd(II), or Ni(II). (B) Structure of the Ternary Complex of GSNO with Ni(II) and L-His, [Ni(L-His)(GSNO)]2-.

here are typical of a high-spin, pseudo-octahedral Ni(II). The positions of d-d bands in such complexes exhibit a rough dependence on the number of nitrogen vs oxygen donors in their coordination sphere, according to a general tendency dictated by the spectrochemical series, i.e., the more nitrogens, the higher transition energies (53). The most characteristic band of these spectra, at 350-400 nm, is obscured in GSNO by much more intense SNO transitions. However, the parameters of the lower energy bands, presented in Table 4, can be also analyzed in this respect. The d-d bands of the NiL complex of GSNO and of the analogous complex of L-Ala, with one nitrogen donor bonded to Ni(II) (1N), are centered at 646-647 nm, while those of their NiL2 complexes, 2N, are centered at 638-642 nm. The NiB2- complex of GSSG, which is expected to have a 2N coordination mode with two Glu donor sets bonded to Ni(II), as a macrochelate species, has this band also at 642 nm. Note that the macrochelate formation in GSSG is supported by the high stability of this complex (Table 3) and by conclusions of previous studies of this system (41, 54). The NiA+ and NiA2 complexes of L-His are of 2N and 4N type, respectively (55). Their middle d-d bands are at 608 and 555 nm, respectively. The discrepancy between band positions of 2N species of L-His an the other ligands is due to a different character of its imidazole nitrogen donor. Altogether, one can safely state that the binding of Ni(II) at

NiL + NiA2 ) NiLA- + NiA+ Log Ke equals 1.03 (5). The logarithmic statistical factor, priviledging the addition of a bidentate L2- to NiA+ vs a tridentate A-, is only log 3 ) 0.48. Therefore, a positive interligand interaction takes place in NiLAas well. The d-d part of the UV-vis spectrum of the solution, containing this complex at pH 7.4 (trace 4 in Figure 6), is difficult to analyze, due to overlapping bands, but the lower energy d-d bands could be tentatively discerned upon subtracting the metal-free GSNO spectrum. The position of the 3A2g f 3T1g(F) band maximum at 595 nm cannot be used as a definitive proof

400


of the ternary complex formation, because it corresponds to the computed weighed average of positions of bands of binary complexes, 600 nm. The confirmation of the formation of a ternary complex is, however, provided indirectly by quantitative analysis of kinetic experiments, below. The structural aspects of formation of ternary Ni(II) complexes of GSNO provide an interesting issue for a separate study. GSNO Decay and Its Inhibition by Zn(II) and Cd(II). One millimolar samples of GSNO in 50 mM HEPES buffer at pH 7.4 decayed very slowly but reproducibly, at a constant rate (Figure 7), which corresponds to a decrease of GSNO concentration by ca. 0.2% per hour. First order rate laws have been reported for many nitrosothiols, including GSNO, under a variety of conditions (19, 25, 26, 57, 58). A 0th order rate law, implied by the apparently linear kinetics of decay, is in fact very difficult to discern from a 1st order rate law at such a low rate. The alternative fitting of GSNO decay to a 1st order rate law is in fact possible but requires an assumption on the reaction endpoint. An assumption of reaction completeness yielded a value of k1 ) 5.19 (3) × 10-7 s-1, with t1/2 of 370 h. When fitting was performed with [GSNO] at equilibrium floating freely, the best fit was k1 ) 3.68 (2) × 10-6 s-1 at merely 14.75% of reaction completion, with t1/2 ) 52.5 h. An uncertainty of this magnitude prompted us to present the apparently linear kinetic processes using pseudo-0th order rate constants. The 1st order rate of decay of 1 mM GSNO, reported in the literature, is ca. 2 × 10-5 s-1 (26), substantially higher than any of our estimates; the t1/2 > 10 h was also reported (57). Both are based on experiments in phosphate buffers at pH 7.4. A likely explanation for the discrepancy in rates is provided by results of many studies, which have indicated catalysis by Cu(I)/Fe(II) as a major source of nitrosothiol instability in solution (1823, 25, 26). It is very probable that contamination of phosphate salts with copper and particularly iron ions is much higher than that of a synthetic organic compound, such as HEPES. The presence of equimolar Zn(II) reduced GSNO decay rate by 50%. The stabilizing effect of Cd(II) coordination was less pronounced, at ca. 16%. The magnitudes of these effects correlate to some extent with the amounts of uncomplexed GSNO under the reaction conditions, as provided in Table 5. A simple interpretation of this decay inhibition in terms of GSNO complex equilibria would, however, be false, because the extent of protection is ca. two times higher than the extent of GSNO complexation. The binding constants of cupric/cuprous or ferric/ferrous complexes of GSNO have not been established, due to their kinetic instability. Instead, the stability constants for complexes of simple amino acids, such as L-Ala, can be discussed, as justified by the discussion of coordination modes above. These indicate that Zn(II) and Cd(II) complexes of GSNO might provide partial protection against Fe(II) (59) but are unlikely to compete with any of Cu(II), Cu(I), and Fe(III) (45, 60, 61). Two further, more direct arguments for this opinion are provided by high reactivity of Cu(I) against GSNO at a pH as low as 3, which indicates high complex stability (21), and by the essential lack of effect of coreleased Zn(II) on Cu(II)related decomposition of GSNO with H2O2-treated superoxide dismutase (56). Therefore, by virtue of chemical equilibrium, the process of decomposition of a Zn(II) and/ or Cd(II) complex of GSNO would proceed only margin-


ally slower, because the complex formation with the above adventitious ions is faster by many orders of magnitude than any GSNO decay process (21). An alternative explanation is a sequestration of GSH by Zn(II) and/or Cd(II). GSH is always present in GSNO solutions, due to the reversibility of GSNO formation (18, 62). This notion is also confirmed by our CE measurements, detecting GSH constantly at ca. 50 µM, or 0.5% of GSNO, during the reaction course (peak 2 in Figure 11). GSH, in its thiol-deprotonated form, also participates in the proposed molecular processes of GSNO decay, both metal-free (63) and copper/iron-assisted ones (20-23, 25, 64, 65). The binding constants for GSH complex formation with both Zn(II) and Cd(II) are much higher than those with GSNO, and moreover, the thiolate function of GSH is the primary metal binding site in both cases (31, 39). A consequence of partial formation of GSNO complexes under the conditions of kinetic experiments is also the availability of free Zn2+ or Cd2+, in excess over the trace of GSH, such that GSH complexation is near 100%. This coordination provides a likely interference in the thiolate-assisted GSNO decomposition pathway. It should be noted that Zn(II) effect on GSNO stability was tested previously and described as nonexistent (19). Cd(II) was not studied before. GSNO Decay Acceleration by Ni(II) and Effects of Competing Ligands. Ni(II) effect on GSNO stability was tested previously as well, and similarly to Zn(II), it was concluded to be none (19). Our experiments clearly demonstrated the contrary. The conditions of previous screening experiments with Ni(II) and other potentially reactive metal ions were similar to those appropriate to study catalytic effects of Cu(II), which decomposes GSNO vigorously. Therefore, the slower Ni(II)-assisted processes could have been lost somehow in those experiments. As presented in Figure 8 and Table 5, Ni(II) accelerated GSNO decomposition in a concentration-dependent fashion. As described in the Results section, the asymptotic (momentary) pseudo-0th rates were calculated at the beginning of each reaction and near the end of the 10 h monitoring time. The changing reaction order was indicative of reaction product inhibition, by the way of Ni(II) binding to GSSG, similarly to that found previously for Cu(II) (25, 26, 56, 65). This concept was verified experimentally. An addition of 0.5 mM GSSG to the reaction mixture resulted in a strong inhibition of GSNO decay, as expected. Also, L-His proved to be a strong inhibitor of Ni(II)-dependent GSNO decay, with full reaction quenching, to the level of metal-free GSNO, at 4-fold excess of L-His over Ni(II). We also attempted to assign the GSNO-destructing activity to individual complex species, by calculating species distributions for each momentary pseudo-0th rate and subjecting these data to multiple linear regression. Average kobs values were used, as provided in Table 5. All experiments with Ni(II), including those with added ternary ligands, were pooled for this calculation. The final result of this procedure is presented in Figure 12. The only correlation found was that between the momentary pseudo-0th rate of GSNO decay and the concentration of the NiL complex. The slope of the correlation line for all 12 experimental points (solid line in Figure 12) is 2.9 (2) × 10-5 s-1, and its intercept is 6 (6) × 10-10 M s-1, with a good quality of fit, R ) 0.964. The correlation is markedly improved, with R ) 0.995, if only the data obtained in the absence of GSSG, that is, the initial


Figure 12. Dependence of momentary rates of GSNO decay in the presence of Ni(II) on the momentary concentration of the Ni(GSNO) complex in reaction mixtures. The solid line represents the linear fit to all experimental points. The data from all experiments with Ni(II) are included, as presented in Table 5; b denotes the absence and O denotes the presence of GSSG as reaction product or added sample. The dotted line represents the linear fit to experimental points in the absence of GSSG (b).

velocities of experiments without GSSG added, are used. These data points are represented in Figure 12 with black circles. The parameters of the correlation line for these data points are altered only marginally: slope 3.1 (1) × 10-5 s-1 and intercept 5 (4) × 10-10 M s-1 (dotted line in Figure 12). There is, however, no further correlation between the amount of GSSG or any particular GSSGcontaining species and the reaction velocity. It seems therefore likely that interligand interactions in ternary complexes with GSSG and L-His, as well as those presumably present in the Ni(GSNO)22- molecule, prohibit GSNO decay. The effect of L-His is prohibitory, by means of depletion of the Ni(GSNO) complex. The erratic low level influence of GSSG is difficult to interpret on the basis of our data. The addition of low amounts of GSH to GSNO in the absence of Ni(II) resulted in the decay acceleration, in agreement with the previous report, in which GSH was proposed to be a catalyst of GSNO decomposition (63). The effects of addition of GSH in the presence of Ni(II) were different: the initial acceleration, as compared to the case without GSH addition, was the same with 20 and 50 µM GSH, but the reactions slowed more rapidly, to assume rates virtually identical to those observed in the absence of Ni(II). Such a behavior suggests the formation of a hypothetical “active Ni(II)-based catalyst”, whose formation is not limited by GSH concentration and which decays at a high rate. The analysis of “difference kinetics”, obtained numerically by subtracting the kinetic curves obtained in the absence of Ni(II) from the ones with Ni(II) present, indicates a 1st order process, with the rate of 1.8 (1) × 10-4 s-1 responsible for decomposing 6 and 10% of initial GSNO at 2 and 5% of added GSH, respectively. Furthermore, while Ni(GSNO) is indicated as an active species, its electronic spectrum provides no evidence for an interaction between the Ni(II) ion and the SNO moiety. The Ni(II) ion in this complex is high-spin, pseudo-octahedral. Such complexes do not undergo oneelectron reductions or oxidations (66). Therefore, the mechanism of Ni(II)-dependent activation of the SNO moiety cannot be direct, such as the one established for Cu(II)/Cu(I), with Cu(I) as the active catalyst (20, 21).


A concept, which is not conflicting with any of the above limitations, includes a formation of a hypothetical low-spin ternary complex, resulting from an addition of one or even two GSH molecules to the coordinatively unsaturated Ni(GSNO), and present at a very low concentration, below 0.1% of the total Ni(II), so that it is not detectable by UV-vis. The formation of such a complex at constant, low GSH will indeed be proportional to the concentration of its parent Ni(GSNO) complex, as seen in Figure 12. However, any direct detection of such a complex will, in general, be difficult, due to its kinetic instability. The further steps of Ni(II)-dependent GSSG formation from GSNO remain to be elucidated, but its obvious complexity is in line with the mechanism of GSH assault on GSNO, proposed by Tannenbaum and coworkers (63).

Conclusion The studies reported above provided the first experimental evidence for the stabilization of GSNO in solution by a metal ion. So far, only acceleration of GSNO decay by metals was reported. This interaction may provide an interesting additional level of control in vivo, e.g., in the process of conversion of NO signal to zinc signal in neurons upon the SNO assault on metallothionein (67). With regard to Ni(II), its ability to decompose GSNO may lead to an impairment of cellular redox signaling, parallel to an ability to deplete cellular stores of GSH (68), thus constituting a possible novel epigenetic mechanism in nickel carcinogenesis, which is worth pursuing further.

Acknowledgment. This work was sponsored by the Polish State Committee for Scientific Research (KBN), Grant No. 4 T09A 030 22.

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