©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
S-Nitrosation of Serum Albumin by Dinitrosyl-Iron Complex (*)

(Received for publication, June 12, 1995; and in revised form, August 15, 1995)

Matthias Boese Peter I. Mordvintcev (§) Anatoly F. Vanin (§) Rudi Busse Alexander Mülsch (¶)

From the Center of Physiology, Johann-Wolfgang-Goethe University Clinic, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)(2)Fe(L-cysteine)(2)], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)(2)Fe(L-cysteine)(2) generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NO(x)-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)(2)Fe(L-cysteine)(2) can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.


INTRODUCTION

S-Nitrosation of protein thiols by L-arginine- and nitrovasodilator-derived NO and/or subsequent further reaction of the S-nitrosothiol has been proposed to initiate widespread biological signal and effector pathways(1, 2, 3) . Direct evidence has been provided that a small fraction of circulating serum albumin in human and dogs is S-nitrosated under physiological conditions(4) . Furthermore, indirect evidence suggests that S-nitrosation modulates the function of proteases, cytoskeletal proteins, membrane receptors(5) , membrane ion channels(6) , GTP-binding proteins(7) , protein kinases(8) , phosphotyrosine protein phosphatases(9) , transcription factors(10, 11) , and glutathione reductase(12) . However, the mechanism by which proteins are S-nitrosated in vivo is unknown. It is likely that nitrosation proceedes by electrophilic attack of a nitrosyl-cation (NO) or a partially positively charged NO (NO) on a sufficiently reactive nucleophilic thiol group within target proteins(1) . It has been proposed that naturally occurring metabolites of NO, such as peroxynitrite (ONOO), nitrogen dioxide (NO(2)), or dinitrogentrioxide (N(2)O(3)), account for the S-nitrosation(1, 2) . However, ONOO was shown to oxidize thiols primarily by a mechanism not involving an S-nitrosothiol as an intermediate(13, 14) . Furthermore, it is not known whether the steady state levels of NO(2) and N(2)O(3) attained in NO generating tissues are sufficiently high to account for biological S-nitrosation. It is therefore possible that other endogenous nitrosating species are operative. Since artificial metal-nitrosyls, like sodium nitroprusside, perform nitrosation of nucleophils (15) we speculated that endogenous iron-nitrosyl complexes might do so as well(16) . These comprise so called dinitrosyl-iron complexes, which are generated by various cells and tissues concomitantly with NO(17, 18, 19, 20, 21, 22, 23, 24) . The paramagnetic dinitrosyl-iron moiety is attached to as yet unidentified proteins(25, 26) , primarily via protein thiols and histidines, as judged from the characteristics of the EPR signals(21, 22, 27, 28, 29) . With regard to a ligand-exchange equilibrium between low mass thiol and protein thiol ligands(13, 21, 22, 30) , it is conceivable that low mass dinitrosyl-iron complexes exist biologically, albeit at low steady state levels. For instance, the addition of cell membrane-permeable low mass thiols effects intracellular mobilization and transmembraneous release of the dinitrosyl-iron moiety from intracellular proteins of NO-generating cells(21, 22, 30, 31) . In oxygenated aqueous media the biological half-life of the low molecular forms is inversely proportional to their concentration, amounting to a few seconds below 1 µM, and to about 10 min at 100 µM, for instance(31) . In contrast, proteinacious dinitrosyl-iron complexes are stable for hours in the absence of low mass thiols.

According to theoretical considerations, the dinitrosyl-iron complex and related di- and tetranucleated structures bear a positive charge on the NO(29, 32) . The overall charge of the complex with two thiolate ligands is -1(32) , if the charge of the thiolate residues is zero or neutralized (at isosbestic pH of L-cysteine, for instance). Thus, its paramagnetism is best explained by assuming a d^7 electron configuration at the central iron ion (effective spin system S = 1/2). However, the formal oxidation state of the iron will be -1 if the NO ligands take pure nitrosyl character (NO). In reality, some back donation of electron density to NO might decrease its electrophilicity, which then carries only a partial positive charge (+), similar to the situation with mononitrosyl-metal complexes (33) . Thus, this complex can be regarded as a natural source of NO or NO, capable of S-nitrosation reactions according to Fig. Z1(RS = low mass thiol; R`SH = low mass or protein thiol). To test this hypothesis we assessed S-nitrosation of the free cysteine residue of bovine serum albumin (BSA) (^1)by the dinitrosyl-iron-di-L-cysteine complex. We observed that this complex S-nitrosates albumin, in support of our hypothesis that dinitrosyl-iron complexes are intermediates in the biological NO:S-nitrosothiol pathway.


Figure Z1: Structure 1




EXPERIMENTAL PROCEDURES

Materials

Bromophenol blue, fatty acid-free BSA (96-99% pure), L-cysteine, diethylpyrocarbonate (DEPC), EDTA sodium salt, glutathione, N-ethylmaleimide, Sephadex G-25 were supplied by Sigma, Deisenhofen, Germany. Xylene cyanole was from Roth, Karlsruhe, Germany. NO gas was obtained by reaction of FeSO(4) times 7H(2)O (Fluka, Buchs, Switzerland) with NaNO(2) in 5 N HCl and was purified by low temperature high vacuum (p = 0.01 mm Hg) distillation (31) .

Dinitrosyl-iron complex was prepared by mixing evacuated (10 min high vacuum) solutions of FeSO(4) (5 mg/ml) and neutralized L-cysteine (72 mM) in a Thunberg-type reaction vessel under an atmosphere of pure NO gas (P 500 mm Hg; the NO was added 3 min before mixing). Upon mixing the solution immediately turned green. After 1 min the solution was evacuated for 2 min by high vacuum to remove excessive NO, immediately frozen, and stored in liquid nitrogen. The molar ratio of Fe to L-cysteine was 1:20.

S-Nitroso-L-cysteine and S-nitroso-BSA were synthetized by mixing at 20 °C either L-cysteine (100 mM) or fatty acid-free BSA (2 mM) with an equimolar amount of sodium nitrite in 0.5 M H(2)SO(4) for 5 min. The S-nitrosothiols were frozen and stored at -70 °C until use. The yields of S-nitroso-L-cysteine and S-nitroso-BSA were 90 and 100% with respect to free thiol added (BSA-thiol/BSA = 0.4 ± 0.04 mol/mol), as calculated from the typical S-nitrosothiol absorbance spectra, using the reported molar absorption coefficients: S-nitroso-L-cysteine, = 940 and = 22 M cm (MeOH; 34); and S-nitroso-BSA, = 870 (H(2)O; 35).

Analytical Methods

BSA-thiols were titrated by 5,5`-dithiobis(2-nitrobenzoic acid)(36) , using the absorbance of the thionitrobenzoate-anion at 412 nm ( = 1.36 times 10^4M cm) as a quantitative measure of thiol concentration. Protein was determined by the Bio-Rad method, using BSA as a standard. UV visible spectra were recorded on a Kontron 941 Plus spectrometer.

Determination of S-Nitrosothiols, Nitrite, and Nitrate

S-Nitrosothiols were assessed by diazotization of beta-naphthol and azo-coupling with N,N-ethylenediamine in the presence and absence of Hg ions (3 mM) according to Saville(37) . Hg releases NO from S-nitrosothiols(37) , which under acid conditions gives a positive Griess reaction, similar to acidified nitrite. The concentration of the red azo-compound was determined after 15 min by measuring the absorption at 570 nm on a microplate reader (MR 600, Dynatech, Alexandria, VA). In each experiment S-nitrosothiol/nitrite were determined in quadruplicate. The content of S-nitrosothiol accounted for the mean difference of absorption readings of Hg-containing versus Hg-free samples. A calibration curve was established in each experiment with freshly synthesized S-nitroso-L-cysteine and sodium nitrite as standards (1-100 µM). The detection limit was 1 µM. In some experiments the mixture of BSA, DNICs and the Griess reagent was frozen at specific time points and analyzed by EPR spectroscopy. Nitrate was determined after its reduction to nitrite by means of a cadmium reductor (38) and then assessed by the Griess reaction as described above.

Electrophoresis

Electrophoresis of DNIC was performed at 5 °C in a horizontal 1% (w/v) agarose slab gel (20 mM KH(2)PO(4) buffer, pH 7.5 or 6.5). 70 mML-cysteine was included in the gel buffer to prevent decomposition of DNIC. Freshly thawed stock solutions of the complex (40 µl) were loaded in slots at equidistant positions from the electrodes in the middle of the gel. The anionic dyes bromphenol blue and xylene cyanole were loaded in separate slots. Electrophoresis was started immediately at 50 V. Electrophoretic mobility relative to the anionic dyes was estimated from the migration of the green band of DNIC after 1 h. Thereafter the band corresponding to DNIC was excised, frozen, and analyzed by EPR spectroscopy.

EPR Spectroscopy

EPR spectra were recorded on a Bruker EPR 300E spectrometer at about 80 K with frozen aqueous solutions (0.6 ml) introduced into a quartz Dewar (5-mm inner diameter) chilled with liquid nitrogen. Some samples (25 µl of aqueous solution) were also recorded at 293 K in a quartz capillary tube (1 mm inner diameter). The EPR instrument was operated at a microwave frequency of 9.60 GHz, microwave power 20 mW, modulation frequency 100 kHz, modulation amplitude 5 Gauss, time constant 0.1-1.3 s. The concentration of DNIC was calculated by comparison with the EPR signal of a DNIC-L-cysteine standard based on double integration of the first derivative EPR signals(31) .

Covalent Modification of Serum Albumin

The single thiol group accessible on BSA was blocked by incubation of the protein (2 mM in 0.1 M potassium P(i) buffer, pH 7.4) for 5 min at 20 °C with either 20 mMN-ethylmaleimide, or for 30 min with 6 mM HgCl(2)(13) . Modification of histidine residues was performed by adding a 10-fold molar excess of DEPC and subsequent incubation for 2 h at 20 °C(39) . In control experiments it was verified that DEPC did not alter the EPR spectrum of low mass DNIC, indicating that both compounds did not directly interact.


RESULTS

Physicochemical Characterization of Low Mass DNIC

In frozen state at 77 K, DNIC exhibited an anisotropic EPR signal of axial symmetry with g factors g = 2.04 and g&cjs0952; = 2.01 (Fig. 1a)(28, 29, 30, 31) . In liquid phase at 20 °C this spectrum was transformed into a narrow isotropic signal at g = 2.03 with a 13 line hyperfine structure (Fig. 1e). The isotropy results from the high tumbling rate of the small paramagnetic molecule in liquid phase, which averages out any anisotropy of g values. The small coupling constant (A = 0.07 millitesla) and the absence of further fine structures points to a low spin configuration (S = 1/2). The unpaired electron is mainly localized on the iron atom(27, 29, 32) . The hyperfine splitting results from the interaction of the unpaired electron with four protons of the methylene groups of both L-cysteines and two nitrogen nuclei of the NO groups. Similar EPR spectra were obtained if DNIC was synthesized with glutathione or N-acetyl-L-cysteine instead of L-cysteine (data not shown).


Figure 1: Representative EPR spectra of dinitrosyl-iron complexes. ESR spectra were recorded at 77 K, modulation amplitude 5 Gauss (a-d), and at 295 K, 0.5 Gauss (e) as described under ``Experimental Procedures.'' The position of the g factors is indicated by arrows. Note that spectra a-d were recorded at 4-fold higher magnetic field sweep than spectrum e, as indicated by the calibration bars (4 and 1 millitesla). a, DNIC-L-cysteine (25 µM); b, desalted DNIC-BSA (30 µM); c, BSA-DNIC (30 µM) from N-ethylmaleimide-treated BSA; d, DNIC (40 µM) in Griess reagent; e, DNIC-L-cysteine (200 µM) recorded at 295 K. For assignment of structures see ``Results.''



In agarose gel electrophoresis conducted at pH 6.5 (the isoelectric pH of L-cysteine) DNIC moved toward the anode, indicating that DNIC carries a net negative charge. The electrophoretic mobility of the complex was 0.38 times that of the anionic dyes bromphenol blue and 0.85 times that of xylene cyanol, which migrated 2.9 and 1.3 cm/h, respectively. The electrophoretic mobility of DNIC increased at pH 7.5, presumably due to increased negative charge on the L-cysteine-carboxylates. EPR spectroscopic analysis of the gel band bearing DNIC revealed the pure EPR spectrum of DNIC. Therefore, DNIC seems to migrate in the electric field as an intact complex anion.

To assess its decomposition kinetics, DNIC was incubated at 37 °C for defined periods and was then quickly frozen for cryogenic EPR analysis. The rate of decomposition of DNIC, as estimated from the decrease in the EPR signal intensity, was inversely correlated to the initial concentration of DNIC (Fig. 2).


Figure 2: Concentration-dependent decay of DNIC assessed by EPR spectroscopy. DNIC-L-cysteine (, 400 µM; , 100 µM; bullet, 20 µM) dissolved in 100 mM KH(2)PO(4) buffer (pH 7.4) was incubated at 37 °C for the indicated periods of time (x axis) and then quickly frozen to assess the concentration of paramagnetic DNIC by EPR analysis. Means + S.E. (error bars) of three experiments.



Interaction of DNIC with BSA

DNIC reacted immediately with BSA to form a paramagnetic protein-bound dinitrosyl-iron complex, amounting to about 0.4 mol of DNIC bound per mol of BSA. In the frozen state this BSA-DNIC exhibited an EPR spectrum identical to that of low mass DNIC (Fig. 1a). The macromolecular nature of the paramagnetic species was confirmed by showing that, in contrast to the EPR signal of low mass DNIC (Fig. 1e), the anisotropy of the BSA-DNIC EPR signal did not change in liquid phase at room temperature (data not shown). This preservation of anisotropy in liquid media is due to the slow rotation rate of paramagnetic macromolecules. According to its characteristic EPR features the dinitrosyl-iron moiety must be attached to two nuclear equivalent ligands(25, 26, 27, 28, 29, 30) . In the case of BSA-DNIC, these ligands are L-cysteines, one of which is the single reduced cysteinyl-thiol available on native BSA (cysteine 34), the other is provided by free L-cysteine. The apparently subequimolar recovery of BSA-DNIC (0.4 mol/mol BSA) resulted from the limited availability of cysteine 34, which is masked by disulfide formation with glutathione, L-cysteine, and BSA dimer formation in commercial BSA preparations(40) . This was confirmed by titration of BSA with 5,5`-dithiobis(2-nitrobenzoic acid).

When BSA-DNIC was passed over a desalting column (Sephadex G-25) to remove excessive L-cysteine (DNIC was synthesized with 20 mol of L-cysteine per mol of iron, although only two L-cysteines are directly included into the complex), the EPR signal of BSA-DNIC changed to rhombic symmetry with g factors g(1) = 2.05, g(2) = 2.04, and g(3) = 2.01 (Fig. 1b). These EPR features were reminiscent of dinitrosyl-iron complexes with one non-thiol ligand, presumably a histidine-imidazole, as described previously(28, 29, 30) .

If the free thiol group of BSA was blocked by N-ethylmaleimide, or the preformed BSA-DNIC was treated with HgCl(2), another rhombic EPR signal was generated, with g factor values g(1) = 2.055, g(2) = 2.035, and g(3) = 2.01 (Fig. 1c). These EPR features were similar to that of imidazole-ligated DNIC(28, 29, 30) . Thus, it can be inferred that the dinitrosyl-iron moiety was attached to the thiol-blocked BSA via histidine nitrogen atoms. If, on the other hand, the histidine-imidazole residues of BSA were carboxylated by DEPC, formation of BSA-DNIC was completely abolished (data not shown). This finding suggests that the carboxylated histidine-imidazole restricts access of DNIC to the free thiol group of BSA and is compatible with structures of the diverse BSA-DNIC isoforms as shown in Fig. Z2.


Figure Z2: Structure 2



Reaction of DNIC in Griess Reagent

In the presence of acid (0.5 N HCl), DNIC and BSA-DNIC rapidly transformed into a paramagnetic complex of rhombic symmetry with g factor values g(1) = 2.06, g(2) = 2.05, and g(3) = 2.02 (Fig. 1d). Formation of this complex was not affected by the Griess reagent. The ``acid'' complex formed by 40 µM DNIC decayed by apparent first order kinetics with a half-life of 20.4 ± 1.3 min (n = 8). HgCl(2) did not affect the formation, stability, and EPR features of this complex (data not shown).

To identify NO(x) from decomposed DNIC the latter was incubated at 37 °C in 100 mM potassium P(i) (pH 7.4) for defined periods and then assessed for nitrate, nitrite, and S-nitrosothiol (see ``Experimental Procedures''). DNIC (20 µM in iron) generated 30 ± 2 µM NO(2) after 3 min and 40 ± 1 µM after 30 min of incubation (n = 3). A freshly thawed stock solution of DNIC (3.6 mM) contained less than 0.5% S-nitrosothiol positive material, which waned within 3 min. Nitrate was not detectable at any time. These findings demonstrate that DNIC generates exclusively NO(2) as a stable NO(x) metabolite.

S-Nitrosation of BSA by DNIC

DNIC (0.75 mM) and BSA (1 mM) at 37 °C rapidly and transiently generated an S-nitrosothiol, with a maximal concentration (85 ± 12 µM; n = 5) achieved at 5-8 min after mixing both reactants (Fig. 3). A similar transient S-nitrosation (maximally 40 ± 8 µMS-nitrosothiol) was observed after mixing DNIC (0.75 mM) with glutathione (20 mM).


Figure 3: Time course of S-nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated in 100 mM potassium phosphate (pH 7.4) with 0.75 mM DNIC. At the indicated times a sample was withdrawn to determine the concentration of S-nitrosothiol by the Saville/Griess reaction. Means + S.E. of five experiments.



To study the influence of L-cysteine introduced with the DNIC stock solution on S-nitrosation of BSA, BSA-DNIC was generated as described before and then desalted by means of a Sephadex G-25 column (1.6 times 8 cm; 0.1 M potassium P(i) buffer, pH 7.0). All manipulations were conducted at 4 °C to decelerate the S-nitrosation reaction, although some S-nitrosation during the procedure could not be avoided. The protein fraction was quickly aliquoted, one aliquot was taken for EPR analysis to confirm the presence of BSA-DNIC, the other aliquot was halved and incubated in the presence and absence of 20 mML-cysteine. A low amount of S-nitrosoprotein co-eluted from the column (t = 0 min value) with BSA-DNIC, but did not further increase upon incubation at 37 °C in the absence of L-cysteine (open symbols; Fig. 4). In contrast, the subsequent addition of L-cysteine largely enhanced formation of S-nitrosothiol by a time course similar to that shown in Fig. 3(closed symbols; Fig. 4).


Figure 4: Excess free L-cysteine is required for S-nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated for 5 min at 4 °C with DNIC (0.75 mM) and desalted at 4 °C by a Sephadex G-25 column. The time course of S-nitrosothiol formation by DNIC-BSA was then assessed at 37 °C in the absence (circle) and presence (bullet) of 20 mML-cysteine. Means + S.E. of three experiments.



Identity of the S-Nitrosation Product

To ascertain that BSA and not a low mass thiol was S-nitrosated by DNIC, a preincubated (5 min, 37 °C) mixture (0.8 ml) of BSA (1 mM) and DNIC (0.4 mM) was fractionated at 4 °C into protein and low mass constituents by the aforementioned desalting column technique. Individual fractions (1 ml) were immediately assessed for S-nitrosothiol and protein. As shown in Fig. 5, the S-nitrosothiol eluted mainly with the protein fraction (27 ± 3% recovery with respect to DNIC), and only a minor part was associated with the low mass (salt) fraction (5 ± 2% recovery).


Figure 5: Separation of S-nitroso-BSA and low mass S-nitrosothiols derived from DNIC by gel chromatography. BSA (0.4 mM free thiol) was incubated with DNIC (0.4 mM) for 5 min at 37 °C. Subsequently S-nitroso-BSA was separated from S-nitroso-L-cysteine by chromatography on Sephadex G-25. Individual fractions (#) of the column eluate were tested for S-nitrosothiol (circle) and protein (bullet). Shown is a representative experiment out of three.



Trans-S-nitrosation

Low mass S-nitrosothiols are principally capable of transnitrosation reactions according to the following equilibrium(35, 41, 42) :

On-line formulae not verified for accuracy

To assess how efficiently low mass S-nitrosothiols could accomplish trans-S-nitrosation, BSA (0.5 mM) was incubated for 5 min with S-nitroso-L-cysteine and S-nitrosoglutathione (0.5 mM). This mixture (0.8 ml) was passed through the Sephadex column to analyze the protein and salt fraction for S-nitrosoprotein and low mass S-nitrosothiol, respectively. Significant trans-S-nitrosation of BSA (54 ± 4 nmol; n = 3) was detected with S-nitroso-L-cysteine, accounting for about 13% of the S-nitroso-L-cysteine added. About 26% of the S-nitroso-L-cysteine was recovered in the salt fraction (Fig. 6), and the remainder (about 60%) of S-nitroso-L-cysteine had decomposed to nitrite. In contrast, S-nitrosoglutathione added was recovered entirely in the salt fraction and did not generate measurable amounts of S-nitroso-BSA (data not shown). Addition of 8 mML-cysteine markedly decreased (5 ± 0.6-fold; n = 3) the extent of trans-S-nitrosation of BSA by S-nitroso-L-cysteine.


Figure 6: Trans-S-nitrosation of BSA by S-nitroso-L-cysteine. BSA (0.5 mM free thiol) was incubated for 5 min with an equal amount of S-nitroso-L-cysteine. The protein was separated from the low molecular compounds by gel chromatography on Sephadex G-25 and S-nitrosothiol (circle) and protein (bullet) was measured in individual fractions. This is a representative experiment out of three.



Effect of Blockade of Histidine Residues of BSA on S-Nitrosation

The following experiments aimed to clarify whether or not carboxylation of BSA histidine with DEPC, which leaves the single BSA thiol in a reduced state, affected S-nitrosation of BSA by DNIC. Although formation of BSA-DNIC was reduced by 90%, S-nitrosation of BSA was altered (Fig. 7). These findings show that S-nitrosation of BSA by DNIC does not necessarily depend on previous formation of BSA-DNIC.


Figure 7: Carboxylation of BSA-histidinyl residues by DEPC does not affect S-nitrosation of BSA by DNIC. BSA (1 mM) was carboxylated at histidinyl residues (see ``Experimental Procedures'') and then incubated with DNIC (0.4 mM; 5 min; 37 °C). The protein was desalted by column chromatography. The protein fraction was assessed for protein content (open columns), paramagnetic BSA-DNIC (hatched), and S-nitroso-BSA (cross-hatched). Formation of BSA-DNIC, not formation of S-nitroso-BSA, was reduced. Means + S.E. of three experiments.



Influence of Oxygen on S-Nitrosation of BSA by DNIC

To exclude the possibility that an oxygenated NO(x) species (14) generated by DNIC-derived NO and molecular oxygen accounted for S-nitrosation of BSA, the influence of reduced ambient oxygen tension on this reaction was analyzed. Buffered (100 mM potassium P(i), pH 7.4) solutions of BSA (1 mM) and DNIC (0.4 mM) were evacuated (p < 0.1 mm Hg) separately in a Thunberg flask until bubbling stopped and then mixed for 5 min. The mixture was removed by means of an airtight syringe and loaded on a desalting column, avoiding contact with air. The column was eluted with deoxygenated and nitrogen-saturated buffer and S-nitrosothiol was determined immediately in the eluting protein and salt fractions. The concentration of S-nitroso-BSA achieved in the anaerobic reaction mixture (70.5 ± 17.6 µM) was not significantly different from that obtained in parallel experiments conducted in the presence of ambient oxygen (63.6 ± 7.6 µM; n = 3).


DISCUSSION

The objective of this investigation was to delineate a molecular mechanism accounting for biological S-nitrosation of protein thiols by NO(1, 2, 3) . NO per se is not capable of this reaction and requires electrophilic activation(1, 2, 14) . As a model reaction we assessed the S-nitrosation of BSA by low molecular weight dinitrosyl-iron-di-L-cysteine complex (DNIC), a representative of naturally occurring iron-nitrosyls generated by the L-arginine-NO pathway in mammalian cells(17, 18, 19, 20, 21, 22, 23, 24) . We (16) and others (32) rationalized that these complexes contain ``activated'' (electrophilic) NO. Although physicochemical evidence for the electrophilic character of NO in our DNIC preparation is not available, related complexes studied by Butler and co-workers (32) were shown to have a linear Fe-N-O geometry, indicative of an NO character of the nitrosyl moiety(33) .

Formation of S-Nitroso-BSA by DNIC

The major finding of the present study was that DNIC readily accomplished a transient S-nitrosation of BSA in the presence of excess L-cysteine. Several lines of evidence indicated that S-nitrosation proceeded by an intermolecular reaction of the free DNIC with the protein thiol, rather than by an intramolecular rearrangement of protein-bound DNIC. 1) Removal of excessive L-cysteine from preformed BSA-DNIC prevented further S-nitrosation. 2) The L-cysteine-depleted BSA-DNIC transformed into S-nitroso-BSA after addition of L-cysteine, presumably because low mass thiols released the dinitrosyl-iron moiety from protein complexes by thiol-ligand exchange(16, 30, 31) . 3) The extent of S-nitrosation of BSA was not influenced by covalent modification of the protein with DEPC, although formation of BSA-DNIC was reduced by 90%. This latter finding shows that an intramolecular reaction does not contribute to a significant extent to S-nitrosation of BSA. Instead, S-nitrosation by a nitrosyl moiety and attachment of the iron by thiol-ligand exchange seem to compete for the same accessible thiol group on BSA. Protein-bound DNIC may be essential for S-nitrosation by providing a reservoir for low mass DNIC, according to the mechanism described in point 2 above.

As expected DNIC did not only nitrosate BSA-thiol, but also glutathione, which forms a rather stable S-nitrosothiol(35, 41, 42) . Some S-nitroso-L-cysteine was also generated by DNIC at one-fifth the rate of S-nitroso-BSA (Fig. 6). Thus, S-nitrosation of BSA by S-nitroso-L-cysteine (35, 41, 42) transiently formed from DNIC had to be considered as a potential nitrosation mechanism. However, under identical incubation conditions authentic S-nitroso-L-cysteine was about 3-fold less efficient than DNIC in S-nitrosating BSA ( Fig. 5and Fig. 6). Therefore, with regard to the low amount of S-nitroso-L-cysteine generated by DNIC, a trans-S-nitrosation mechanism appeared unlikely. This mechanism was finally excluded by the demonstration that addition of 8 mML-cysteine to BSA decreased trans-S-nitrosation by S-nitroso-L-cysteine, while S-nitrosation by DNIC increased (Fig. 4). These findings reveal different requirements for S-nitrosation of BSA by DNIC and S-nitroso-L-cysteine.

In principal DNIC could accomplish S-nitrosation by direct attack of coordinated NO on the protein thiol, similar to nitrosation of nucleophils by sodium nitroprusside and other metal-nitrosyls(1, 33) . Alternatively, S-nitrosation could proceed via an unidentified intermediate NO(x) species, similar to that reported to account for S-nitrosation of low mass thiols by NO/O(2) mixtures(14) . However, generation of such an NO(x) by DNIC was unlikely, since S-nitrosation by DNIC was not influenced by oxygen. This is in line with the finding that nitrite was the only stable NO(x) metabolite detected after aerobic decomposition of DNIC. Two routes could lead to formation of nitrite from DNIC: (i) oxygenation of released NO to N(2)O(3) and subsequent hydrolysis and (ii) direct nucleophilic attack of OH on coordinated NO(33) . No matter which of both reactions dominated, the presence of considerable amounts of NO(2), N(2)O(4), or ONOO in the aerobic reaction mixtures is excluded(43) , because these NO(x) would generate nitrate. Furthermore, the lack of any influence of oxygen on S-nitrosation excludes participation of N(2)O(3) possibly generated by pathway (ii). Therefore, a very likely mechanism of S-nitrosation is a direct attack of the electrophilic NO of DNIC on the protein thiol.

Interestingly the diamagnetic non-heme-iron-nitrosyl, bis-methylthio-di-iron-tetranitrosyl (Roussin's red methyl ester), which was detected in pickled vegetables consumed in northern China and is suspect to account for the abundance of oesophageal cancer in this region, has been reported to perform N-nitrosations in vitro and in vivo and to be a co-mutagen of N-nitrosamines(32, 44, 45) . It has since been shown that N-nitrosation by this complex was accomplished by an unidentified oxidation product (46) formed upon standing in the presence of oxygen. The infrared stretching frequency of NO in the fresh and in the oxidized complex was below 1850 cm, which is not compatible with the characteristics of NO(33) as derived from the linear geometry at the Fe-N-O bond revealed by x-ray crystallography (32) . However, further infrared absorbances were exhibited by the oxidized complex assigned to metal-coordinated nitrate or peroxonitrite, an efficient N-nitrosating species(47) . On the other hand, thiols readily transform Roussin's red methyl ester into paramagnetic DNIC(32) , which according to our findings exhibit S-nitrosating activity and might as well explain the N-nitrosation observed in vivo.

Biological Significance of S-Nitrosation by DNIC

In vitro S-nitrosation of proteins can be accomplished by various NO(x)(14) , low mass S-nitrosothiols and NO coordinated to transition metals, including DNIC. Which of these NO species is most relevant for S-nitrosation in vivo and under which conditions remains to be investigated. DNIC with low mass ligands is a favorable candidate, because its precursor protein-DNIC can accumulate in high concentrations in tissues. For instance, in parasite-infected rabbit liver up to 100 µM (!) protein-bound DNIC can be detected, (^2)which is the highest concentration observed so far among the nitrosating species known. We also detected up to 4 µM protein-DNIC in the periphery of human liver tumors.^2 The highest concentration of S-nitrosothiols reported in vivo was 4.4 µM, present in bronchial secretion of pneumonia patients(48) . Although DNIC was not detectable in cellular constituents of the bronchial secretion, these rather stable S-nitrosothiols might have originated from DNIC present in lung tissue.

A judgment on the biological role of other NO(x) (NO(2), N(2)O(4), N(2)O(3), HNO(2), and ONOO) for S-nitrosation is difficult, because there exist no reliable data on the steady state levels of these species in tissues. They may also exhibit biological target profiles different to DNIC. For example, peroxynitrite (49) and NO(2)(50) are capable of nitration of protein tyrosyl residues.

It is necessary to comment on some pecularities of S-nitrosation by DNIC. It is conceivable that the DNIC pathway of S-nitrosation predominates during limited oxygen availability, such as in ischemic tissues, since DNIC does not require oxygen for S-nitrosation. This still has to be shown in vivo.

A more general consideration is that DNIC might be more selective than other NO(x) in nitrosating protein thiols, because DNIC with low mass ligands is a bulky molecule compared to other NO(x). Certainly, the molecular microenvironment of the protein thiol will control the access of and the interaction with the nitrosating species. The ligands attached to DNIC (L-cysteine and glutathione) may serve for target-specific recognition.

In conclusion, we have shown that low mass DNIC S-nitrosates BSA via a mechanism requiring an excess of low mass thiols, but which is independent of trans-S-nitrosation, oxygen, and the formation of protein-bound DNIC. Besides providing a mechanistic route for formation of S-nitroso-BSA in body fluids this reaction appears to be of general importance for covalent post-translational modification of proteins.


FOOTNOTES

*
This work was supported by a doctoral stipend (to M. B.) and a grant from Land Hessen (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Visiting scientist from the Institute of Chemical Physics, Russian Academy of Science, Kosyginstr. 4, 117334 Moscow, Russia.

To whom correspondence should be addressed: Zentrum der Physiologie, Klinikum der Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Tel.: +49(0)69-6301-6995; Fax: +49(0)69-6301-7668.

(^1)
The abbreviations used are: BSA, bovine serum albumin; DEPC, diethylpyrocarbonate; DNIC, dinitrosyl-iron-di-L-cysteine.

(^2)
A. Mülsch, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Ingrid Fleming for expert editorial help.


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