(Received for publication, June 12, 1995; and in revised form, August 15, 1995)
From the
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)Fe(L-cysteine)
], a
compound similar to naturally occurring iron-nitrosyls. Within
5-10 min, (NO)
Fe(L-cysteine)
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
-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)
Fe(L-cysteine)
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.
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
), or
dinitrogentrioxide (N
O
), 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
and
N
O
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 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) (
)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
Dinitrosyl-iron complex was prepared by mixing
evacuated (10 min high vacuum) solutions of FeSO (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 HSO
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
O; 35).
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;
, 20
µM) dissolved in 100 mM KH
PO
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.
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 = 2.05, g
= 2.04, and g
= 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, another rhombic EPR signal
was generated, with g factor values g
= 2.055,
g
= 2.035, and g
= 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
To
identify NO from decomposed DNIC the latter was incubated
at 37 °C in 100 mM potassium P
(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
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
as a stable NO
metabolite.
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 8 cm; 0.1 M potassium P
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 () and
presence (
) of 20 mML-cysteine. Means +
S.E. of three experiments.
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 () and protein (
). Shown is a
representative experiment out of three.
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 () and protein (
) was measured in
individual fractions. This is a representative experiment out of
three.
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.
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) .
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
species, similar to that reported to account for S-nitrosation of low mass thiols by NO/O
mixtures(14) . However, generation of such an NO
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
metabolite detected after aerobic
decomposition of DNIC. Two routes could lead to formation of nitrite
from DNIC: (i) oxygenation of released NO to N
O
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
, N
O
, or
ONOO
in the aerobic reaction mixtures is
excluded(43) , because these NO
would generate
nitrate. Furthermore, the lack of any influence of oxygen on S-nitrosation excludes participation of N
O
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.
A judgment on the biological role of other
NO (NO
, N
O
,
N
O
, HNO
, 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
(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 in nitrosating protein thiols,
because DNIC with low mass ligands is a bulky molecule compared to
other NO
. 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.