(Received for publication, August 31, 1995; and in revised form, November 15, 1995)
From the
Iron regulatory protein (IRP) is a cytosolic bifunctional
[Fe-S] protein which exhibits aconitase activity or binds
iron responsive elements (IREs) in untranslated regions of specific
mRNA. The modulators of these activities are the intracellular
concentration of iron and, as recently described, NO synthase activity.
In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO, the
product of the reaction between NO and
O
), as well as oxygen-derived radicals
(O
and H
O
) and
various NO donors, allow IRP to bind IREs using cytosol extract of
macrophage-like RAW 264.7 cells. Neither the addition of a bolus of
ONOO
or H
O
nor
O
generation significantly affected IRE
binding even though they inhibited its aconitase activity. Moreover, we
show that 3-morpholinosydnonimine (SIN-1), a chemical which releases
both NO and O
, enhanced IRE binding
activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus glutathione (GSH)
activated IRE binding by IRP whereas oxyhemoglobin prevented
enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, an aconitase substrate, also abolished the
effect of SIN-1/SOD on IRE binding by IRP. These results imply that
neither O
nor ONOO
can convert [4Fe-4S] IRP into IRE-binding protein but
rather suggest that an active redox form of NO converts IRP into its
IRE binding form by targeting the [Fe-S] cluster.
Iron regulatory protein (IRP) ()controls iron
homeostasis by regulating the expression of ferritin and transferrin
receptor through binding to iron-responsive elements (IREs) in the 5`-
and 3`-untranslated regions of their respective mRNAs. In iron-depleted
cells, IRP has high affinity for IRE(s) and both stabilizes transferrin
receptor mRNA and prevents ferritin translation. Conversely, in
iron-replenished cells, it functions as an [Fe-S]
cluster-containing aconitase which converts citrate into isocitrate in
the cytosol(1, 2) . These activities are mutually
exclusive, because the fully assembled [4Fe-4S] cluster
required for enzymatic activity at the active site sterically inhibits
IRE binding (2, 3) . We previously reported that
induction of nitric-oxide synthase in activated murine macrophages
results in loss of mitochondrial enzyme
activity(4, 5) . In addition, we showed that
inhibition of mitochondrial aconitase was rapidly
reversible(6) . Subsequently, we and others demonstrated that
NO synthesis increases IRE binding in several cell
types(7, 8, 9) . Conversely, NO synthesis
causes loss of aconitase activity of IRP as shown for mitochondrial
aconitase(7, 9) . Therefore, the functional behavior
of IRP, either aconitase or IRE-binding protein, can be determined by
the intracellular iron concentration (1, 2) and the NO
flux into the cell(7, 8, 9, 10) .
Another line of research showed previously that the activities of
bacterial and mammalian aconitases are inhibited by
O
(11, 12, 13) , whereas the activity of
plant aconitase which exhibits a strong homology with mammalian IRP (14) is inhibited by H
O
(15) .
As loss of the aconitase activity of IRP generally correlates with an
increase in IRE binding(2, 16) , it seems reasonable
to postulate that reactive oxygen intermediates (ROS) enhance IRE
binding of IRP as well. Further, more recent findings support the view
that peroxynitrite (ONOO
), a reactive molecule
derived from NO, is responsible for inhibiting both cytosolic and
mitochondrial aconitases, rather than NO
itself(12, 17) . All these results and considerations
led us to investigate IRE binding by IRP to ascertain whether it is
directly sensitive to O
,
H
O
(ROS), and ONOO
. For this
purpose, we compared the direct effect of these three species on IRP
activities to that of various NO donors on IRP activities. The results
showed that even though ROS and ONOO
inhibited
aconitase activity of IRP, contrary to NO donors, they did not activate
its IRE binding activity.
Aconitases are [Fe-S] enzymes sensitive to
oxidation. Even though aconitase activity of IRP is more stable than
that of mitochondrial aconitase, it tends to decrease progressively
with time when exposed to air. Likewise, IRE binding activity also
responds to the effects of oxidoreduction. Hence, in accordance with
the results of preliminary experiments, we avoided incubating IRP too
long in vitro. Cell lysates were therefore exposed to
additives for 1 h or less before the activities of IRP were determined.
IRE binding activity was analyzed by gel shift experiments, and, in
some experiments, the aconitase activity of IRP was also measured by
spectrophotometry. In our experiments, two bands corresponded to
specific IRE-binding proteins since they were displaced by an excess of
specific IRE probe (not shown): a main band, indicated by an arrow, and
a faint and somewhat elusive faster migrating band. This second IRP
(called IRP or IRP2, to distinguish it from the main band
IRP, designed IRP1) was recently
characterized(23, 24, 25, 26) . This
second IRP has no aconitase activity, and it is still not clear whether
it possesses an [Fe-S] cluster(24) , but, according
to a recent report, it also responds to iron starvation and to NO
synthesis(26) . However, IRP2 is far less stable than IRP1 and,
contrary to the latter, its ability to bind IRE does not result from a
post-translational mechanism(27) . In our system, IRP2
exhibited little activity and did not respond consistently to any
stimulus applied. Therefore, in the results given below, only IRP1,
referred to as IRP, will be considered.
SIN-1 is an NO generator
commonly used in vivo as a vasodilator. It also releases
O which reacts with NO to yield
ONOO
(28, 29, 30) .
Therefore, SOD, an O
scavenger, is
usually added along with SIN-1 to increase the NO half-life. As shown
in Fig. 1A, the combination of SIN-1 plus SOD
significantly increased IRE binding by IRP with time. According to
phosphorimaging quantification, IRE binding activity by IRP from
SIN-1/SOD-treated cytosol reached up to 50% of the full activity
expressed in the presence of 2% 2-ME within 1 h. Concomitantly,
aconitase activity dropped drastically (Fig. 1B). In
another set of experiments, cytosolic extracts were also exposed to
increasing concentrations of SIN-1 (plus SOD) for 30 min. Nitrite and
nitrate, taken as indicators of NO production, were released by 0.5, 1,
5, and 10 mM SIN-1 at constant rates of 1, 1.6, 4.3, and 6
µM/min, respectively. Fig. 2shows that, during
this incubation, IRE binding activity markedly increased with the
amounts of SIN-1 applied. Neither SIN-1C, which is a by-product of
SIN-1 unable to release NO, nor nitrite and nitrate had any effect (not
shown).
Figure 1:
Time course of the
modulation by SIN-1 of aconitase and IRE binding activities of IRP in
RAW 264.7 cell cytosol. Cytosol (20 µg of protein) was treated with
1 mM SIN-1 and 750 units/ml SOD for increasing periods of time
at 37 °C. The reaction was stopped on ice. A, P-labeled human ferritin H-chain IRE probe (60,000 cpm)
was added to an aliquot of 2 µg of protein, and the IRE
IRP
complex was analyzed by gel retardation as described under
``Experimental Procedures.'' Where indicated (bottom
lanes), 2% 2-ME was added before the probe to reveal the full
extent of IRE binding. Sections of autoradiograms of native gel are
shown, and the position of the free probe was cut off from the bottom
of the autoradiogram. The IRE
IRP complex is visualized by an arrow. B, IRE binding activity quantified by
phosphorimaging and expressed as percent of the value obtained with 2%
2-ME. % IRE binding activity, as well as aconitase activity, were
plotted versus the time of cell cytosol exposure to SIN-1/SOD.
Measurement of aconitase activity (100 µg of protein) was based on
the disappearance of cis-aconitate, detected at 240 nm. One
unit is defined as nanomoles of cis-aconitate consumed per
min.
Figure 2: Dose dependence of the enhancement of the IRE binding activity of IRP upon SIN-1 treatment of RAW 264.7 cell cytosol. This cytosol (50 µg of protein) was treated with increasing doses of SIN-1 for 30 min in the presence of 750 units/ml SOD, and IRE binding was analyzed as in Fig. 1.
The rate constant of the reaction between NO and
O was re-evaluated recently (30) and was found to be similar to that of the dismutation of
O
by SOD: 6.7
10
M
s
versus 2
10
M
s
(31) . Accordingly, it is likely that
even in the presence of modest amounts of SOD, SIN-1 releases
O
which, by combining with NO, yields
ONOO
(29, 30) . This is the reason
why we decided to compare carefully the IRE binding of IRP exposed to
SIN-1 either alone or along with large amounts of SOD (3000 units/ml),
in order to make certain that NO was produced in larger amounts than
O
(30) . As testified by the
rhodamine assay (22) , ONOO
release from
SIN-1 was reduced by 95-98% in the presence of 3000 units/ml SOD.
As shown in Table 1, 1 mM SIN-1 inhibited the aconitase
activity of IRP either in the presence of SOD or in its absence, but
catalase abrogated the inhibitory effect of SIN-1/SOD. These results
are consistent with the fact that O
and
ONOO
, two products released from SIN-1, as well as
H
O
, are known to inhibit aconitase
activity(12, 15, 17) . Regarding IRE binding,
SIN-1 by itself was hardly active at all but note that it was far more
active in the presence of 3000 units/ml SOD (Fig. 3). The
potency of the combination SIN-1/SOD was not due to increased
production of NO as judged by nitrite and nitrate release (not shown).
Further, the addition of catalase reduced IRE binding only slightly,
thus suggesting that H
O
which might be produced
by the combination SIN-1/SOD, did not play a major part in this play.
Oxyhemoglobin, which binds NO with high affinity, protected IRP against
the effect of SIN-1/SOD in a dose-dependent manner (Fig. 4),
thus confirming that any O
released
from SIN-1 cannot per se make IRP bind IRE.
Figure 3: The effect of SOD on the IRE binding activity of IRP exposed to the NO donor SIN-1. RAW 264.7 cell cytosol was treated with 1 mM SIN-1 for 1 h at 37 °C in the presence of 3000 units/ml SOD and 100 units/ml catalase in PBS or in their absence. IRE binding was then analyzed as in Fig. 1.
Figure 4: The effect of hemoglobin on the IRE binding activity of IRP induced by the NO donor SIN-1. RAW 264.7 cell cytosol was treated with 5 mM SIN-1 for 1 h in the presence of 3000 units/ml SOD (lanes 3-7) and increasing amounts of hemoglobin (lanes 4-7). Samples were then desalted on P-6 Bio-Spin columns and analyzed for IRE binding activity as in Fig. 1.
Aconitase substrates such as citrate and cis-aconitate bind to an [4Fe-4S] cluster at the active site of aconitases (16, 32) and protect IRP from the effect of 2-ME(33) . To see whether the reactive molecule released by SIN-1 targets the iron-sulfur center of IRP, we incubated cell cytosol with both SIN-1 (plus SOD) and cis-aconitate and then measured both aconitase and IRE binding. The results (Fig. 5A) show that the presence of cis-aconitate prevented SIN-1 from inducing IRP to bind IRE. As previously documented(33) , cis-aconitate also protected IRP from the effect of 2-ME (Fig. 5A, bottom lanes). In another set of experiments, incubation of cis-aconitate with SIN-1 also prevented the inhibition of aconitase activity (Fig. 5B).
Figure 5: The effect of cis-aconitate on the activities of IRP exposed to SIN-1. RAW 264.7 cell cytosol was incubated for 30 min at 37 °C with 5 mM SIN-1 and 750 units/ml SOD along with cis-aconitate where indicated. Samples were then analyzed for IRE binding activity (A) or desalted on P-6 Bio-Spin columns and analyzed for aconitase activity (B), as described in Fig. 1.
ROS inhibit aconitase activity in various
systems (13, 15) and interact with the
[Fe-S] cluster of several dehydratases(34) . These
species were therefore good candidates for converting IRP from the
aconitase form to the IRE binding form. To test this possibility, RAW
264.7 cell cytosol was exposed either to the
O generator system
hypoxanthine/xanthine oxidase or directly to
H
O
. Under these conditions, the
hypoxanthine/xanthine oxidase system generated significant flow of
O
(3 to 5 µM/min for at
least 15 min) as determined by the SOD-inhibitable reduction of
ferricytochrome c. H
O
was probably
also produced following spontaneous dismutation of
O
. IRP lost its aconitase activity upon
the generation of ROS (Table 2), and inhibition of this activity
was completely prevented by the addition of both SOD and catalase.
Regarding IRE binding, Fig. 6shows that exposure of IRP to the
O
generator system had no significant
effect (left panel). Likewise, direct exposure of cell cytosol
to high doses of H
O
reduced aconitase activity
by about 50% (Table 2) without increasing the IRE binding
activity of IRP (Fig. 6, right panel). In contrast, in
both sets of experiments, the combination of SIN-1 plus SOD, taken as a
positive control, significantly increased IRE binding by IRP (Fig. 6, lanes 6 and 12, respectively).
Figure 6:
Comparative effects of ROS and of the NO
donor SIN-1 on the IRE binding activity of IRP. RAW 264.7 cell cytosol
was incubated for 1 h at 37 °C with the hypoxanthine/xanthine
oxidase O generating system (left
panel) or with H
O
(right panel).
In the same sets of experiments, cytosol was also exposed to 2 mM SIN-1 (left panel) or 5 mM SIN-1 (right
panel) to which 3000 units/ml SOD had been added. Samples were
then analyzed for IRE binding activity as described in Fig. 1.
When cell cytosol was exposed to a directly applied bolus of
ONOO, the aconitase activity of IRP was significantly
inhibited by ONOO
concentrations from around 25
µM (Fig. 7A), thus confirming previous
published data(12, 17) . However, Fig. 7B shows that at concentrations of up to 250 µM,
ONOO
had no effect on IRE binding. In several other
similar experiments, ONOO
, even at 1 mM, had
no effect either. Yet in parallel experiments, ONOO
was able to oxidize dihydrorhodamine into rhodamine (not shown).
These experiments indicate that ONOO
was able to
inhibit IRP aconitase activity significantly but did not affect its IRE
binding activity.
Figure 7:
Effect of ONOO on IRP
activities. RAW 264.7 cell cytosol was incubated for 10 min at room
temperature with increasing concentrations of ONOO
.
In the first set of experiments (A), aconitase activity was
assessed spectrophotometrically by measuring the initial rate of
disappearance of cis-aconitate at 240 nm. Experiments were
representative of 4 which gave similar results. In the second set of
experiments (B), cytosol incubated with ONOO
was analyzed for IRE binding using gel retardation as in Fig. 1. Where indicated, peroxynitrite was allowed to decompose
in buffer before the cytosol was added (reverse-order addition control
or ROA).
Besides SIN-1, two other types of chemicals that
release NO but not oxygen-derived molecules were tested for their
capacity to enhance IRE binding by IRP: S-nitrosothiols and
the secondary amineNO complex, spermine-NONOate (sper/NO). Two S-nitrosothiols were routinely assayed: S-nitrosoglutathione (GSNO), a product derived from
physiologically relevant thiol, and an S-nitrosothiol derived
from cysteamine and further referred to below as S-nitrosocysteamine (SNC). GSNO is fairly stable and required
the presence of a reducing agent, e.g. ascorbate, to release
significant amounts of NO (as testified by nitrite production) during
its 1-h incubation with IRP. SNC rapidly decomposes into NO and is
kinetically predictable. Indeed, Roy et al.(35) reported that millimolar range of SNC rapidly yields
micromolar concentrations of NO. SNC was therefore suitable for
short-time incubation with IRP. Under our conditions, GSNO (+
ascorbate) and SNC released
4 and 8 µM nitrite/min,
respectively, during the 1-h incubation. Fig. 8shows that both S-nitrosothiols made IRP bind IRE. S-Nitroso-DL-penicillamine, another well-known S-nitrosothiol, was also assayed but gave, with time, gave
inconsistent results, and the experiments were not pursued. We also
used sper/NO, another fast NO-releasing compound(36) .
Half-time of sper/NO is
40 min at pH 7.5, so that it was suitable
for a 1-h incubation with IRP. Exposure of cytosol to 1 mM sper/NO consistently inhibited aconitase activity by 88% (mean of
6 experiments). Inhibition was prevented by oxyhemoglobin (not shown).
The results depicted in Fig. 9(lane 6) show that sper/NO
increased IRE binding by IRP moderately, but did so far more in the
presence of reduced glutathione (GSH) (lane 7). Ascorbate also
boosted the capacity of sper/NO to activate IRE binding activity of IRP
(not shown). It was interesting to observe that oxyhemoglobin prevented
the increase in IRE binding induced by the combination of sper/NO and
GSH (lane 8), but methemoglobin did not (lane 9).
Figure 8: Effect of nitrosothiols on IRE binding activity of IRP. RAW 264.7 cytosol (30 µg of protein) was incubated for 1 h at 37 °C with SNC (left panel) or 5 mM GSNO plus 1 mM ascorbate (right panel). Samples (2 µg) were then analyzed for IRE binding, with or without 2% 2-ME, as described in Fig. 1.
Figure 9: The effect of sper/NO on the IRE binding activity of IRP. RAW 264.7 cell cytosol was incubated for 1 h at 37 °C with 1 mM sper/NO, either alone (lane 6) or together with 1 mM glutathione (lane 7), and either 100 µM oxyhemoglobin (lane 8) or 100 µM methemoglobin (lane 9). The cytosol was also treated with 1 mM spermine (Sper, lane 5) or 1 mM spent sper/NO, i.e. sper/NO which had been allowed to decompose for several hours before its addition to the cytosol (lane 10).
Several lines of evidence indicate that as well as
oxygen-derived molecules, NO, which diffuses freely away from its site
of generation, may be a biological messenger used by the cell to react
with redox-sensitive gene trans-regulators. Thus, it was
reported that exposure to O or
H
O
or to exogenous NO activates of the
eukaryotic trans-activators AP-1 (37, 38) and
NF-
B(39, 40) , as well as the bacterial Sox-RS
regulon system(41, 42) . Furthermore, we and others
previously supplied evidence that endogenous and exogenous NO modulates
the cellular iron metabolism by increasing IRP binding to specific
sequence(s) in their mRNA, i.e. to
IREs(7, 8, 10, 26, 43) .
Conversely, the aconitase activity of IRP is lost as a consequence of
NO synthesis. The redox-sensitive site of IRP responsive to NO synthase
product(s) is probably a [4Fe-4S] cluster which when intact,
both allows enzymatic activity and prevents IRE
binding(2, 3, 16) . Besides,
O
and ONOO
were
identified as likely products of brain NO
synthase(43, 44) . In addition,
O
is known to interact with iron-sulfur
clusters and has been reported to inhibit aconitase in E. coli and mammals(11, 13, 33) whereas
H
O
inhibits plant aconitase(15) .
Recently, it was further proposed that O
represses ferritin translation by activating IRE binding through
the interaction of IRP with IRE(46) . Consequently, it was
plausible to envisage that either of these two products could be
responsible for the converse modulation of IRP exhibited by cells
expressing NO synthase. Accordingly, to gain insight into the
physiologically relevant mechanism that allows NO-producing cells to
express IRP, crude IRP from macrophage extracts was exposed to ROS,
ONOO
, and various NO-releasing compounds, to see
whether these species increased IRP binding to IRE and, if so, which
one(s).
In the presence of oxygen, SIN-1 was found to release both
NO and O(28) and, in the
absence of SOD, it is often considered as a source of
ONOO
(29, 47) . It is therefore a
useful tool for studying the effect of ONOO
produced
at a low rate and by adding SOD and catalase, the respective
contributions of O
and NO to the effect
of SIN-1. We showed here that incubation of IRP with SIN-1 decreased
its aconitase activity. Inhibition was completely abolished by a
combination of SOD and catalase, suggesting at first glance that
H
O
had a role in this inhibition. However,
direct addition of high doses of H
O
to cell
cytosol did not completely inhibit aconitase activity. Taken together,
these results imply that H
O
alone is not
sufficient to inhibit the aconitase activity of IRP and may require a
combination with NO to be potent. Such interplay between
H
O
and NO has already been suggested to play a
part in cell cytotoxicity(48, 49) . SIN-1 alone did
not increase IRE binding by IRP significantly, but it must be
emphasized that the presence of SOD consistently increased the IRE
binding of IRP, thus confirming our previous results obtained with
recombinant human IRP (7) . The reason why SOD enhances the
SIN-1-stimulated IRE binding activity is still not clear. The simplest
explanation is that SOD prolongs NO half-life by preventing its
reaction with O
. Alternatively, SOD has
been suggested to favor the conversion of NO into certain redox forms
independently of dismutation(50, 51) . Here, the
presence of catalase in addition to SOD led to a slight but consistent
reduction of IRE binding. However it is doubtful that
H
O
was directly involved in this reduction
because addition of large doses of H
O
had no
significant effect on IRE binding activity of IRP. Further, these
results are consistent with those of Pantopoulos and Hentze (52) who recently showed that even though H
O
enhanced IRE binding in a fibroblast cell line, it had no direct
effect on this activity. It is possible that the protection afforded by
catalase of aconitase activity and IRE binding against SIN-1 plus SOD
was due to the scavenging of nitric oxide by catalase, a
heme-containing enzyme.
Aconitase substrates stabilize the
[Fe-S] cluster of mitochondrial aconitase and presumably that
of IRP (3, 16, 32) by binding to one iron
atom, known as Fe. We showed here that cis-aconitate attenuated the increase in IRE binding induced
by SIN-1 plus SOD. The protective effect of cis-aconitate is
thus consistent with the proposal that a SIN-1-derived reactive
molecule and cis-aconitate compete for the same site of
ligation, i.e. the [Fe-S] cluster. Further, the
protective effect of citrate raises the possibility that the cellular
concentration of citrate may exert a feedback mechanism toward the
stimulation by NO synthase of IRE binding.
The exposure of cell
cytosol to the generation of ROS by the hypoxanthine/xanthine oxidase
system led to the inhibition of IRP aconitase activity without allowing
IRP binding to IRE. However, when SOD, even at high doses, was added in
parallel experiments, SIN-1 greatly enhanced IRE binding. The fact that
the O generating system was innocuous
and that the presence of SOD increased the effect of SIN-1 rules out
the possibility that O
in itself makes
a major contribution to the switch of IRP into IRE-binding protein.
In addition, there is growing evidence that ONOO,
which results from the reaction of O
with NO, may play an important part in several pathophysiological
effects formerly attributed to NO(53) . In the same line of
research, it was shown that ONOO
is produced by rat
alveolar macrophages triggered in vitro by phorbol
12-myristate 13-acetate (54) and can inhibit the mitochondrial
respiration complexes I and II containing [Fe-S]
clusters(55) , as well as both mitochondrial and cytosolic
aconitase(12, 17) . Consequently, it has been proposed
that ONOO
, rather than NO, is the physiological
effector molecule which in cells expressing NO synthase activity,
inhibits aconitase activity. As aconitase activity and the IRE binding
activity of IRP are reciprocally modulated in response to changes in
the iron concentration (2) or to NO
synthesis(7, 10) , it was tempting to conclude that
ONOO
increases the IRE binding activity of IRP. Our
results provided evidence that this is unlikely because
ONOO
when applied as a bolus or released from SIN-1
has little or no effect on IRE binding by IRP. One may wonder whether
absence of effect of ROS and ONOO
resulted from
absence of an adequate ``redox status'' in the cell extract.
This deserves further investigation, but the marked difference between
the effect of SIN-1 alone (which releases ONOO
) and
SIN-1 plus large amounts of SOD (which release NO) seems to call for
another explanation.
Lastly, to improve our knowledge of the precise
mechanism by which the NO synthesis can change the functional response
of IRP, we exposed cell cytosol to two S-nitrosothiols which
release NO but not O: exposure to SNC
and GSNO (the latter in the presence of ascorbate), resulted in
enhancement of IRE binding. GSNO was previously shown by Castro et
al.(17) to inhibit aconitase activity, but these authors
concluded that the release of NO was not responsible for this
inhibition, and S-nitrosothiols indeed react with protein
thiols, e.g. by trans-nitrosation or by thiyl radical
release, independently of their capacity to yield NO. Therefore,
another chemical able to release NO was also tested, the
``NONOate'' sper/NO. Provided it was used with GSH, sper/NO
gave results similar to those obtained with S-nitrosothiols.
The boosting effect of reducers in triggering IRE binding was not
evaluated, but it was not related to an enhanced capacity to release
NO, as testified by nitrite production. Rather, it suggests that an
adequate redox environment within the cell favors IRE binding resulting
from NO production. Further, since oxyhemoglobin, an NO-scavenger,
reduced IRE binding resulting from the action of sper/NO plus GSH, we
inferred that the formation of the IRP
IRE complex resulted from
NO release rather than to a side effect of sper/NO.
The fact that
ROS and ONOO inhibit the enzymatic activity of IRP
without triggering IRE binding may raise an important issue. These
products may affect the iron-sulfur cluster of IRP only to a limited
extent, so that they only convert the [4Fe-4S] form, which
allows aconitases to be active, into the [3Fe-4S] form which
possesses neither aconitase activity nor IRE binding
ability(2, 3, 16) . Alternatively, we cannot
exclude the possibility that ONOO
both disrupts the
cluster and prevents binding to IRE, by promoting the oxidation of some
free sulfhydryl groups required for binding
IREs(3, 21) . It is noteworthy that two cysteine
residues involved in iron-sulfur cluster assembly, namely Cys-503 and
Cys-506, are located in the IRE binding domain(56) .
It must be recalled that some NO synthase product(s) can alter IRP in living cells sufficiently to change both its functions reciprocally(7, 9) . The exact nature of the NO synthase product which metamorphoses IRP into IRE-binding protein is still open to question. Whether NO itself is this molecule is controversial(7, 12, 17) , but this issue was not addressed here. However, according to the results of our experiments with NO donors (performed here in aerobiosis), we feel justified in proposing that the right ``track'' to follow in order to identify the molecule responsible for NO synthase-dependent conversion of IRP into IRE-binding protein, is probably to look for nitrosating species resulting from the reaction between NO and oxygen (NOx, NxOy, etc.) (57) and to determine their adequate redox environment. Finally, the differential effects of ROS and NO (or NO-oxygenated products) on IRP imply that the latter is a biosensor able to discriminate between each of these classes of reactive molecule.
In conclusion, if the results of the in vitro manipulations performed in our study indeed reflect the in
vivo situation, our data support the assumption that neither
O nor ONOO
is per
se the physiological molecule which converts IRP from its
aconitase form into its IRE binding form. In contrast, as several
NO-releasing products promoted IRE binding in cell cytosol, we conclude
that NO, or certain oxidized or redox-activated species derived from NO
but different from ONOO
, are the most likely
molecules responsible for modulating IRP functions in living cells
expressing NO synthase.