The mechanism of nitrogen monoxide (NO)-mediated iron mobilization
from cells
NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron
from ferritin in a glutathione-dependent manner
Ralph N. Watts and Des R. Richardson
The Iron Metabolism and Chelation Group, The Heart Research Institute, Camperdown, Sydney, New South Wales, Australia
Nitrogen monoxide (NO) is a cytotoxic effector molecule
produced by macrophages that results in Fe mobilization
from tumour target cells which inhibits DNA synthesis and
mitochondrial respiration. It is well known that NO has a
high affinity for Fe, and we showed that NO-mediated Fe
mobilization is markedly potentiated by glutathione (GSH)
generated by the hexose monophosphate shunt [Watts, R.N.
& Richardson, D.R. (2001) J. Biol. Chem. 276, 4724–4732].
We hypothesized that GSH completes the coordination shell
of an NO–Fe complex that is released from the cell. In this
report we have extended our studies to further characterize
the mechanism of NO-mediated Fe mobilization. Native
PAGE
59
Fe-autoradiography shows that NO decreased
ferritin-
59
Fe levels in cells prelabelled with [
59
Fe]transferrin.
In prelabelled cells, ferritin-
59
Fe levels increased 3.5)fold
when cells were reincubated with control media between 30
and 240 min. In contrast, when cells were reincubated with

branch of coordination chemistry [2]. Apart from the
regulatory role of NO, its cytotoxic actions are found when
it is produced in large quantities by cells such as activated
macrophages [3]. Interestingly, NO produced by such
systems inhibits the proliferation of intracellular pathogens
and tumour cells [3–5]. These effects can be explained by the
reactivity of NO with Fe in the [Fe–S] centres of critical
proteins, including aconitase and complex I and II of the
electron transport chain [4–6]. The high affinity of NO for
Fe probably results in both the removal of Fe from [Fe–S]
centres and the formation of dinitrosyl Fe species within
[Fe–S] proteins (reviewed in [7]).
It has already been shown that NO forms complexes with
a range of Fe-containing proteins including ferritin [8],
ribonucleotide reductase [9], haem-containing proteins
[10–12], and ferrochelatase [13]. Further, it has been
suggested that ferritin can act as a store of NO [8], and
NO-mediated Fe release from isolated and purified ferritin
has been demonstrated [14]. When activated macrophages
are cocultured with tumour cells, this inhibits target cell
DNA synthesis and results in the release of 64% of cellular
59
Fe within 24 h [15]. This loss of Fe may be due to the
NO-mediated release of Fe from enzymes such as mito-
chondrial aconitase [4,16–18]. Others have suggested that
NO can also target loosely bound pools of nonhaem Fe [19].
Nonetheless, the identification of Fe–nitrosyl complexes
(Fe–dithiol dinitrosyl complexes and haem–nitrosyl com-
plexes) by EPR spectroscopy in activated macrophages and
their tumour cell targets show the importance of the Fe–NO

59
Fe from prelabelled
cells as, or more, effectively than the clinically used Fe
chelator desferrioxamine (DFO) [29]. In contrast, the
precursor compounds of these latter NO-generators,
namely N-acetylpenicillamine (NAP), glutathione (GSH),
and spermine (Sper), respectively, had no effect [29].
Previous studies have suggested that NO may be released
from cells as a complex composed of NO, Fe, and thiol-
containing ligands such as cysteine or GSH [23,30,31].
Considering this and the other data described above, we
recently examined the energy-dependency of NO-mediated
Fe release from cells [32]. Our investigation showed that
metabolism of
D
-glucose potentiates NO-mediated Fe efflux
from a variety of cell types. Further, we demonstrated that
the metabolism of
D
-glucose by the hexose monophosphate
shunt (HMPS) and the maintenance of GSH levels was
essential for NO-mediated Fe mobilization [32]. However,
we are not proposing a direct coupling between glucose
import/metabolism and NO metabolism. Rather, our ex-
periments suggested that the generation of GSH after
incubation with
D
-glucose could result in GSH acting as a
ligand which together with NO would complete the coordi-
nation shell of Fe [32]. Such a Ômixed Fe complexÕ with both

L
-buthionine-(S,R)-
sulphoximine (BSO), diethylenetriaminepentaacetic acid
(DTPA), E
`
DTA, GSH, GSNO, horse spleen ferritin and
Sper were obtained from Sigma. SperNO was obtained from
Cayman Chemicals. Eagle’s minimum essential medium
(MEM) was obtained from Gibco BRL. DFO was obtained
from Novartis Pharmaceutical Co. Pyridoxal isonicotinoyl
hydrazone (PIH) and its analogue, 2-hydroxy-1-naphthylal-
dehyde isonicotinoyl hydrazone (311), were synthesized by
standard techniques [34]. Both PIH and 311 are strong Fe-
binding ligands [34] and were used as positive Fe chelation
controls. Apolactoferrin was from Calbiochem. A polyclonal
rabbit anti-(human ferritin) Ig was from Roche Diagnostics.
All other chemicals were of analytical reagent quality. The
NO-generators and other compounds were dissolved in
media immediately prior to an experiment [29,35].
Cell culture
Human SK-N-MC neuroepithelioma cells, SK-Mel-28
melanoma cells, and MCF-7 breast cancer cells were from
the American Type Culture Collection. The mouse LMTK
–
fibroblast cell line was from the European Collection of Cell
Cultures. The BE-2 neuroblastoma cell line was a gift from
G. Anderson, Queensland Institute of Medical Research
(Brisbane, Australia). All cell lines were grown in MEM
containing 10% foetal calf serum (Gibco), 1% (v/v) non-
essential amino acids (Gibco), 100 lgÆmL

Standard techniques were used to examine the effect of NO
and other agents on the efflux of
59
Fe from prelabelled cells
[29,32,34]. Briefly, cells were labelled with
59
Fe-Tf (0.75 l
M
)
for 3 h at 37 °C in MEM. After this incubation, the cell
culture dishes were placed on a tray of ice, the medium
aspirated, and the cell monolayer washed four times with
ice-cold balanced salt solution (BSS). The cells were then
reincubated for various incubation times up to 240 min at
37 °C. After this incubation, the overlying supernatant
(efflux medium) was transferred to c-counting tubes. The
cells were removed from the petri dishes after adding 1 mL
BSS and by using a plastic spatula to detach them.
Radioactivity was measured in both the cell pellet and
supernatant using a c-scintillation counter (LKB Wallace
1282 Compugamma, Finland).
Determination of intracellular iron distribution
using native-PAGE-
59
Fe-autoradiography
Native-PAGE-
59
Fe-autoradiography was performed using
standard techniques in our laboratory [38]. Bands on X-ray
film were quantified by scanning densitometry using a Laser

one freeze-thaw cycle and then detached from the flask
using a Teflon spatula in the presence of the nonionic
detergent Triton X-100 (1.5%) at 4 °C. These samples were
then centrifuged at 21 300 g for 30 min at 4 °Candthe
cytosol removed and assessed for radioactivity using the
c-counter described above. The cytosolic samples were then
incubated for 3 h at 37 °C with DFO (0.5 m
M
)orGSNO
(0.5 m
M
). The generation of nitrite by GSNO was used as a
control to ensure that the NO-donor was producing NO in
the lysate. After this incubation, the samples were then
subjected to centrifugation at 4 °C through a 5-kDa M
r
exclusion filter (Vivaspin 500, Sartorius AG). After centri-
fugation, the eluent, eluate, and membrane were taken to
estimate
59
Fe levels. Examination of
59
Fe levels on the
membrane were considered important to assess the possi-
bility of adsorption of the
59
Fe-complex.
Statistics
Experimental data were compared using Student’s t-test.
Results were considered statistically significant when

GSNO
GSH
SNAP
NAP
SperNO
Sper
DFO
311
Control
GSNO
GSH
SNAP
NAP
SperNO
Sper
DFO
311
59
Fe-Ferritin Levels
Relative Density (% Control)
0
25
50
75
100
125
150
Anti-Ferritin
Antibody
Control

washed four times on ice. The cells were then reincubated for 3 h at 37 °C with control media, GSNO (0.5 m
M
), GSH (0.5 m
M
), SNAP (0.5 m
M
),
NAP (0.5 m
M
), SperNO (0.5 m
M
), Sper (0.5 m
M
), DFO (100 l
M
), 311 (25 l
M
) or polyclonal anti-human ferritin antibody (1 : 10 dilution). The
overlying media and cells were then separated and the
59
Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE-
59
Fe-
autoradiography (see Materials and methods). (B) Native PAGE-
59
Fe-autoradiographs of cellular cytosols treated as described above and
densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of three performed.
The data shown in (B) are a representative experiment of three performed.
Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3385
to 240 min at 37 °C in the presence or absence of the agents

Examining intracellular
59
Fe distribution in SK-N-MC
cells (Fig. 1B), the most pronounced band identified comi-
grated with purified horse spleen ferritin (data not shown).
Experiments incubating the lysate with an anti-ferritin
antibody demonstrated that only this band could be super-
shifted (Fig. 1B), again indicating that it was ferritin. As
described previously in neoplastic cells [41], a faint and very
diffuse band below ferritin was present which comigrated
with low M
r
Fe complexes (
59
Fe-citrate) (Fig. 1B). We
previously showed that this low M
r
Fe appeared to be
59
Fe
bound from the lysate by the low M
r
chelators in the gel
running buffer e.g. Tris [41]. In the present study, the low M
r
band will not be considered in detail as this component
remains undefined and its relevance uncertain.
In each case, GSNO, SNAP and SperNO, decreased
ferritin-
59

59
Fe release
from prelabelled cells (Fig. 2A) and ferritin-
59
Fe levels
(Fig. 2B) was examined. These experiments showed that
GSNO appreciably increased
59
Fe mobilization from pre-
labelled cells at a GSNO concentration of 0.05 m
M
,and
then plateaued at 0.5 m
M
(Fig. 2A). When assessing the
effect of NO on intracellular
59
Fe distribution, a GSNO
concentration of 0.05 m
M
decreased ferritin-
59
Fe levels to
26% of the control value (Fig. 2B). Higher concentrations
of the NO-donor were no more effective at reducing
ferritin-
59
Fe levels (Fig. 2B).
Studies were performed to determine the effect of
reincubation time in the presence and absence of GSNO

18
GSNO Concentration (mM)
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
120
59
Fe-Ferritin Levels
Relative Density (% Control)
Control
0.01 mM
0.05 mM
0.1 mM
0.5 mM
1 mM
GSNO Concentration
Fig. 2. The effect of GSNO concentration on (A) the mobilization of
59
Fe from prelabelled cells and on (B) intracellular ferritin-
59
Fe levels. (A) SK-N-
MC neuroepithelioma cells were labelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
), washed four times on ice and then reincubated for 3 h at

ferritin gradually increasing as a function of reincubation
time (Fig. 3B). In contrast, in cells treated with GSNO,
ferritin-
59
Fe levels decreased after 90 min of reincubation.
In fact, after 240 min, ferritin-
59
Fe levels were 10-fold less
than those of control cells at the same reincubation time
(Fig. 3B). These data suggest that NO directly or indirectly
results in
59
Fe mobilization from ferritin and also intercepts
59
Fe before it is deposited within this protein.
NO reduces ferritin-
59
Fe levels in a variety of cell types
TheeffectofNOatreducingferritin-
59
Fe levels was found
in a number of cell types including SK-N-MC neuroepi-
thelioma cells, MCF-7 breast cancer cells, LMTK
–
fibro-
blasts, BE-2 neuroblastoma cells and SK-Mel-28 melanoma
cells. However, there was marked variation in the effect of
NO between cell types, with a 15–74% decrease in
ferritin-
59

Fe levels, while BSO
treatment totally prevented this decrease (Fig. 4B). These
results indicate that GSH is required for the effect of NO at
decreasing ferritin-
59
Fe levels.
It is of interest that incubation of BSO-treated LMTK
–
and SK-N-MC cells with GSNO resulted in an increase in
the amount of ferritin-
59
Fe (Fig. 4B). These data were in
contrast to the decrease observed after treatment of control
cells with GSNO (Fig. 4B).
Cell membrane-impermeable or -permeable iron
chelators do not increase NO-mediated
59
Fe efflux
It was possible that passive diffusion may be involved in
NO-mediated
59
Fe release from cells. Previous studies have
shown that Fe mobilization in the absence of NO is
increased by incubation with apoTf and extracellular
chelators, presumably due to the ability of these agents to
act as an extracellular Fe ÔsinkÕ to increase the concentration
gradient across the cell membrane [42–44]. Considering this,
and the fact that a NO–Fe–GSH complex may be released
from cells [32], experiments were designed to investigate the
effects of 0.1 mgÆmL

Time (min)
0 60 120 180
240
Cellular Iron Released (% Total)
0
2
4
6
8
10
12
14
Control
GSNO
Time (min)
0 60 120 180 240
0
2
4
6
8
10
Control
GSNO
59
Fe-Ferritin Levels
Relative Density (Arbitrary Units)
Fig. 3. The effect of GSNO on (A) the mobilization of
59
Fe from cells, and (B) ferritin-

Fe mobilization from prela-
belled cells (Fig. 5B).
As NO acted like an Fe chelator to mobilize
59
Fe from
prelabelled cells [29,32], studies were performed to deter-
mine if the same Fe pool bound by the permeable chelators,
DFO (Fig. 6A) or PIH (Fig. 6B), was bound by NO. In
these studies, cells were prelabelled with
59
Fe-Tf for 3 h at
37 °C, washed, and then reincubated for 3 h at 37 °Cwith
either increasing concentrations of DFO (0.05–1 m
M
)or
PIH (1–50 l
M
) or these chelators combined with GSNO
(0.5 m
M
). The addition of increasing concentrations of PIH
or DFO to GSNO had no significant effect on cellular
59
Fe
mobilization (Fig. 6A and B). These results suggested that
(A) (B)
Cellular Iron Released (% Total)
0
5
10

Control
BSO
SK-N-MC
59
Fe-Ferritin Levels
Relative Density (% Control)
0
40
80
120
160
Fig. 4. The depletion of GSH prevents (A) NO-mediated
59
Fe mobilization from prelabelled cells, and (B) the decrease in intracellular ferritin-
59
Fe
levels seen in the presence of GSNO. (A) SK-N-MC neuroepithelioma cells and LMTK
–
fibroblasts were pretreated for 20 h at 37 °C in the presence
or absence of the specific GSH inhibitor BSO (0.1 m
M
). The cells were then prelabelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
), washed four
times on ice, and then reincubated with control media or media containing GSNO (0.5 m
M
)for3hat37°C. The media and cells were separated
and the

and cells were then separated and the
59
Fe levels in each assessed (see Materials and methods). These results are mean ± SD (three replicates) in a
representative experiment of three performed.
3388 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002
GSNO and the chelators were acting on the same intracel-
lular compartment of
59
Fe. In contrast, when added without
GSNO, increasing concentrations of DFO or particularly
PIH, resulted in enhanced
59
Fe mobilization from cells
(Fig. 6A,B).
Examination of the direct effect of NO
and iron chelators on iron pools in cytosolic lysates:
comparison with intact cells
As NO could form an intracellular low M
r
Fe–dithiol
dinitrosyl complex [23], experiments were performed to
determine if NO or DFO could mobilize
59
Fe from lysates
prepared from cells labelled with
59
Fe-Tf for 3 h at 37 °C
(Fig. 7A). The lysates were centrifuged to obtain cytosols
and then incubated for 3 h at 37 °C with DFO (0.5 m
M

), washed four times on ice, and then reincubated
with increasing concentrations of desferrioxamine (DFO; 0.05–1 m
M
) or pyridoxal isonicotinoyl hydrazone (PIH; 1–50 l
M
) either alone or in the
presence of GSNO (0.5 m
M
) for 3 h at 37 °C. The overlying media and cells were then separated and the
59
Fe levels in each assessed (see Materials
and methods). These results are mean ± SD (three replicates) in a representative experiment of three performed.
Fig. 7. GSNO, in contrast to DFO, does not mobilize
59
Fe from (A) cytosolic lysates, derived from prelabelled cells, while (B) GSNO is more effective
than DFO at mobilizing
59
Fe from prelabelled intact cells. (A) Cells were labelled with
59
Fe-Tf for 3 h at 37 °C and washed four times on ice and
lysates prepared. The lysates were centrifuged to obtain cytosols and then incubated for 3 h at 37 °C with DFO (0.5 m
M
)orGSNO(0.5m
M
). The
cytosols were then subjected to ultracentrifugation through a 5-kDa cut-off filter. (B) Cells were prelabelled with
59
Fe-Tf (0.75 l
M
)for3hat37 °C,

DISCUSSION
Previous studies have clearly demonstrated that NO has a
marked effect on cellular Fe metabolism [25–27,32]. Indeed,
NO-mediated Fe depletion of tumour target cells by
activated macrophages could play an important role in
immune surveillance [3–5,15,16,45]. Our previous studies
have shown that NO-mediated Fe mobilization is potenti-
ated by incubating cells with
D
-glucose due to the
subsequent generation of GSH [32]. In the present study
we have significantly extended our knowledge of this
process. For the first time, we demonstrate in a cellular
system that NO intercepts Fe before it is incorporated into
ferritin and appeared to indirectly mobilize Fe from this
protein.
NO could remove Fe from ferritin by two possible
mechanisms: (a) by directly chelating ferritin-bound Fe, or
(b) by chelating a cellular Fe pool which leads to ferritin
releasing its Fe. Of these two possibilities our evidence
favours the second mechanism, as NO could not remove
59
Fe from ferritin in cellular lysates (Fig. 8). Furthermore,
the processes resulting in cellular Fe mobilization and Fe
release from ferritin were dependent on cellular metabolism
(Fig. 7) and the generation of GSH (Fig. 4 and [32]). These
latter observations indicate that active cellular metabolism
was required for Fe mobilization rather than direct chela-
tion of ferritin-Fe by NO.
A previous in vitro study by Reif & Simmons [14] using

appropriate lipophilicity and charge to diffuse or be
transported from the cell. Previous studies using EPR
spectroscopy have demonstrated the presence of dithiol
dinitrosyl–Fe complexes within cells [23,30]. Further, Rogers
and Ding [48] have shown that
L
-cysteine is necessary for the
removal of dinitrosyl–Fe complexes from [Fe–S]-containing
proteins in Escherichia coli. Interestingly, these authors
showed that GSH was able to perform the same function but
Con
DFO
311
SNAP
NAP
GSNO
GSH
GSH + GSNO
Fig. 8. The direct effect of incubating GSNO and Fe chelators on
59
Fe-containing molecules in cytosolic lysates derived from prelabelled
cells. SK-N-MC neuroepithelioma cells were labelled for 3 h at 37 °C
with
59
Fe-transferrin (0.75 l
M
) and washed four times on ice. Cells
were then lysed and the cytosols incubated with DFO (0.5 m
M
), 311

ATP
TCA
HMPS
Glucose
G-6-P
GSH
Glucose
Transporter
Fe-Protein
NO
NO
NO-Fe-Protein
Transporter
Diffusion
Fig. 9. Hypothetical model of
D
-glucose-dependent NO-mediated Fe
mobilization from cells.
D
-Glucose is transported into cells and is used
by the tricarboxylic acid cycle for the production of ATP and by the
HMPS for the generation of reduced GSH. Nitrogen monoxide (NO)
either diffuses or is transported into cells where it intercepts and binds
Fe bound to proteins or Fe on route to ferritin. The high affinity of NO
for Fe results in the formation of an NO–Fe complex and GSH may
either be involved as a reductant to remove Fe from endogenous lig-
ands or may complete the Fe coordination shell along with the NO
ligand(s). This complex may then be released from the cell by an active
process requiring a transporter (see text for details).
3390 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002

complex.
Based upon the results presented in this and our previous
study [32], we suggest in Fig. 9 a hypothetical model of
D
-glucose-dependent NO-mediated Fe mobilization from
cells.
D
-Glucose is transported into cells and is used by the
tricarboxylic acid cycle (TCA) for the production of ATP
and by the HMPS for the generation of GSH. NO diffuses or
is transported [51] into cells where it intercepts Fe on route to
ferritin and binds Fe bound to proteins (Fig. 9). The high
affinity of NO for Fe [2] results in the formation of an NO-Fe
complex and GSH may either be involved as a reductant to
remove Fe from endogenous ligands [48] or may complete
the Fe coordination shell along with NO [17,20,23,30]. This
complex may then be transported out of the cell by an energy-
dependent transporter such as ferroportin 1 [52], or
alternatively, the ATP-binding cassette (ABC) transporter
family (e.g. glutathione-S-conjugate export pump), which
are known to mediate the efflux of glutathione-conjugates
[53,54] (Fig. 9). Further studies aimed at identifying the exact
molecular nature of the Fe released by NO and the
transporter involved are underway. Finally, out current
results may be important in understanding the cytotoxic
actions of NO produced by activated macrophages.
ACKNOWLEDGEMENTS
The authors thank J. Kwok for her excellent suggestions on this
manuscript prior to submission. This work was supported by an
Australian Research Council Large Grant and Grants 970360 and

ferritins. Biochemistry 33, 3679–3687.
9. Lepoivre, M., Fieschi, F., Coves, J., Thelander, L. & Fontecave, M.
(1991) Inactivation of ribonucleotide reductase by nitric oxide.
Biochem. Biophys. Res. Commun. 179, 442–448.
10. Ignarro, L.J. (1991) Heme-dependent activation of guanylate
cyclase by nitric oxide: a novel signal transduction mechanism.
Blood Vessels 28, 67–73.
11. Khatsenko, O.G., Gross, S.S., Rifkind, R.R. & Vane, J.R. (1993)
Nitric oxide is a mediator of the decrease in cytochrome P450-
dependent metabolism caused by immunostimulants. Proc. Natl
Acad. Sci. USA 90, 11147–11151.
12. Griscavage, J.M., Fukuto, J.M., Komori, Y. & Ignarro, L.J.
(1994) Nitric oxide inhibits neuronal nitric oxide synthase by
interacting with the heme prosthetic group. Role of tetra-
hydrobiopterin in modulating the inhibitory action of nitric oxide.
J. Biol. Chem. 269, 21644–21649.
13. Kim, Y M., Bergonia, H.A., Muller, C., Pitt, B.R., Watkins,
W.D. & Lancaster, J.R. Jr (1995) Loss and degradation of
enzyme-bound heme induced by cellular nitric oxide synthesis.
J. Biol. Chem. 270, 5710–5713.
14. Reif, D.W. & Simmons, R.D. (1990) Nitric oxide mediates iron
release from ferritin. Arch. Biochem. Biophys. 283, 537–541.
15. Hibbs, J.B. Jr, Taintor, R.R. & Vavrin, Z. (1984) Iron depletion:
possible cause of tumor cell cytotoxicity induced by activated
macrophages. Biochem. Biophys. Res. Commun. 123, 716–723.
16. Hibbs, J.B. Jr, Taintor, R.R., Vavrin, Z. & Rachlin, E.M. (1988)
Nitric oxide: a cytotoxic activated macrophage effector molecule.
Biochem. Biophys. Res. Commun. 157, 87–94.
17. Lancaster, J.R. & Hibbs, J.B. Jr (1990) EPR demonstration of
iron-nitrosyl complex formation by cytotoxic activated macro-

25. Drapier, J C., Hirling, H., Wietzerbin, J., Kaldy, P. & Ku
¨
hn, L.C.
(1993) Biosynthesis of nitric oxide activates iron regulatory factor
in macrophages. EMBO J. 12, 3643–3649.
26. Weiss, G., Goossen, B., Doppler, W., Fuchs, D., Pantopoulos, K.,
Werner-Felmayer,G.,Wachter,H.&Hentze,M.W.(1993)
Translational regulation via iron-responsive elements by the nitric
oxide/NO-synthase pathway. EMBO J. 12, 3651–3657.
27. Richardson, D.R., Neumannova, V., Nagy, E. & Ponka, P. (1995)
The effect of redox-related species of nitrogen monoxide on
transferrin and iron uptake and cellular proliferation of erythro-
leukemia (K562) cells. Blood 86, 3211–3219.
28. Kennedy, M.C., Antholine, W.E. & Beinert, H. (1997) An EPR
investigation of the products of the reaction of cytosolic and
mitochondrial aconitases with nitric oxide. J. Biol. Chem. 272,
20340–20347.
29. Wardrop, S.L., Watts, R.N. & Richardson, D.R. (2000)
Nitrogen monoxide activates iron regulatory protein 1 RNA-
binding activity by two possible mechanisms: effect on the [4Fe-4S]
cluster and iron mobilization from cells. Biochemistry 39,
2748–2758.
30. Vanin, A.F., Bliumenfel’d, L.A. & Chetverikov, A.G. (1967) EPR
study of non-heme iron complexes in cells and tissues. Biofizika
[Russian] 12, 829–841.
31. Woolum, J.C., Tiezzi, E. & Commoner, B. (1968) Electron spin
resonance of iron-nitric oxide complexes with amino acids, pep-
tides and proteins. Biochim. Biophys. Acta 160, 311–320.
32. Watts, R.N. & Richardson, D.R. (2001) Nitrogen monoxide (NO)
and glucose: unexpected links between energy metabolism and

131–138.
38. Richardson, D.R., Ponka, P. & Vyoral, D. (1996) Distribution of
iron in reticulocytes after inhibition of heme synthesis with suc-
cinylacetone. Examination of the intermediates involved in iron
metabolism. Blood 87, 3477–3488.
39. Sedlack, J. & Lindsay, R. (1968) Estimation of total protein-
bound and nonprotein sulfhydryl groups in tissue with Ellman’s
reagent. Anal. Biochem. 25, 192–205.
40. Griffith, O.W. & Meister, A. (1979) Potent and specific inhibition
of glutathione synthesis by buthionine sulfoximine (S-n-butyl
homocysteine sulfoximine). J. Biol. Chem. 254, 7558–7560.
41. Richardson, D.R. & Milnes, K. (1997) The potential of iron
chelators of the pyridoxal isonicotinoyl hydrazone class as
effective antiproliferative agents II. The mechanism of action of
ligands derived from salicylaldehyde benzoyl hydrazone and
2-hydroxy-1-naphthylaldehyde benzoyl hydrazone. Blood 89,
3025–3038.
42. Baker, E., Page, M. & Morgan, E.H. (1985) Transferrin and iron
release from rat hepatocytes in culture. Am. J. Physiol. 248,
G93–G97.
43. Baker, E., Morton, A.G. & Tavill, A.S. (1980) The regulation of
iron release from the perfused rat liver. Br. J. Haematol. 45,
607–620.
44. Young, S.P., Fahmy, M. & Golding, S. (1997) Ceruloplasmin,
transferrin and apotransferrin facilitate iron release from human
liver cells. FEBS Lett. 411, 93–96.
45. Nestle, F.P., Greene, R.N., Kichian, K., Ponka, P. & Lapp, W.S.
(2000) Activation of macrophage cytostatic effector mechanisms
during acute graft-versus-host disease: release of intracellular iron
and nitric oxide-mediated cytostasis. Blood 96, 1836–1843.