Originally published In Press as doi:10.1074/jbc.M303158200 on May 5, 2003
J. Biol. Chem., Vol. 278, Issue 33, 31340-31351, August 15, 2003
Yeast Frataxin Sequentially Chaperones and Stores Iron by Coupling Protein Assembly with Iron Oxidation*
Sungjo Park 
,
Oleksandr Gakh ,
Heather A. O'Neill **,
Arianna Mangravita 
,
Helen Nichol ¶,
Gloria C. Ferreira and
Grazia Isaya ||
From the
Departments of Pediatric & Adolescent
Medicine and Biochemistry & Molecular Biology, Mayo Clinic and Foundation,
Rochester, Minnesota 55905, the Department of
Biochemistry & Molecular Biology, College of Medicine and H. Lee Moffitt
Cancer Center and Research Institute, University of South Florida, Tampa,
Florida 33612, and the ¶Department of Anatomy and
Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon S7N
5E5, Canada
Received for publication, March 27, 2003
, and in revised form, May 1, 2003.
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ABSTRACT
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We have investigated the mechanism of frataxin, a conserved mitochondrial
protein involved in iron metabolism and neurodegenerative disease. Previous
studies revealed that the yeast frataxin homologue (mYfh1p) is activated by
Fe(II) in the presence of O2 and assembles stepwise into a
48-subunit multimer (
48) that sequesters >2000 atoms of
iron in 2–4-nm cores structurally similar to ferritin iron cores. Here
we show that mYfh1p assembly is driven by two sequential iron oxidation
reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first
assembly step ( 
3), followed by a slower
autoxidation reaction that promotes the assembly of higher order oligomers
yielding 48. Depending on the ionic environment, stepwise
assembly is associated with accumulation of 50–75 Fe(II)/subunit.
Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized
by chelators or made available to the mitochondrial enzyme ferrochelatase to
synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in
the presence of citrate, a physiologic ferrous iron chelator, suggesting that
the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is
not transferred to a ligand, iron oxidation, and mineralization proceed to
completion, Fe(III) becomes progressively less accessible, and a stable
iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a
novel mechanism by which yeast frataxin can function as an iron chaperone or
an iron store.
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INTRODUCTION
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Mitochondria require micromolar concentrations of iron to support the heme
and the iron-sulfur cluster biosynthetic pathways
(1,
2). Making this iron
bioavailable while limiting its participation in free radical reactions is an
essential function accomplished by mechanisms that remain largely
uncharacterized
(2–4).
The importance of these mechanisms is exemplified by Friedreich ataxia (FRDA),
a severe neuro- and cardio-degenerative disease
(5) in which mitochondria lack
the ability to handle iron properly (reviewed in Ref.
6). FRDA is caused by defects
in frataxin, a conserved nucleus-encoded mitochondrial protein of as yet
unknown function (6,
7). Studies in
Saccharomyces cerevisiae have shown that the loss of frataxin results
in accumulation of iron in mitochondria, widespread oxidative damage to
mitochondrial and nuclear DNA via Fenton chemistry, and impaired respiration
(8–11).
This phenotype can be explained by new findings that yeast frataxin is
required for the biosyntheses of iron-sulfur clusters
(12–16)
and heme (17), two processes
critical for maintenance of mitochondrial iron homeostasis
(18,
19).
An open question is how frataxin influences two different iron-dependent
pathways and also provides protection from iron toxicity. We have proposed
that such diverse roles could be reconciled if the basic function of frataxin
were to bind and store iron in a bioavailable and nontoxic form
(20). Our studies with
recombinant yeast frataxin have shown that the protein is activated by Fe(II)
in the presence of O2 and forms an oligomeric species
(3) that catalyzes Fe(II) oxidation
(21). When the Fe(II)
concentration exceeds the iron-loading capacity of 3,
stepwise assembly of 3 oligomers yields a 48-subunit
multimer (48) that sequesters 
2,400 atoms of ferric
iron. The multimer is a regular spherical particle with a hydrodynamic radius
of 11 nm and contains small iron cores of 2–4 nm
(22) with Fe-O and Fe-Fe
interactions similar to those found in ferritin iron cores
(23). Similarly, recombinant
human frataxin assembles during expression in Escherichia coli
yielding regular spherical particles of 1 MDa and ordered polymers of
these particles that sequester up to 10 atoms of iron per subunit in small
cores structurally identical to the yeast frataxin iron cores
(23). High molecular weight
forms of frataxin can be detected by gel filtration and Western blotting in
yeast cells or mouse cardiac tissue, and the native protein binds
stoichiometric amounts of 55Fe in metabolically labeled yeast cells
(24,
25). These previous findings
support the idea that frataxin, like ferritin, has an iron storage role. Here,
we have tested if frataxin might also serve as a reservoir of bioavailable
iron. We describe the coupled stepwise-assembly/iron-oxidation reaction of
yeast frataxin and show that this mechanism is compatible with both iron
chaperone and storage functions.
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EXPERIMENTAL PROCEDURES
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Reagents, Solutions, and Purified Proteins—HEPES, ferrous
ammonium sulfate, potassium chloride, -'-bipyridine
(BIPY),1 EDTA,
dithionite, deuteroporphyrin IX, pyridine, sodium citrate, and bovine serum
albumin were from Sigma, and bovine brain calmodulin from Calbiochem. All
buffers and solutions were made with milli-Q-deionized water (18
M
). Stock solutions of ferrous ammonium sulfate (2–10
mM) were freshly prepared in water previously deaerated by purging
with argon gas (<0.2 ppm O2). Calmodulin and albumin were
desalted into the appropriate buffer using NAP-25 columns (Amersham
Biosciences). The mature forms of yeast frataxin (mYfh1p and mYfh1p[C98A]) and
yeast ferrochelatase were expressed in E. coli
(24,
26) and purified as previously
described (22,
27). The construct for
expression of mYfh1p[C98A] was created via PCR-mediated site-directed
mutagenesis as previously described
(24). Human H- and L-chain
apoferritin homopolymers (28)
(designated H- and L-apoferritin) were a generous gift of P. Arosio (Brescia
University, Brescia, Italy) and S. Levi (Ospedale San Raffaele, Milano,
Italy). Protein concentration was determined from the absorbance and
extinction coefficient (
280 nm = 20,000/44,200/27,900/34,000
M–1 cm–1
for mYfh1p, bovine serum albumin, H- and L-chain apoferritin, respectively,
and 276 nm = 3,000 M–1
cm–1 for calmodulin). Iron concentration was
either directly measured by inductively coupled plasma mass spectrometry
(ICP-MS) at the Mayo Metals Laboratory or deduced from the concentration of
Fe[BIPY]32+ (520 nm = 9,000
M–1 cm–1)
(21).
Electrode Oximetry, Ultrafiltration, Gel Filtration, and Fluorescence
Measurements—Measurements of dissolved O2 concentration
were performed with a MI-730 micro-O2 electrode (Microelectrodes,
Inc.) (21). The drift of the
electrode was 2 µM/60 min at 30 or 20 °C. Iron binding
by mYfh1p and other proteins were measured by ultrafiltration with a molecular
mass cutoff of 5 kDa (21). To
analyze stepwise assembly of 48, independent samples
containing identical concentrations of mYfh1p and Fe(II) were incubated at 30
°C for different periods of time. Each sample was rapidly cooled down to 4
°C to stop assembly, and analyzed by Superdex 200 or Sephacryl 300 gel
filtration (22). Tryptophan
fluorescence intensity was measured in a Quanta Master fluorimeter (Photon
Technology International, Ontario, Canada) with a monochromator bandwidth of
2–4 nm and a pathlength of 4 mm. Excitation was at 294 nm, and
tryptophan emission was quantitated from the area under the emission band
integrated from 300 to 400 nm.
Fe[BIPY]32+ and
Ferrochelatase Assays—Fe(II) was added to purified mYfh1p monomer,
H- or L-apoferritin, or calmodulin in 10 mM HEPES-KOH, pH 7.3, and
each sample (8 ml) was incubated at 30 °C. Two aliquots were withdrawn at
the indicated time points. BIPY was added to the first aliquot (500 µl) at
a final concentration of 2 mM, and after 5 min of incubation at
room temperature, the concentration of
Fe[BIPY]32+ was determined
(21). Ferrochelatase and
deuteroporphyrin IX were added to the second aliquot (300 µl) at final
concentrations of 2 and 118 µM, 2 and 200 µM, or 4
and 400 µM, respectively, and incubation was continued for an
additional 20 min at 30 °C. The ferrochelatase reaction was stopped by
adding 1 M NaOH and pyridine (176 µl each), and
iron-deuteroporphyrin was measured by the pyridine hemochromogen method
(29) with a
(545–530) nm = 15.3
mM–1 cm–1
for the (reduced – oxidized) difference spectrum
(30). Competition assays were
designed to test if transfer of Fe(II) from mYfh1p to ferrochelatase can occur
in the presence of a Fe(II) chelator, as was done by others to study the
transfer of copper from a metallochaperone to its target protein
(31). Unlike in the transfer
equilibrium between a copper chaperone and its target protein, we measured the
end product of the transfer reaction, i.e. heme. Thus, upstream and
downstream steps that are also susceptible to iron chelation had to be
considered in choosing an appropriate chelator. We have shown that mYfh1p
assembles stepwise into an 840-kDa molecule sequestering up to 50–75
Fe(II) atoms per subunit; this iron is accessible to direct chelation until it
is oxidized and incorporated into a stable ferrihydrite mineral (Refs.
22 and
24 and this study). Yeast
ferrochelatase is a homodimer of 80 kDa containing one Fe(II) substrate
binding site and one protoporphyrin binding cleft per subunit; heme synthesis
requires the insertion of the Fe(II) atom into the porphyrin ring
(32). A second site in each
ferrochelatase subunit is thought to be involved in the initial Fe(II) binding
or enzyme regulation (32).
Citrate is a relatively weak Fe(II) chelator (Fe(II)-binding constant =
104 M–1)
(33) believed to represent one
of the most abundant ligands to the "free" iron pool in
vivo (Refs. 19 and
34 and Refs. therein). In
ferrochelatase assays performed under strictly anaerobic conditions, Fe(II)
can be provided as a ferrous citrate salt
(35). Thus, citrate should not
be able to remove the Fe(II) ion from ferrochelatase after the transfer or to
destabilize heme, as has been shown to occur with thiol reagents
(36). Moreover, at the neutral
pH and under the aerobic conditions used in our assays, citrate will promote
rapid autoxidation of Fe(II)
(37) such that any
mYfh1p-bound Fe(II) mobilized by citrate will be rapidly oxidized and excluded
from the reaction. Both citrate and a stronger chelator, EDTA (EDTA
Fe(II)-binding constant = 1014 M–1)
(38), were used in competition
assays. We used citrate/total iron ratios ranging from 0.06:1 to 166:1, and
citrate/ferrochelatase ratios ranging from 1:1 to 2500:1, which encompass and
exceed the ratios used in anaerobic ferrochelatase assays (citrate/Fe = 1:1;
citrate/ferrochelatase = 28:1)
(35). EDTA/total iron ratios
ranged from 0.33:1 to 7:1 and EDTA/ferrochelatase ratios from 5:1 to
100:1.
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RESULTS
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Stepwise Assembly of mYfh1p Is Coupled with Two Sequential Iron
Oxidation Reactions—At Fe(II)/mYfh1p ratios 
0.5, mYfh1p
catalyzes Fe(II) oxidation with a stoichiometry of 2 Fe(II)/O2
and production of H2O2 (ferroxidase reaction)
(21). A 50-kDa oligomer
(3) is responsible for this activity suggesting that three
mYfh1p subunits may form one binuclear ferroxidation site
(21). Here, we have analyzed
the iron oxidation reaction of mYfh1p at concentrations of iron (40–75
Fe(II)/mYfh1p) that encompass the iron loading capacity of mYfh1p (50–75
Fe(III)/mYfh1p depending on the ionic environment) and result in stepwise
assembly of 3 to yield iron-loaded 48
(22,
24).
Fig. 1A shows
representative O2 consumption curves recorded when 100
µM Fe(II) was incubated in 10 mM HEPES-KOH, pH 7.3,
in the absence or presence of 2 µM mYfh1p (Fe(II)/mYfh1p =
50/1). In buffer without protein there was an initial lag phase due to the
time required to generate sufficient hydrolyzed Fe(III) to initiate
autoxidation of Fe(II) (39)
(Fig. 1A, black
plot). The final Fe(II)/O2 stoichiometry was 3.7 ± 0.3
(n = 3) as expected for autoxidation
(39). In the presence of
mYfh1p, the initial rate of O2 consumption was faster compared with
buffer, consistent with ferroxidase activity, but became slower after the
first 4 min (Fig. 1A,
red plot). The final Fe(II)/O2 stoichiometry was 3.6
± 0.3 (n = 3), indicating that ferroxidation was rapidly
overcome by autoxidation. We were unable to detect any
H2O2 released into the solution, which should be
expected given the high Fe(II)/mYfh1p ratio used in these experiments
(21,
40).
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FIG. 1.
Stepwise assembly of mYfh1p is coupled with a twophase iron oxidation
reaction. A, O2 consumption curves were recorded in 10
mM HEPES-KOH, pH 7.3, at 30 °C for 2 µM mYfh1p or
buffer only (red and black plot, respectively) upon addition
of 100 µM Fe(II). The inset shows the initial faster
phase; the arrow indicates the postulated transition from
ferroxidation to autoxidation. B, samples containing 4
µM mYfh1p and 200 µM Fe(II) were incubated in 10
mM HEPES-KOH, pH 7.3, at 30 °C for different times. Each sample
was cooled down on ice to stop assembly, and immediately centrifuged for 5 min
at 20,000 x g and chromatographed through a Superdex 200 column
(16 mm x 50 cm) at 4 °C. A sample containing 4 µM
mYfh1p but no added Fe(II) was similarly analyzed (). The elution
profiles of the mYfh1p samples are superimposed on that of molecular weight
standards. V, vitamin B12 (1.4 kDa); M, myoglobin
(17 kDa); O, ovalbumin (44 kDa); G, gamma globulin (158
kDa); T, thyroglobulin (669 kDa). The A280 of
mYfh1p and molecular weight standards is shown on the left- and
right-hand y-axes, respectively. V0
denotes void volume as determined by the elution volume of blue dextran (2
MDa).
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In Fig. 1B, gel
filtration was used to determine the speciation of mYfh1p during the iron
oxidation reaction described above. Experimental conditions were similar to
those employed for electrode oximetry in
Fig. 1A except that
both the protein and the iron concentrations were increased 2-fold to enable
detection of mYfh1p by absorbance measurements. Such an increase is not
expected to change the rate of 48 assembly to a significant
degree (22,
24), and therefore the gel
filtration data (Fig.
1B) can be correlated with the two phases in the
O2 consumption curve of mYfh1p
(Fig. 1A). The initial
faster phase (Fig. 1A,
0–4 min, red plot) was associated with the assembly of an
oligomer of 50 kDa (Fig.
1B, 3 min), while the subsequent slower phase
(Fig. 1A, 4–50
min, red plot) was associated with stepwise assembly of higher order
oligomers (Fig. 1B, 6,
10, and 30 min), in agreement with the previously described progression,
3 6
12 24 48
(22,
24). The
A280 of assembled mYfh1p species increased in a
time-dependent manner (Fig.
1B). At the end of the iron oxidation reaction, the
A280 of 48 was much higher than the
A280 of monomer analyzed in the absence of any added
Fe(II) (Fig. 1B,
peak ). These results are consistent with progressive
oxidation of Fe(II) to Fe(III) and formation of a ferrihydrite-like mineral
(which, unlike Fe(II), absorbs at 280 nm) within the assembled protein
(23).
We showed previously that the mature form of Yfh1p is generated by cleavage
of an N-terminal mitochondrial targeting signal between residues 51–52
(24,
41). This cleavage eliminates
one cysteine residue at position 32. The mature form of the protein (amino
acids 52–174), which is the form used in our experiments, contains one
cysteine residue at position 98. Thus, an alternative explanation for the
results in Fig. 1B
could be that chelation of iron by cysteine residues from different mYfh1p
subunits leads to formation of metal-thiolate aggregates
(42). However, others have
reported that when yeast frataxin is treated with iodoacetamide to block any
exposed cysteine residues and subsequently incubated with 20 equivalents of
Fe(II), iron-dependent oligomerization is not affected
(43). We obtained similar
results using a mYfh1p variant in which cysteine 98 was replaced by an alanine
residue (data not shown). We therefore conclude that mYfh1p assembly is driven
by iron oxidation: A ferroxidase reaction catalyzed by mYfh1p is associated
with the first assembly step ( 3), followed by
a slower autoxidation reaction associated with assembly of higher order
oligomers to ultimately yield 48.
The Ferrous Iron Sequestered by mYfh1p Is Bioavailable— The
time required to complete the iron oxidation reaction of mYfh1p is in the
order of hundreds of seconds (Fig.
1A), much longer than the iron oxidation reaction of
ferritin, which is in the order of tens of seconds (Ref.
40 and data not shown). At the
beginning of its reaction, however, mYfh1p rapidly sequesters up to
50–75 Fe(II)/subunit, which are then progressively oxidized within the
assembled protein (Ref. 21,
22, and
24 and data presented below).
Given that mobilization of iron from ferritin is inefficient in the absence of
reducing agents (44), we
hypothesized that the coupling of a slow iron autoxidation reaction with
stepwise assembly might enable mYfh1p to serve as a temporary reservoir and a
chaperone for Fe(II). We therefore measured iron mobilization during mYfh1p
assembly by use of ,'-bipyridine (BIPY), a chelator that
preferentially binds Fe(II)
(45), or purified yeast
ferrochelatase, a mitochondrial enzyme that catalyzes the insertion of Fe(II)
into protoporphyrin IX to yield heme (reviewed in Ref.
46). Iron mobilization from
human H- or L-apoferritin (28)
was analyzed in parallel. These two proteins are pre-assembled 24-subunit
shells with a negatively charged inner surface that promotes iron autoxidation
and mineralization; in addition, H-apoferritin has 24 dinuclear ferroxidation
sites (28,
47). As a negative control we
used calmodulin, a calcium-binding protein with a molecular mass and an
isoelectric point similar to those of mYfh1p (17 versus 14 kDa, and
4.09 versus 4.34). Reactions were started by addition of a fixed
concentration of Fe(II) (30 µM) to buffer in the absence or
presence of protein (0.4 µM; Fe(II)/subunit = 75:1 for all
proteins tested). At successive time points, an aliquot was withdrawn and
divided in two parts that were immediately incubated with either BIPY (2
mM) or ferrochelatase (2 µM) and deuteroporphyrin IX
(118 µM). The half-life of BIPY-accessible iron estimated from a
single exponential fitting was 21.5 min in the presence of mYfh1p compared
with 1.0, 4.0, 6.7, and 8.8 min in the presence of H-apoferritin,
L-apoferritin, buffer only, and calmodulin, respectively
(Fig. 2A). Similarly,
ferrous iron was more accessible to ferrochelatase in the presence of mYfh1p
relative to buffer or calmodulin (Fig.
2B). BIPY can bind Fe(III) and/or reduce Fe(III) to
Fe(II) although with lower affinity compared with Fe(II)
(48–50),
suggesting that the BIPY accessible iron mobilized from mYfh1p could represent
a mixture of both ferrous and ferric iron. However, the concentrations of
BIPY-accessible iron at successive time points in the presence of mYfh1p
(Fig. 2A) were in the
same order as the concentrations of ferrochelatase-accessible iron measured
under similar conditions (Fig.
2B). We therefore conclude that the iron mobilized by
direct chelation (i.e. BIPY accessible iron) during mYfh1p assembly
is largely in ferrous form, becoming progressively less accessible as it is
oxidized to Fe(III). The Fe(II) that can be mobilized by direct chelation can
also be donated to ferrochelatase. This should involve a direct
mYfh1p-ferrochelatase interaction given that there was no deuteroheme
synthesis in samples containing Fe(II), mYfh1p, and deuteroporphyrin IX but
not ferrochelatase (data not shown).
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FIG. 2.
Yeast frataxin promotes Fe(II) availability. Time courses of
A, Fe[BIPY]32+ and B,
deuteroheme synthesis were started by adding 30 µM Fe(II) to
samples containing 0.4 µM mYfh1p, calmodulin, L-apoferritin, or
H-apoferritin, or buffer without added protein. Conditions were 10
mM HEPES-KOH, pH 7.3, at 30 °C. BIPY was added at a final
concentration of 2 mM. Yeast ferrochelatase and deuteroporphyrin IX
were added at final concentrations of 2 µM and 118
µM, respectively. The levels of
Fe[BIPY]32+ and deuteroheme were determined
as described under "Experimental Procedures." The bars
represent the mean ± S.D. of 3 (each protein) or 5 (buffer) independent
measurements. The traces show single exponential fittings to the
data. C, heme synthesis assays were performed under the conditions
used in B. Three sets of assays were analyzed: mYfh1p-Fe(II) + FC +
[Cit + PPIX], mYfh1p, and Fe(II) were incubated for 15 min, ferrochelatase
(FC) was added for 5 min, followed by citrate (Cit) and protoporphyrin IX
(PPIX), and the incubation continued for 20 min (n = 2);
mYfh1p-Fe(II) + Cit + [FC + PPIX], same as above except that citrate was added
to mYfh1p-Fe(II) for 5 min, followed by ferrochelatase and protoporphyrin IX
(n = 1); mYfh1p-Fe(II) + Cit + FC + PPIX, mYfh1p and Fe(II) were
incubated for 20 min, then citrate, ferrochelatase, and protoporphyrin IX were
added in this order (n = 1). Heme levels measured at the end of each
assay are plotted versus the citrate concentration.
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Transfer of Fe(II) from mYfh1p to Ferrochelatase Occurs in the Presence
of Citrate—Copper transfer from a metallochaperone to its target
protein is not affected by the presence of the physiologically relevant Cu(I)
binding agent, glutathione, indicating that the transfer occurs via an
intermolecular interaction
(31). We investigated if
transfer of Fe(II) from mYfh1p to ferrochelatase can occur in the presence of
a physiologically relevant Fe(II) binding agent, citrate (Refs.
19 and
34 and Refs. therein). In
Fig. 2C, reactions
were started by addition of a fixed concentration of Fe(II) (30
µM) to buffer in the presence of 0.4 µM mYfh1p,
under the conditions used in the time courses of Fe(II) availability in
Fig. 2, A and
B. Three different sets of additions followed. In set 1,
2 µM ferrochelatase was added to mYfh1p-Fe(II) after 15 min of
incubation, and the incubation continued for another 5 min to allow putative
protein-protein interactions to take place. Then, citrate and deuteroporphyrin
IX (120 µM) were added in rapid sequence and the incubation
continued for an additional 20 min, after which deuteroheme levels were
measured. In set 2, Fe(II) was incubated with mYfh1p for 15 min as above,
after which citrate was added and the incubation continued for 5 min to allow
the chelator to access mYfh1p-Fe(II) prior to the putative docking of
ferrochelatase onto mYfh1p. Then, ferrochelatase and deuteroporphyrin IX were
added in rapid sequence. In set 3, Fe(II) was incubated with mYfh1p for 20 min
after which citrate, ferrochelatase, and deuteroporphyrin IX were added in
rapid sequence. The time courses in Fig. 2,
A and B indicate that 20 min after addition of
30 µM Fe(II) to 10 mM HEPES-KOH, pH 7.3, at 30
°C, little residual Fe(II) is present in buffer without mYfh1p (<3
µM; Fig.
2A, black plot) while 16 µM
Fe(II) is still available in the presence of mYfh1p
(Fig. 2A, red
plot). Therefore, in all the three sets described above, heme synthesis
will largely depend on 16 µM mYfh1p-Fe(II). In all cases,
the small size of citrate (<9 Å) may allow this compound to penetrate
the protein and directly access mYfh1p-Fe(II), similar to mobilization of
ferritin-iron by direct-chelation
(44). However, at neutral pH
and in the presence of atmospheric O2, which are the conditions
used in these assays, citrate will promote rapid autoxidation of Fe(II)
(37). Therefore, any
mYfh1p-Fe(II) mobilized by citrate will be rapidly oxidized and excluded from
the reaction. If ferrochelatase has a high affinity for binding to mYfh1p, as
would be expected for a specific intermolecular interaction, the yield of the
transfer reaction should be the same between set 1 and set 3. If docking of
ferrochelatase onto mYfh1p hampers the ability of citrate to penetrate mYfh1p
and directly chelate Fe(II), the yield of the transfer reactions in set 2
should be lower compared with set 1 and set 3. In set 1, the addition of
2–500 µM citrate (Fe(II)-binding constant = 104
M–1) resulted in a 6–25% drop in heme
levels; however, increasing the citrate concentration to 2 and 5 mM
(corresponding to a 66–166-fold molar excess over the total iron
concentration and a 1000–2500-fold molar excess over the ferrochelatase
concentration) did not cause any significant additional decrease
(Fig. 2C, gray
plot). Compared with set 1, heme synthesis was further decreased in set 2
(Fig. 2C, pink
plot) but remained unchanged in set 3
(Fig. 2C, blue
plot). These results can be explained by two parallel reactions: (i)
transfer of Fe(II) from mYfh1p to ferrochelatase yielding heme; (ii) direct
chelation of mYfh1p-Fe(II) by citrate. It appears that mobilization of
mYfh1p-Fe(II) by citrate increased with increasing chelator concentrations up
to a maximum limited value that was augmented if the chelator was added to
mYfh1p-Fe(II) 5 min before the addition of ferrochelatase
(Fig. 2C, pink
plot). We will show below that a fraction of mYfh1p-Fe(II) can be
released into the solution during ultrafiltration. The fraction of
mYfh1p-Fe(II) accessible to citrate most likely corresponds to this labile
mYfh1p-Fe(II) pool (see Table
II, 10 min). Thus, in the three sets of assays shown in
Fig. 2C, the Fe(II)
consumed by citrate affected the yield of the transfer reaction. However, once
chelation of Fe(II) by citrate reached its maximum, heme levels did not change
significantly even in the presence of a large excess of citrate. This
indicates that the transfer reaction is mostly independent of the presence of
a physiologic ferrous iron chelator, suggesting that the transfer occurs via a
specific intermolecular interaction. Two alternative ways by which Fe(II)
transfer might occur include (i) release of Fe(II) from mYfh1p into the
solution, and (ii) release of Fe(II) from mYfh1p via general pores on the
protein surface. In these scenarios, ferrochelatase and citrate would directly
compete for the same Fe(II) pool or the same docking sites. Under either
condition, heme synthesis would be expected to decrease proportionally to an
increase in the citrate concentration which is not observed in
Fig. 2C. Therefore,
the results in Fig. 2C
better fit a model where ferrochelatase and citrate access mYfh1p-Fe(II) via
different paths. Furthermore, the higher levels of heme detected in set 1 and
set 3 compared with set 2 suggest that docking of ferrochelatase onto mYfh1p
may hamper the ability of citrate to penetrate mYfh1p. Addition of
10–200 µM EDTA (Fe(II)-binding constant = 1014
M–1)
(38) resulted in a marked
inhibition of heme synthesis (30% heme synthesized relative to assays
without EDTA). However, this strong chelator is expected to mobilize most
mYfh1p-bound Fe(II) as is the case for BIPY
(Fig. 2A; see also
Fig. 3). EDTA could also
chelate Fe(II) from ferrochelatase after the transfer and/or destabilize heme,
as has been reported for thiol reagents
(36).
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FIG. 3.
Iron oxidation stabilizes mYfh1p assemblies. Samples containing 40
µM mYfh1p and 1.6 mM Fe(II) were incubated in 10
mM HEPES-KOH, pH 7.3, at 30 °C for different times. Each sample
was cooled down on ice, and immediately centrifuged for 5 min at 20,000
x g and chromatographed through a Superdex 200 column (10 mm
x 30 cm) at 4 °C (A–F, black plots). For each time
point, a duplicate sample was incubated for the appropriate time at 30 °C,
20 mM EDTA was added, and the incubation continued for 1 h at 30
°C (A–D, red plots). In other duplicate samples, 16
mM dithionite was added instead of EDTA, and the incubation
continued for 10 min at 30 °C(E–F, orange plots). Each
sample was then treated and analyzed by gel filtration as described above. An
equal amount of monomer without any added iron was similarly analyzed
(green plot). The peaks denoted by asterisks probably
represent dithionite and small levels of ferric oxides.
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FIG. 5.
Salt effects on the rate of iron autoxidation and mYfh1p assembly.
A–C, O2 consumption curves for mYfh1p (red
plots) or buffer (black plots), and D, time course of
mYfh1p assembly. Conditions were as described in the legend of
Fig. 1, A and
B, respectively, except that assays were carried out in
(A, D) 10 mM HEPES-KOH, pH 7.3, 150 mM KCl,
(B) 10 mM HEPES-KOH, pH 8.0, 150 mM KCl, or
(C) 10 mM HEPES-KOH, pH 7.3, 10 mM
MgCl2. In A and B, in the presence of mYfh1p
there is some initial overshoot of the electrode of 3 µM
O2, indicating that the electrode response time (half-life = 6 s)
is not adequate to monitor the fast reaction under these conditions.
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FIG. 6.
Salt effects on the availability of mYfh1p-Fe(II). A, time
courses of Fe[BIPY]32+ synthesis were started
by adding 100 µM Fe(II) to samples containing 2 µM
mYfh1p or buffer without added protein. Conditions were 10 mM
HEPES-KOH, pH 7.3, at 30 °C in the absence or presence of increasing
concentrations of KCl. The bars represent the mean ± S.D. of
at least three independent measurements. The traces show single exponential
fittings to the data. B, tryptophan fluorescence intensity was
measured at 25 °C for 2 µM mYfh1p in 10 mM
HEPES-KOH, pH 7.3, in the absence (F0) or
presence (F) of increasing concentrations of KCl.
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The Ferrous Iron Sequestered by mYfh1p Is Loosely Associated with the
Protein—To analyze the interaction between mYfh1p and iron during
stepwise assembly, Fe(II) (30 µM) was incubated in the absence
or presence of mYfh1p or L-apoferritin (Fe(II)/subunit = 75:1) under
conditions similar to those used in the BIPY and ferrochelatase assays
described above. After 10 or 60 min of incubation, each sample was subjected
to ultrafiltration with a molecular mass cutoff of 5 kDa
(21). In the absence of
protein, extensive precipitation of insoluble ferric oxyhydroxides was
observed at both time points as expected
(Table I). In the presence of
L-apoferritin, most iron was recovered in a protein-bound form after either 10
or 60 min of incubation and only very little free iron was observed
(Table I), reflecting rapid
oxidation of Fe(II) within the protein shell as also seen in
Fig. 2A. After 10 min
of incubation in the presence of mYfh1p, similar levels of iron (12
µM) were detected in protein-bound and free form
(Table I). After 60 min of
incubation, the mYfh1p-bound iron increased to 19 µM,
corresponding to a Fe/mYfh1p stoichiometry of 50/1
(22,
24), while free iron decreased
to 3 µM (Table
I). Under the conditions used in this experiment, the iron
sequestered by mYfh1p consists of mostly Fe(II) after 10 min of incubation and
Fe(III) after 60 min (see Fig. 2,
A and B). Therefore, a significant proportion of
the Fe(II) sequestered by mYfh1p was released into the solution during
ultrafiltration (Table I, 10
min), indicating that this Fe(II) is loosely bound to the protein. However, if
Fe(II) was allowed to oxidize inside mYfh1p prior to ultrafiltration, iron
release was greatly reduced (Table
I, 60 min). We conclude that Fe(II) is loosely bound to mYfh1p but
it is not released into the solution unless the binding equilibrium is
perturbed as it occurs during ultrafiltration. Once Fe(II) is oxidized to
Fe(III), the iron is more tightly bound to the protein.
Iron Oxidation Stabilizes mYfh1p Assemblies—To analyze iron
mobilization from 48, samples containing 40 µM
protein and 1.6 mM Fe(II) were incubated at 30 °C. These
concentrations changed the reaction kinetics as compared with
Fig. 1B, such that
48 was formed within 2 min of incubation. At different time
points, one sample was rapidly cooled down to 4 °C to stop assembly
(22) and immediately analyzed
by gel filtration. A duplicate sample was first treated with EDTA (20
mM; EDTA/Fe(II) = 12.5), a chelator that binds both Fe(II) and
Fe(III) with high affinities (1014 and 1025
M–1, respectively)
(38), incubated for an
additional 60 min at 30 °C, and finally analyzed by gel filtration. In
samples that had not been treated with EDTA, mYfh1p monomer ()
assembled into 48 and there was a progressive increase in
the A280 of this species at successive time points
(Fig. 3, A–F,
black plots). Addition of EDTA after 2 min of incubation resulted in
disassembly of 48 back to smaller assembly intermediates,
with a concomitant decrease in the A280 due to
mobilization of mYfh1p-bound iron by direct chelation
(Fig. 3A, red
plot). A similar result was obtained upon addition of EDTA after 10 min
of incubation, although the shift from 48 to smaller
assembly intermediates was less pronounced and the levels of EDTA-accessible
iron were significantly decreased compared with the 2-min sample
(Fig. 3B, red
plot). Upon addition of EDTA after 1 or 16 h of incubation, protein
disassembly was no longer observed and there was a time-dependent decrease in
the levels of EDTA-accessible iron (Fig. 3,
C–D, red plots). These results are
consistent with a model in which iron oxidation and mineralization are an
integral part of mYfh1p assembly
(23). The Fe(II) sequestered
by 3 is progressively oxidized and incorporated into a
ferrihydrite crystallite. As the crystallite increases in size, stepwise
assembly of trimers is promoted by the alignment and binding of one
crystallite to another. As mineralization proceeds, the proportion of iron
that can be mobilized by direct chelation decreases, while the stability of
mYfh1p assemblies increases. In agreement with this model, EDTA caused
time-dependent disassembly of 48 into smaller oligomers,
whereas the reducing agent, dithionite, caused quantitative disassembly of
48 back to monomer in a time-independent manner
(Fig. 3, E and
F, orange plots). Others and we have found that
the cysteine residue present in the mYfhp sequence is not required for
stepwise assembly (Ref. 43 and
data not shown). These observations exclude the possibility that the
disassembly induced by treatment with dithionite was due to reduction of
metal-thiolate aggregates.
mYfh1p Catalyzes Oxidation of Fe(II) in Different Ionic
Environments—To investigate a recent report that iron binding by
frataxin takes place only at very low ionic strength
(43), we analyzed whether
mYfh1p exhibits ferroxidase activity in the presence of 150 mM KCl,
close to the concentration believed to exist in mitochondria
(43,
51).
Fig. 4 shows representative
O2 consumption curves recorded when 48 µM Fe(II) was
incubated in 10 mM HEPES-KOH, pH 7.0, 150 mM KCl, in the
absence or presence of 96 µM mYfh1p (Fe(II)/mYfh1p = 0.5).
Except for the presence of 150 mM KCl, these are standard
conditions to detect the ferroxidase activity of mYfh1p
(21). O2
consumption was facilitated in the presence of mYfh1p compared with buffer
without added protein (Fig. 4).
This was not the case for samples containing 96 µM albumin
instead of mYfh1p (Fig. 4,
inset). A stoichiometric Fe(II)/O2 ratio of 2.1 ±
0.3 (n = 3) was determined for the completed reaction of mYfh1p
(Fig. 4 and data not shown),
consistent with the presence of ferroxidase activity
(47,
52). Stoichiometric
Fe(II)/O2 ratios of 3.8 ± 0.3 (n = 2) and 4.6
± 0.2 (n = 2), consistent with autoxidation, were otherwise
measured for the completed reactions of albumin and buffer without added
protein, respectively (Fig. 4
and data not shown). These results demonstrate that mYfh1p binds and catalyzes
oxidation of Fe(II) at physiologic concentrations of salt.
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FIG. 4.
mYfh1p catalyzes oxidation of Fe(II) at physiologic ionic strength.
Oxygen consumption curves were recorded in 10 mM HEPES-KOH, pH 7.0,
150 mM KCl, at 20 °C for 96 µM mYfh1p or buffer
(red and black plot, respectively), or 96 µM
albumin (inset) upon addition of 48 µM Fe(II).
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In aqueous solution, the rate of spontaneous Fe(II) oxidation is influenced
by the ionic strength as well as the interaction of Fe(II) with different
anions
(53–55).
Upon addition of 100 µM Fe(II) to 10 mM HEPES-KOH, pH
7.3, O2 consumption was significantly slowed down in the presence
of 150 mM KCl compared with buffer without added salt (compare
black plots in Figs.
1A and
5A) as expected
(55).
Fig. 5A shows a
representative O2 consumption curve for 2 µM mYfh1p
upon addition of 100 µM Fe(II) (Fe(II)/mYfh1p = 50) in 10
mM HEPES-KOH, pH 7.3, 150 mM KCl. There is an initial
phase (0–2 min) during which Fe(II) oxidation is much faster compared
with buffer without protein, consistent with ferroxidase activity, followed by
a prolonged phase during which Fe(II) oxidation proceeds at a similar slow
rate as in buffer without protein (Fig.
5A). The Fe(II)/O2 stoichiometry for the
completed reactions was 3.7 ± 0.3 (n = 4) for mYfh1p and 4.4
± 0.8 (n = 4) for buffer. A two-phase O2
consumption curve was similarly recorded for 2 µM mYfh1p upon
addition of 100 µM Fe(II) in 10 mM HEPES-KOH, pH 8.0,
150 mM KCl (Fig.
5B)or10mM HEPES-KOH, pH 7.3, 10 mM
MgCl2 (Fig.
5C), conditions that were previously reported to prevent
iron binding by frataxin (43).
These results confirm that two sequential iron oxidation reactions take place
at high Fe(II)/mYfh1p ratios in different ionic environments: A faster
reaction catalyzed by mYfh1p is followed by a slower autoxidation reaction. In
addition, a comparison of the O2 consumption curves for mYfh1p in
Fig. 1A and
5, A–C indicates
that both reactions are influenced by the ionic environment, which may depend
on salt effects on the protein fold (see below) and the reactivity of Fe(II)
toward O2
(53–55).
Salt Affects the Rate of Stepwise Assembly but Not the Iron Binding
Capacity of mYfh1p—Evidence reported above (Figs.
1, A and B
and 3) and elsewhere
(23) is consistent with a
model in which iron oxidation and biomineralization are integral parts of
mYfh1p assembly. Thus, the report that iron-dependent self-assembly of
frataxin is inhibited at physiologic concentrations of salt
(43) might be explained by
salt effects on the kinetics of iron oxidation, and thus on the kinetics of
mYfh1p assembly. We analyzed a time course of mYfh1p assembly under conditions
similar to those used in Fig.
1B except for the presence of 150 mM KCl
during assembly. At the end of a 10-min incubation we could only detect low
levels of 3 and larger oligomers, with 48
becoming detectable at 20 min (Fig.
5D). The rate of ferrihydrite mineral accumulation
(estimated from the increase in the A280 of
48 at successive time points) was slower in the presence of
150 mM KCl compared with buffer without added salt (compare Figs.
1B and
5D), consistent with
the respective O2 consumption curves (Figs.
1A and
5A, red
plots). In addition, the levels of residual monomer (peak )
decreased only minimally at successive time points in the presence of 150
mM KCl (Fig.
5D). One possible interpretation of these results is that
the presence of salt inhibits iron binding by mYfh1p, hence iron-dependent
protein aggregation is also inhibited
(43). On the other hand, iron
binding by mYfh1p is expected to occur efficiently in the assembly reactions
analyzed in Fig. 5D
because the protein exhibits ferroxidase activity under similar experimental
conditions (Figs. 4 and
5A). Thus, an
alternative explanation is that the salt slows down stepwise assembly of
3 to 48 due to the inhibitory effect of
KCl on the rate of Fe(II) autoxidation as discussed above. This results in the
release of Fe(II) during gel filtration leading to protein disassembly,
similar to what we observed in Table
I and Fig. 3. If
this explanation is valid, the iron loading capacity of 48
assembled in the presence of 150 mM KCl should not be impaired. We
therefore analyzed assembly of 48 under conditions similar
to those employed in Fig.
5D except that the protein and the iron concentration
were increased 10- and 8-fold, respectively, to enable determination of the
Fe/mYfh1p stoichiometry. Upon gel filtration, fractions corresponding to
48 were analyzed for protein concentration by SDS/PAGE and
for iron concentration by ICP-MS, and a stoichiometric ratio of
70–75 Fe/mYfh1p was determined (n = 2), which is higher
than the 50/1 ratio determined for 48 samples assembled
in the absence of added salts (Refs.
22 and
24 and
Table I, 60 min). Fractions
from Superdex 200 gel filtration (fractionation range 10–600 kDa)
corresponding to 48 were further analyzed by Sephacryl 300
gel filtration (fractionation range 10 kDa to 1.5 MDa) and the same
macromolecular species of 1 MDa was observed for samples assembled in the
absence or presence of 150 mM KCl (data not shown). We conclude
that physiologic KCl concentrations
(43) slow down the rate of
Fe(II) autoxidation and thereby influence the rate at which
3 assembles into 48, but do not impair and
may even improve the iron loading capacity of mYfh1p. An analogous effect has
been described for ferritins, where the more slowly incorporating L-rich
variants have larger iron cores than H-rich ferritins
(56).
The Iron Chaperone Properties of mYfh1p Are Enhanced by Its Stepwise
Assembly—A possible advantage of being able to control Fe(II)
autoxidation and stepwise assembly via changes in the ionic environment might
be to prolong the availability of the Fe(II) bound to mYfh1p. To test this,
salt effects on iron mobilization from mYfh1p were analyzed by incubating 100
µM Fe(II) in the absence or presence of 2 µM
mYfh1p in 10 mM HEPES-KOH, pH 7.3, at increasing KCl
concentrations. Fig.
6A shows that more iron is accessible to BIPY and for a
longer time in the presence of mYfh1p compared with buffer. Moreover, this
effect is enhanced at increasing salt concentrations. This is not the case for
buffer without protein where added salt has little or no effect on iron
accessibility to BIPY after the first 10 min of incubation
(Fig. 6A). There was a
progressive increase in tryptophan fluorescence intensity when mYfh1p monomer
was incubated in the presence of increasing KCl concentrations, with a maximum
change between 100 and 150 mM KCl
(Fig. 6B), suggesting
that salt concentrations close to physiologic conditions may influence the
fold of the protein and improve its function.
Table II further shows that
mYfh1p keeps more iron available to BIPY or ferrochelatase and for a longer
time relative to buffer even though the concentration of Fe(II) decreases at a
similar rate in samples with or without mYfh1p
(Table II). These results
demonstrate that mYfh1p does not simply provide a passive storage compartment
for Fe(II), but also enhances the availability of the stored Fe(II), possibly
by keeping it at a high concentration within the protein. At any given time
point in the presence of mYfh1p, the concentration of BIPY-accessible iron
reflects the estimated concentration of residual Fe(II)
(Table II). In contrast, the
concentration of ferrochelatase-accessible iron is lower at 10 min but becomes
comparable to the estimated Fe(II) concentration at later time points
(Table II, 30 and 60 min).
Nearly identical results were obtained with two different concentrations of
ferrochelatase and deuteroporphyrin IX (2 or 4 µM and 200 or 400
µM, respectively), which should exclude the possibility that
these two reagents were limiting relative to the available Fe(II). A similar
effect is seen in Fig. 2, A and
B, where the concentration of deuteroheme is
significantly lower than that of Fe[BIPY]32+
at the two earliest time points but not at later time points. From these data
it would appear that the transfer of Fe(II) from mYfh1p to ferrochelatase
becomes more efficient as mYfh1p assembles into progressively larger
oligomers.
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DISCUSSION
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The need to maintain a supply of bioavailable iron while avoiding iron
toxicity is a central problem in biology
(57). In aqueous solutions
under aerobic conditions, iron toxicity depends on its tendency to catalyze
production of free radicals as illustrated by the iron-catalyzed Haber-Weiss
reaction (57) shown in
Equations
1,2,3.
 | (Eq. 1) |
 | (Eq. 2) |
 | (Eq. 3) |
Our work indicates that the mature form of the yeast frataxin homologue
(mYfh1p) has a mechanism to control iron oxidation and availability in
vitro. Initial studies showed that in the presence of Fe(II) and
O2, mYfh1p monomer assembles stepwise yielding 3
6 12
24 48. The assembly intermediates
in this progression were found to accumulate increasing Fe(III) levels,
reaching an apparent maximum loading capacity of 50 Fe(III) per subunit in
48 (22,
24). We have recently shown
that two different iron oxidation reactions take place during the initial
assembly step ( 3). A ferroxidase reaction
with a stoichiometry of 2 Fe(II)/O2 is evident at
Fe(II)/mYfh1p ratios 0.5, while an autoxidation reaction with a
stoichiometry of 4 Fe(II)/O2 becomes predominant at ratios
between 0.5 and 1.5 (21). At
Fe(II)/mYfh1p ratios 0.5, only a small fraction of the
H2O2 expected from the ferroxidase reaction
(47,
52) is detected in solution,
and concomitant oxidative degradation of mYfh1p suggests that most
H2O2 reacts with the protein itself
(21). Here, we have analyzed
the reaction of mYfh1p at saturating concentrations of iron (40–75
Fe(II)/mYfh1p). These ratios were chosen because they encompass the iron
loading capacity of mYfh1p and result in stepwise assembly of
3 to yield iron-loaded 48
(22,
24). Under these conditions, a
faster initial phase is rapidly overcome by a slower phase, with a final
stoichiometric Fe(II)/O2 ratio of 4 and no detectable
H2O2 released in the solution, consistent with
ferroxidase activity being rapidly overcome by autoxidation (Figs.
1A,
5, A–C, and data
not shown). The initial phase correlates with assembly of 3,
while autoxidation is associated with assembly of higher order oligomers
yielding 48 (Figs.
1B and
5D). Oligomerization
enables mYfh1p to rapidly sequester up to 50–75 Fe(II)/subunit depending
on the ionic environment. Initially, Fe(II) is loosely bound to the protein
and can be readily mobilized, whereas ferric iron is more tightly bound
(Tables I and
II; Figs.
2,
3, and
6A). We therefore
postulate that the mYfh1p reaction is as follows (at Fe(II)/mYfh1p ratios 
0.5) in Equation 4,
 | (Eq. 4) |
where (mYfh1p)n represents any of the species formed during
stepwise assembly.
This reaction would appear to provide two main advantages compared with
spontaneous Fe(II) oxidation in solution (see
Equation 1): (i) H2O
is expected to represent the predominant product of O2 reduction;
(ii) the iron bound to mYfh1p is in a readily accessible form until it is
converted to a water-soluble ferrihydrite mineral
(23), which is stored within
the assembled protein.
One limitation is that our analyses were carried out under controlled
conditions of pH (7, 7.3, or 8), temperature (20 or 30 °C), and ionic
strength (0.01–0.15 N), which are different from the much
more complex mitochondrial matrix environment in living cells. The
mitochondrial matrix has an alkaline pH of 8
(58) and contains significant
concentrations of certain salts (2 µM CaCl2, 0.8
mM MgCl2, and 100 mM KCl) and iron-chelating
molecules (51,
59). These factors are known
to influence the rate of Fe(II) oxidation
(33,
60) and could interfere with
the iron oxidation reaction of mYfh1p. However, the ferroxidase activity of
mYfh1p (Figs. 4 and
5, A–C), as well
as its iron loading capacity and ability to enhance iron availability
(Fig. 6A) were
conserved at salt concentrations and pH values close to those believed to
exist in the mitochondrial matrix. This suggests that what the protein can do
in vitro most likely reflects its function in vivo.
Current reports strongly suggest that yeast frataxin controls the iron
required for the in vivo biosyntheses of iron-sulfur clusters
(13–16)
and heme (17). A recent study
further shows that human frataxin functions as an iron donor for assembly of
[2Fe-2S] clusters in ISU-type proteins in vitro
(61). Here, we performed
deuteroheme synthesis assays to assess the availability of the Fe(II) bound to
mYfh1p using a physiologic Fe(II) chelator, i.e. the mitochondrial
enzyme ferrochelatase. Given that there was no deuteroheme synthesis in
samples lacking ferrochelatase (data not shown), we postulate that Fe(II) is
near the outer surface of mYfh1p whereby it is transferred to ferrochelatase.
We have shown that this transfer can occur in the presence of an excess of a
physiologic ferrous iron chelator (Fig.
2C) and at physiologic ionic strength
(Table II), and becomes more
efficient as mYfh1p assembles into progressively larger oligomers
(Table II). These data strongly
suggest that transfer of Fe(II) from mYfh1p to ferrochelatase involves an
intermolecular interaction. This conclusion is in accord with a recent report
showing that: (i) zinc-protoporphyrin, not heme, is synthesized in yeast cells
lacking Yfh1p, consistent with a specific role of frataxin in making iron
available to ferrochelatase, and (ii) Yfh1p and ferrochelatase physically
interact with each other in Biacore experiments
(17).
The ability of yeast frataxin to provide iron to such diverse proteins as
ISU-type proteins and ferrochelatase indicates that frataxin is different from
copper chaperones, which deliver copper ions to specific partners
(62). Copper chaperones
contain the motif, M(T/H)CXXC, also present in their target proteins,
that bind metal ions via the two cysteine residues
(62). Metal transfer requires
the docking of the chaperone and target protein with their metal binding
domains close to each other, followed by metal exchange via formation of
intermediates that involve the cysteine residues from both proteins
(62). It would appear that
yeast frataxin is a different type of metallochaperone, acting as a general
reservoir of Fe(II) atoms and making them available to different users perhaps
via hydrophobic interactions mediated by the conserved neutral surface found
on the protein (43).
Importantly, if the Fe(II) is not transferred to iron users, it is oxidized
and stored in a water-soluble mineral within the assembled protein
(23).
Our previous findings
(21–24)
together with the results reported here support the following mechanism for
yeast frataxin (Fig. 7).
Monomer is activated by Fe(II) in the presence of O2 and forms an
oligomer, 3, with a negatively charged inner surface
(63,
64) that sequesters Fe(II)
from the solution (Fig.
7A). The oligomer catalyzes oxidation of Fe(II) to
Fe(III) and further promotes nucleation of a ferrihydrite crystallite at the
negatively charged surface. If the Fe(II) concentration exceeds that of the
ferroxidase sites on the protein (>0.5 Fe(II)/subunit), ferroxidation is
rapidly overcome by a slower autoxidation reaction at the surface of the
growing crystallite. At high Fe(II)/mYfh1p ratios, the initial ferrihydrite
crystallite grows into a larger particle
(Fig. 7B). Nichol
et al. (23) have
proposed that the iron core of frataxin forms via a process similar to
bacterial biomineralization
(65). According to this model,
alignment and binding of one ferrihydrite particle to another leads to the
interaction of trimers, which further facilitates biomineralization
(Fig. 7B). During this
process, frataxin acts as a chaperone, donating residual Fe(II) to
ferrochelatase or ISU-type proteins to support heme and iron-sulfur cluster
biosynthesis, respectively (Fig.
7B). Interestingly, the Fe(II) chaperone function appears
to have been separated from the Fe(III) storage function in human frataxin.
Ongoing studies in our laboratory show that the human frataxin monomer acts as
a Fe(II) donor to ferrochelatase, consistent with a recent report that human
frataxin monomer acts as an iron donor to ISU-type proteins in vitro
(61). On the other hand, the
assembled form of human frataxin
(25) has ferroxidase
activity2 and is able
to store Fe(III) in iron cores structurally identical to the yeast frataxin
iron cores (Ref. 23).
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FIG. 7.
Postulated mechanism of action of mYfh1p. A, assembly at
low Fe(II)/mYfh1p ratios that do not exceed the iron loading capacity of
3. B, assembly at saturating Fe(II)/mYfh1p ratios.
Red and black diamonds symbolize Fe(II) and Fe(III),
respectively. Yeast ferrochelatase is shown as a soluble dimer with one
molecule of protoporphyrin IX (IX) and one Fe(II)-binding site per subunit
(32); in vivo,
ferrochelatase is bound to the inner mitochondrial membrane with the iron
binding site exposed on the matrix side
(67).
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|
Observations we have made previously in vivo support this model.
When native frataxin is analyzed by gel filtration, the protein is detected as
a distribution of species over a broad molecular mass range (from 13 to
>600 kDa) corresponding to monomer and progressively larger molecules
(24,
25). This suggests that
stepwise assembly occurs in mitochondria and that monomer may be in
equilibrium with higher order oligomers. Further support comes from structural
studies. Scanning transmission electron microscopy data
(22) and extended x-ray
absorption fine structure analysis
(23) indicate that yeast and
human frataxin iron cores are composed of small ferrihydrite crystallites. The
three-dimensional structures of human and bacterial frataxin show a highly
conserved negatively charged surface similar to the anionic surface involved
in the iron storage mechanism of ferritin
(63,
64,
66). This surface could be
involved in iron oxidation and nucleation, and facilitate biomineralization by
keeping the growing ferric iron crystallites in a soluble form. Point
mutations of carboxylate residues in this surface compromise stepwise assembly
of bacterial frataxin (43) as
well as the ferroxidase activity and stepwise assembly of yeast
frataxin.2 A second
highly conserved uncharged surface predicted to be involved in protein-protein
interactions (63,
64) could mediate interactions
between frataxin and iron users. Indeed, yeast frataxin and ferrochelatase
were found to interact with each other by Biacore studies
(17). This evidence and our
work support the hypothesis that frataxin could work both as a chaperone for
Fe(II) when mitochondrial iron is limiting, and as a storage compartment for
Fe(III) when iron is in excess.
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FOOTNOTES
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|---|
* This work was supported by Grants RPG-96-051-04-TBE (to G. C. F.), from the
American Cancer Society, ROP58337 (to H. N.), from the Canadian Institutes of
Health Research, and AG15709 (to G. I.) from the National Institute on Aging,
NIA, National Institutes of Health. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to the memory of our colleague and friend Frank
Rusnak. 
** Supported by Fellowship NRSA 44748 from NINDS, National Institutes of
Health.
||
To whom correspondence should be addressed: Mayo Clinic and Foundation, 200
First St. SW, Stabile 7-52, Rochester, MN 55905. Tel.: 507-266-0110; Fax:
507-266-9315; E-mail:
isaya{at}mayo.edu.
1 The abbreviation used is: BIPY, -'-bipyridine.
2 H. A. O'Neill, S. Park, O. Gakh, and G. Isaya, unpublished results.
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ACKNOWLEDGMENTS
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We thank P. Arosio and S. Levi for H- and L-apoferritin, T. Burgardt for
sharing of the equipment, and P. Rinaldo for critical reading of the
manuscript.
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ABSTRACT
INTRODUCTION
