PERSPECTIVE

Mitochondrial iron trafficking and the integration of iron

metabolism between the mitochondrion and cytosol
Des R. Richardsona,1, Darius J. R. Lanea, Erika M. Beckera, Michael L.-H. Huanga, Megan Whitnalla,
Yohan Suryo Rahmantoa, Alex D. Sheftelb, and Prem Ponkac,d,1
a
Iron Metabolism and Chelation Program, Discipline of Pathology, University of Sydney, NSW 2006, Australia; bInstitut für Zytobiologie,
Philipps-Universität Marburg, Marburg 35037, Germany; cLady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC,
Canada H3T 1E2; and dDepartments of Physiology and Medicine, McGill University, Montreal, QC, Canada H3A 2T5
Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 30, 2010 (received for review March 15, 2010)

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron
metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative
phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial path-
ways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the
regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g.,
lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been
stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In
addition, recent work examining mitochondrial diseases (e.g., Friedreich’s ataxia) has established that communication exists between iron
metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-
processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-
processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

iron sulfur cluster | heme | Friedreich’s ataxia | frataxin | sideroblastic anemia

T
he mitochondrion is mostly ap-
preciated for its role in energy
transduction. However, it is less
well known that this organelle
can be considered a focal point when it
comes to the metabolism of the most
common transition metal in cells, namely
iron (1). It is the reversible oxidation states
of iron that enable the mitochondrion to
catalyze electron transport via heme- and
iron sulfur cluster (ISC)-containing pro-
teins and use this process in energy trans-
duction. Considering this alone, it is no
wonder that the mitochondrion plays such
a critical role in iron metabolism. In fact,
the mitochondrion is the sole site of heme
synthesis and a major generator of ISCs,
both of which are present in mitochondria
and cytosol (2). Although the molecular
pathways involved in the generation of
heme and ISCs are well known, only more
recently have some of the molecular
players responsible for mitochondrial iron
transport been identified. Clearly, these
molecular circuits are vital for the supply
of the metal ion that is needed for gen-
erating the final biologically important
end-products, namely heme and ISCs. In
contrast to the mitochondrion, at the
whole-cell level the molecular pathways
and regulation of iron uptake and storage
have been well characterized. Hence, these
will be first briefly described to provide
basic background on the field of iron me-
tabolism (3, 4) before examining what is
known regarding the mitochondrion.
Cellular Iron Metabolism and Transport
Iron is an essential metal for the organism
because of its unparalleled versatility as
a biological catalyst. Consequently, iron is
a crucial element required for growth.
However, the very chemical properties of
iron that allow this versatility also create
a paradoxical situation, making acquisition
by the organism very difficult. Indeed, at
pH 7.4 and physiological oxygen tension,
the relatively soluble iron(II) is readily
oxidized to iron(III), which upon hydrolysis
forms insoluble ferric hydroxides. As a
result of this virtual insolubility and po-
tential toxicity because of redox activity,
iron must be constantly chaperoned. In
fact, specialized molecules for the acqui-
sition, transport, and storage of iron in
a soluble, nontoxic form have evolved to
meet the organism’s iron requirements.
Over the last 15 years there has been a
wide variety of unique molecules discov-
ered that play a role in iron metabolism,
and the most relevant of these to this re-
view are listed in Table S1.
Because of its redox properties, iron can
catalyze the production of reactive oxygen
species (ROS) that can be highly toxic
(3). Therefore, under normal physiological
conditions, iron is specifically transported
in the blood by diferric transferrin (Tf)
(4, 5). All tissues acquire iron by the
binding of Tf to the transferrin receptor 1
(TfR1), with this complex then being in-
ternalized by receptor-mediated endocy-
tosis (4, 5) (Fig. 1A). Recent studies have
demonstrated that the internalization of
the Tf-containing endosome via the cyto-
skeleton is under control of intracellular
iron levels (6, 7). In part, this uptake
mechanism is mediated by myotonic dys-
trophy kinase-related Cdc42-binding ki-
nase alpha (MRCKα) (6). This molecule
plays a role in organizing the actin cyto-
skeleton and is up-regulated by iron-
depletion through the iron regulatory
protein (IRP)–iron-responsive element
(IRE) interaction (see below) (6). MRCKα
colocalizes with Tf-TfR1 complexes fol-
lowing their internalization and it has been
shown that attenuation of MRCKα ex-
pression causes a significant decrease in
Tf-mediated iron uptake (7). Additionally,
it is known that Sec15l1, which is involved
in the mammalian exocyst complex (8),
plays a role in iron uptake from Tf via its
role in exocytosis (9, 10). In fact, Sec15l1 is
linked to the Tf cycle through its inter-
action with Rab11 (a GTPase involved in
vesicular trafficking) and a mutation in
Sec15l1 leads to the anemia found in he-
moglobin-deficit mice (9, 10).
Within the endosome, the affinity of Tf
for iron is decreased by the low pH gen-
erated through the activity of a proton
pump (11, 12). Importantly, the TfR1 fa-
cilitates liberation of iron from Tf in the
pH range attained by the endosome (pH
5–5.5) (13). In vitro, iron release from
Tf requires a “trap,” such as pyrophos-
phate (13), but a physiological chelator
serving this role has not been identified. In
erythroid cells, once iron(III) is released
Author contributions: D.R.R., D.J.R.L., E.M.B., M.L.-H.H., M.W.,
Y.S.R., A.D.S., and P.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence may be addressed. E-mail: prem.
ponka@mcgill.ca or d.richardson@med.usyd.edu.au.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.0912925107/-/
DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0912925107 PNAS | June 15, 2010 | vol. 107 | no. 24 | 10775–10782

from Tf in the endosome, it is thought to
be reduced to iron(II) by a ferrireductase
in the endosomal membrane known as the
six-transmembrane epithelial antigen of
the prostate 3 (14, 15) (Fig. 1A). After this
step, iron(II) is then transported across
the endosomal membrane by the divalent
metal transporter-1 (DMT1) (16) and
forms, as generally believed, the cytosolic
low Mr labile or chelatable iron pool (17).
This pool of iron is thought to supply the
metal for storage in the cytosolic protein
ferritin and for metabolic needs, including
iron uptake by the mitochondrion for
heme and ISC synthesis.
Iron can also be released from the cell
by the transporter, ferroportin1 (18) (Fig.
1A). Ferroportin1 expression can be reg-
ulated by the hormone of iron metabo-
lism, hepcidin. Hepcidin is a key regulator
of systemic iron metabolism (18) and is
transported in the blood bound to α2-
macroglobulin (18). Hepcidin secretion by
the liver is stimulated by high iron levels
and also inflammatory cytokines, such as
interleukin-6 (19).
Regulation of Cellular Iron Homeostasis
The chelatable iron pool is thought to
control the activity of IRPs-1 and -2
(Fig. 1A). The IRPs are RNA-binding
proteins that bind to IREs in the 3′- and
5′-untranslated regions in mRNAs of
molecules playing crucial roles in the up-
take, utilization, export, and storage of
iron (e.g., TfR1, ferritin, etc.) (20).
Fig. 1. Schematics of cellular iron uptake. (A) The process or iron uptake and utilization. (B) The “kiss
and run” hypothesis (see text).
10776 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107
IRP-1 performs two functions: (i) regu-
lating iron homeostasis via binding IREs
and (ii) having cytosolic aconitase activity
when containing an [4Fe-4S] cluster (21).
IRP-2 is thought to be the principal RNA-
binding protein in vivo and is regulated by
iron-dependent proteasomal degradation
(22–24). Although the IRP-IRE mecha-
nism plays crucial roles in the regulation of
iron metabolism, complete understanding
of the homeostatic mechanisms involved
in iron metabolism in relation to commu-
nication between the cytosol and mito-
chondrion have yet to be deciphered, and
are considered in the following sections.
Intracellular Iron Transport and Communication
with the Mitochondrion. Once iron is trans-
ported out of the endosome via DMT1, it
enters the chelatable or labile iron pool (Fig.
1A) that traditionally was thought to consist
of low Mr complexes (e.g., iron-citrate) (17,
25, 26). The only strong evidence that such
a pool exists comes from studies with che-
lators that mobilize iron from cells (27, 28).
However, it is just as likely that these com-
pounds remove iron from organelles and
proteins as it is that they chelate iron from
genuine cytosolic low Mr complexes (29).
Studies using reticulocytes, which are
highly active in terms of iron uptake,
demonstrate that these cells contain very
little iron as low Mr complexes (30). In fact,
the only low Mr iron present had kinetics
of iron uptake consistent with an end-
product rather than an intermediate (30).
Furthermore, the inhibitor of heme syn-
thesis, succinylacetone, led to a reduction
in this low Mr iron, suggesting it was heme
or a heme-containing molecule (30). These
studies, coupled with the findings in pre-
vious investigations by others, led to a hy-
pothesis that iron is always transported, at
least in erythroid cells, bound within hy-
drophobic pockets of proteins that act as
intermediates (or chaperones) and prevent
cytotoxic redox chemistry (30).
As yet, such iron chaperone molecules
remain elusive, although Vyoral et al. (31)
identified a high Mr intermediate that ap-
peared to donate its iron to ferritin after
incubation of K562 cells with Tf. More
recently, a protein known as poly (rC)-
binding protein 1 has been identified that
donates iron to ferritin and may play an
important role in this process (32). Al-
though work identifying chaperones that
transport iron remain preliminary, it is
notable that for copper, which is the sec-
ond most-abundant transition metal ion in
mammalian tissues, much evidence of
chaperone-mediated transport has been
described (33, 34). Like iron, copper is
also cytotoxic because of its redox activity,
and is never found free at significant con-
centrations, but is always bound to trans-
porters, chaperone molecules, and target
proteins (33–36). In fact, copper uptake
and efflux involves chaperones as well as
organelle interactions (33–36).
Considering the arguments above, it has
been shown that highly efficient iron tar-
geting to the mitochondrion is evident in
erythroid cells where ferrochelatase inserts
iron(II) into protoporphyrin IX [PPIX
(11)]. Because Tf-bound iron is efficiently
used for heme synthesis (11, 30) and no
low Mr cytoplasmic iron-transport inter-
mediate has been found in reticulocytes,
an intimate direct transfer of iron from Tf
to the mitochondrion was proposed to
occur (37, 38). This idea has developed in
more recent years and has led to the “kiss
and run” hypothesis (11) (Fig. 1B). This
model suggests that a direct transfer of
iron from the Tf-containing endosome to
the mitochondrion occurs, by-passing the
cytosol (11, 28, 30, 39). The precise mo-
lecular details involved in a possible con-
tact between the endosome and mito-
chondrion remain unknown. However,
molecular motors, docking complexes,
myosin Vb (40), and molecules involved
in regulating the cytoskeleton, namely
MRCKα (5), and also vesicular docking
[e.g., Sec15l1 (9, 10)], point to mechanisms
that may facilitate kiss and run (29).
Communication Between the Cytosol and
Mitochondrion: Regulation of Iron Uptake. If
a direct system of iron transfer exists be-
tween the endosome and mitochondrion,
regulation of iron metabolism by the cell
must be coordinated with iron uptake and
Richardson et al.
transport to the mitochondrion. Such ho-
meostatic mechanisms are not fully un-
derstood, but may work in concert with the
IRP-IRE mechanism to regulate iron ho-
meostasis. Evidence that regulatory pro-
cesses exist that couple cytosolic and
mitochondrial iron metabolism can be de-
duced from studies in vitro and in vivo. For
example, when heme synthesis is inhibited in
the mitochondrion using succinylacetone,
iron continues to enter the organelle (30, 41,
42). This finding could be interpreted in two
ways. First, it may indicate little communi-
cation and coupling between the cytosolic
and mitochondrial iron-metabolism ma-
chineries, as iron continues to be trans-
ported into the organelle irrespective of the
lack of heme synthesis. Second, it could
suggest that iron continues to enter the mi-
tochondrion because of a failure to generate
heme, which leads to a signal-to-import iron
in an effort to rescue heme synthesis.
The latter concept suggests a coupling
between the cytosolic- and mitochondrial
iron-processing pathways and is supported
by several lines of evidence. For example,
Tf and Tf-dependent iron uptake are in-
creased when heme synthesis is inhibited
using reticulocytes in vitro (41, 42), and
this is accomplished by an increase in
TfR1 cycling rate (42). This response oc-
curs despite on-going mitochondrial iron-
loading because of the inhibition of heme
synthesis. Hence, the lack of heme gener-
ation appears to result in a compensatory
increase in Tf-bound iron uptake. Other
evidence comes from studies in vivo using
the muscle creatine kinase conditional
frataxin knockout mouse (43), where con-
ditional frataxin-deletion in cardiomyocytes
leads to depressed ISC and heme synthesis,
mitochondrial iron-loading, TfR1 up-regu-
lation, and thus increased iron uptake from
Tf (44, 45) (Fig. 2).
Under these latter conditions, in the
absence of frataxin, the defect in ISC and
heme synthesis appears to lead to a “res-
cue response,” where iron uptake is in-
creased and targeted away from the
cytoplasm and ferritin toward the mito-
chondrion. This response leads to a rela-
tive cytosolic iron-deficiency and mito-
chondrial iron-loading (44, 45). In the
latter case, it was hypothesized that a sig-
nal (potentially caused by decreased ISC
or heme levels) was sent from the mito-
chondrion to the cytoplasm to up-regulate
iron uptake to compensate for the de-
pressed ISC and heme synthesis resulting
from frataxin-deficiency (45). Although
increased directional targeting of iron to
the mitochondrion occurred, leading to
iron-loading in this organelle, it does not
lead to an effective reversal of the mito-
chondrial defect (44, 45), because frataxin,
which plays a crucial role in ISC and heme
synthesis, is deleted in cardiomyocytes
(43, 46–48). Similarly, Li et al., using cul-
Richardson et al.
Fig. 2. Model of alteration in iron uptake in
Friedreich’s ataxia. Frataxin-deficiency results in
increased mitochondrial-targeted iron uptake and
cytosolic iron-deficiency (see text).
tured cells in vitro, have also demon-
strated that there is a cytosolic iron-deficit
in fibroblasts and lymphoblasts from
Friedreich’s ataxia (FA) patients, as shown
by increased IRP1/2-RNA-binding activ-
ity (49). Finally, overexpression of mito-
chondrial ferritin (Ftmt) in the mitochon-
drion leads to mitochondrial iron-loading
and cytosolic iron-deprivation (50). Hence,
there is evidence that alterations in mito-
chondrial iron homeostasis lead to changes
in cellular iron metabolism, suggesting
communication between compartments
and a modulating influence of the mi-
tochondrion.
The discussion above demonstrates that
cytosolic iron metabolism is regulated, at
least in part, by the mitochondrial demand
for iron that is critical for heme and ISC
synthesis. This finding appears logical,
because the mitochondrion is a focal point
of iron processing. Such compensatory
alterations in iron trafficking, demon-
strating communication between the cy-
tosol and mitochondrion, are also found
in other conditions where mitochondrial
iron-processing is disturbed (51). For ex-
ample, similar mitochondrial iron-loading
occurs in patients with a splice mutation
in the ISC scaffold protein, Iscu (52), or
with a deficiency in glutaredoxin-5 that is
involved in ISC/heme synthesis (53, 54).
Moreover, in a patient with a glutaredoxin-
5 deficiency, this problem led to decreased
ferritin and increased TfR1 expression
(54), which was a similar metabolic phe-
notype to that found in frataxin knockout
mice (44, 45). Further studies are necessary
to define at the molecular level how this
communication occurs between the cytosol
and the mitochondrion, and may be facili-
PNAS | June 15, 2010 | vol. 107 | no. 24 | 10777
tated by a better understanding of the
mechanisms of mitochondrial iron import
and release.
Considering the regulation of whole-cell
iron metabolism via molecular defects
in the mitochondrion described above, it is
intriguing to consider the possibility that
similar mechanisms of communication
exist for other organelles that play key roles
in iron metabolism. For example, the ly-
sosome is involved in ferritin degradation,
recycling of stored iron, and autophagy of
other organelles, including the iron-rich
mitochondrion (55). The rupture of a
small number of lysosomes is an early
upstream event in many cases of apopto-
sis, particularly oxidative stress-induced
apoptosis; necrosis results from a major
lysosomal rupture. Consequently, it has
been suggested that regulation of the ly-
sosomal content of redox-active iron ap-
pears essential for cell survival (56).
Mitochondrial Iron Import
Although DMT1 is responsible for the
export of iron(II) from the endosome, the
itinerary of the metal from the cytosol to
the inner mitochondrial membrane is
not well understood. However, it is known
that the inner membrane of the mito-
chondrion contains proteins capable of
transporting iron into the mitochondrial
matrix. Foury and Roganti suggested a role
for the eukaryotic, mitochondrial solute
carriers, Mrs3 and Mrs4, in mediating
mitochondrial iron metabolism in yeast
(57). A further study demonstrated that
these proteins are essential for yeast
growth under iron-limiting conditions,
suggesting that, at least in this organism,
an additional iron transport mechanism is
present (58). A recent investigation of the
function of Mrs3 and -4 in small mito-
chondrial particles showed these proteins
transport iron(II) along a concentration
gradient (59).
Studies with the zebrafish mutant with
profound anemia, frascati, led to the dis-
covery of the Mrs3 and -4 homolog,
termed mitoferrin-1 (SLC25A37) and
mitoferrin-2 (SLC25A28) (60). These ho-
mologous proteins are important for mi-
tochondrial iron uptake in erythroid and
nonerythroid cells, respectively (60, 61)
(Fig. 1A). Mitoferrin-1 is localized on the
inner mitochondrial membrane and func-
tions as an essential importer of iron for
mitochondrial heme and ISC in erythro-
blasts and is necessary for erythropoiesis
(60). Mitoferrin-1 is highly expressed in
erythroid cells and in low levels in other
tissues, whereas mitoferrin-2 is ubiqui-
tously expressed (61). The half-life of
mitoferrin-1 (but not mitoferrin-2) in-
creases in developing erythroid cells and
this may be part of a regulatory mecha-
nism aiding mitochondrial iron uptake
(61). Recently, Abcb10, a mitochondrial
inner-membrane ATP-binding cassette
transporter, was found to physically inter-
act with mouse Mfrn1, and thereby en-
hance the stability of the protein and
increase mitochondrial iron-import (62).
Interestingly, Abcb10 has been suggested
to play some role in heme synthesis and
can be rapidly induced by the transcription
factor, GATA-1, which plays a role in
erythroid differentiation (63).
It is still unknown how iron is trans-
ported across the outer mitochondrial
membrane and clearly other transporters
may yet be identified. Considering this,
a large-scale computational screen identi-
fied three potential transporters that may
be involved in mitochondrial iron metab-
olism, namely SLC25A39, SLC22A4, and
TMEM14C (64). In fact, targeted knock-
down of these genes in zebrafish resulted
in profound anemia. Furthermore, silenc-
ing of Slc25a39 in murine erythroleukemia
cells impaired iron incorporation into
PPIX to form heme (64).
It is also notable that a mutation in the
transmembrane mitochondrial protein,
sideroflexin1, is responsible for the skeletal
abnormalities and hematological phenotype
in the flexed-tail mouse model, namely
hypochromic, microcytic anemia, and side-
rotic granules in erythrocytes (65). Because
of its predicted five-transmembrane do-
mains, this molecule has been suggested
to be a transporter essential for mitochon-
drial iron metabolism. Indeed, it has been
speculated to transport molecules into or out
of the mitochondrion for iron utilization or
heme synthesis (65). Sideroflexin may not
necessarily transport iron and could mediate
the uptake of other metabolites essential for
heme synthesis (e.g., pyridoxine) that is
necessary for the formation of pyridoxal
phosphate, a coenzyme required for the first
step in heme production (65).
Mitochondrial Iron Metabolism
Three Major Pathways: Heme Synthesis, ISC
Synthesis, and Storage. Once iron is trans-
ported into the mitochondrion it can then
be used for heme synthesis, ISC synthesis,
or stored in Ftmt. It is essential that mi-
tochondrial iron is maintained in a safe
form to prevent oxidative damage, as mi-
tochondria are a major source of cytotoxic
ROS (3). Hence, it is likely that as in the
cytosol, iron is carefully transported within
the mitochondrion in a form distal to the
aqueous environment, deep in the hydro-
phobic pockets of communicating proteins
that form iron transport pathways. Al-
though the molecular nature of these cir-
cuits remains unclear, the pathways that
use iron are well known and are discussed
below.
Mitochondrial iron storage: mitochondrial ferritin.
Like a typical ferritin, Ftmt shows ferrox-
idase activity and binds iron (66). In contrast
to cytoplasmic ferritin, Ftmt is encoded
10778 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107
by an atypical intronless gene (66). How-
ever, its role is unclear, particularly con-
sidering its tissue distribution pattern.
Indeed, Ftmt was found at the highest lev-
els in the testes and the erythroblasts of
sideroblastic anemia patients (67, 68). Mi-
tochondrial ferritin has also been detected
in the heart, brain, spinal cord, kidney, and
pancreas (67). Unlike cytosolic ferritin,
Ftmt is not highly expressed in the liver and
spleen, suggesting that it plays a distinct
role. These findings led to the hypothesis
that Ftmt plays a role in protection from
iron-dependent oxidative damage (67).
The role of Ftmt in iron metabolism was
examined by employing a stably transfected
cell line that hyper-expresses Ftmt (50).
These studies showed that overexpression
of Ftmt resulted in increased IRP-1/2-
RNA-binding activity, decreased cytosolic
and mitochondrial aconitase activity (sug-
gesting decreased ISC synthesis), de-
creased cytoplasmic ferritin, increased
TfR1 expression, decreased heme synthe-
sis, decreased frataxin expression, and in-
creased iron-loading of Ftmt (50). Hence,
Ftmt overexpression leads to partitioning
of iron away from heme and ISC synthesis
in the mitochondrion. This effect not only
alters mitochondrial iron metabolism, but
also whole-cell iron metabolism (50), lead-
ing to a cytosolic iron-deficiency that re-
duces the proliferation of neoplastic cells
hyper-expressing Ftmt in vivo (69). As al-
ready discussed, very similar alterations of
gene expression also occur after ablation of
frataxin expression (44, 45) and indicate
communication between the cytosol and
mitochondrion.
Iron-sulfur cluster biosynthesis. Being a major
site of ISC assembly, the mitochondrion
plays a pivotal role in the biosynthesis of
ISC proteins (1, 2). ISCs consist of iron
and sulfide anions (S2-), which assemble to
form [2Fe-2S] or [4Fe-4S] clusters (2).
These clusters form cofactors in proteins
that perform vital functions, such as elec-
tron transport, redox reactions, metabolic
catalysis, and other such functions (70).
In eukaryotic cells, more than 20 mole-
cules facilitate maturation of ISC proteins
in the mitochondria, cytosol, and nucleus
(2). Functional defects in ISC proteins
or components involved in their biogenesis
lead to human diseases (71, 72). Bio-
synthesis of ISCs and their insertion into
apo-proteins requires both the mitochon-
drial and cytosolic machinery. The first
molecule identified in the ISC machinery
was the enzyme NifS from Azobacter vin-
landii, which is a cysteine desulfurase that
participates in ISC assembly as a sulfur do-
nor (73). This enzyme is highly conserved
and in humans is known as Nfs1 (74).
ISCs are assembled on scaffold proteins
and then transferred to apo-proteins. In
Escherichia coli, this scaffold protein is
known as ISC assembly protein U (IscU)
and leads to sequential assembly leading
to [2Fe-2S] units that then form a [4Fe-4S]
cluster (75). In yeast cells, these reactions
are accomplished by Isu1 and -2 (76),
but in humans, the function of IscU is
performed via the splicing of IscU mRNA
to lead to two transcripts which generate
a cytosolic (Iscu1) or mitochondrial (Is-
cu2) isoform. The maturation of extra-
mitochondrial ISC proteins requires the
mitochondrial ISC assembly system (77).
The mitochondrion contributes a yet-to-be
discovered compound necessary for the
biogenesis of ISCs outside of this com-
partment (i.e., in the cytosol or other or-
ganelles) (70).
The mechanism involved in iron delivery
to Iscu1 is not clear, but it has been sug-
gested to involve frataxin as an iron donor
(2). Moreover, frataxin has been shown to
play a critical role in the early stages of
ISC genesis (78), and this is discussed in
detail below. In yeast, Isu1 only provides
a cluster for de novo cluster production
from which an HSP70-type chaperone
system transfers the new clusters to apo-
proteins (2). Yet another molecule,
ABCB7, appears to mediate cytosolic ISC
biogenesis (79). The maturation of cyto-
solic ISCs is inhibited by mutations in
ABCB7 and this causes the disease, X-
linked sideroblastic anemia with cerebellar
ataxia (XLSA/A). This condition is char-
acterized by loss of cytosolic ISC proteins,
defects in heme metabolism, and in-
creased mitochondrial iron levels (79).
Heme biosynthesis. The third major mito-
chondrial metabolic pathway that utilizes
iron is that of heme synthesis that is ex-
clusive to this organelle (1, 11). Heme is
synthesized by a pathway composed of
eight sequential reactions in the mito-
chondrion and cytoplasm (1, 11). The first
and last three steps in the heme biosyn-
thesis pathway take place in the mito-
chondrion. The first enzyme in the path-
way, 5-aminoleuvulinate synthase (ALAS),
catalyzes the condensation of glycine and
succinyl CoA to form 5-aminolevulinate
(1, 11). There are two genes for ALAS, the
ubiquitously expressed ALAS1 and the
erythroid-specific ALAS2, which is under
the regulation of iron via the IRP system
(1). In nonerythroid cells, the rate of
heme synthesis is dependent on the rate
of 5-aminolevulinate synthesis via ALAS1
(11). In contrast, in erythroid cells, the rate-
limiting step may be the delivery of Tf-
iron (11).
The subsequent four steps of heme
biosynthesis take place in the cytosol, fol-
lowing which coproporphyrinogen III
is decarboxylated and then oxidized in
the mitochondrial intermembrane
space to produce PPIX (1, 11). Cop-
roporphyrinogen may be transported into
mitochondria by either peripheral-type
benzodiazepine receptors (80) or
Richardson et al.
potentially ABCB6 (81). The seventh step,
which is catalyzed by protoporphyrinogen
IX oxidase generates PPIX. In the final
reaction of the pathway, iron(II) is inserted
into PPIX by the ISC protein, ferrochela-
tase, in the mitochondrial matrix (11).
Mitochondrial Iron Export. Apart from
importing iron, the mitochondrion synthe-
sizes heme and ISCs that subsequently are
transported out to the cytosol. These export
processes are important to understand, as
decreased release of iron as heme or ISCs
—or their precursors—can contribute to
mitochondrial iron-loading, as found in the
frataxin knockout mouse (45). A candidate
iron exporter has been identified, namely
mammalian mitochondrial ABC protein 3
(MTABC3; also known as ABCB6) (82).
This protein can rescue mitochondrial iron-
loading, respiratory dysfunction, and mito-
chondrial DNA damage in atm1-deficient
yeast cells (82). Of relevance, the human
ortholog of atm1 is ABCB7, which can
complement atm1 deficiencies in yeast
cells, enabling the maturation of ISC-con-
taining proteins in the cytosol (79). It has
been suggested that ABCB7 transports
ISCs to the cytoplasm (79), although this
has not been directly shown.
It is unknown how heme is exported
from the mitochondrion. However, its low
solubility and highly toxic nature suggest an
efficient heme-carrier must be involved.
Considering this, heme-binding protein 1
has been identified as a candidate for this
carrier (83). The expression of this mole-
cule is ubiquitous, but it is also increased
during erythroid differentiation and high
levels are found in the liver (83). Heme-
binding protein 1 binds one heme per
mole of protein (83) and, although it could
be a candidate for heme transport, direct
evidence for this is lacking.
Diseases of Mitochondrial Iron
Metabolism
Mechanisms of iron transport across mi-
tochondrial membranes have evolved to
supply the necessary iron to mitochondria
and also maintain the balance of cytosolic
iron (1). Mitochondrial iron levels must
be tightly controlled as iron induces ROS,
which can damage the organelle (84).
The importance of these tight controls is
highlighted by the fact that alterations in
mitochondrial iron homeostasis have
pathological consequences (1, 70). In
recent years, cellular and animal models of
mitochondrial iron dysfunction have pro-
vided valuable information in identifying
new proteins to elucidate the pathways
involved in mitochondrial iron homeosta-
sis. Interesting examples of mitochondrial
diseases that have provided important
insight into the processes of mitochondrial
iron metabolism are Friedreich’s ataxia
and sideroblastic anemia. These condi-
Richardson et al.
tions are discussed in detail in the follow-
ing sections.
Friedreich’s Ataxia and the Metabolic Role of
Frataxin. FA is a rare disease leading to
severe neuro- and cardio-degeneration and
is caused by a deficiency of frataxin (85).
The decrease in frataxin expression is
caused by an intronic GAA-repeat ex-
pansion in intron-1 of FRDA. Frataxin is
a vital protein that is highly expressed in
tissues rich in mitochondria (e.g., heart
and neurons) (86), with deletion leading to
lethality (87).
The suggested functions for frataxin all
relate to iron metabolism (Fig. 3) and in-
clude ISC and heme biogenesis, as well
as iron storage. Frataxin was linked to ISC
proteins by the observation that there was
a deficiency in ISC cluster proteins in
knock-out mice, FA patients, and yeast
(43, 46–48). Frataxin has also been impli-
cated in iron scavenging (88), regulating
oxidative stress (89), and as an iron chap-
erone (90). To date, the cumulative evi-
dence suggests frataxin is involved in the
maintenance of iron homeostasis (44, 45).
Frataxin is an inner mitochondrial mem-
brane and mitochondrial-matrix protein
(85). However, as there does not appear to
be any structural feature that would an-
chor frataxin to the mitochondrial mem-
brane, it is possible that membrane-
associated frataxin is bound to other pro-
teins in a macromolecular complex con-
taining ferrochelatase (91).
Recent insight into frataxin function has
come from studies of frataxin orthologs
in yeast (i.e., Yfh1) and bacteria (i.e.,
CyaY) that share a high degree of sequence
identity to the human protein (92). A
consensus is that the functions of frataxin
share a requirement for iron-binding (92–
94). Although there are several propo-
sals, the exact function of frataxin remains
controversial and is discussed in the
following sections.
Frataxin and iron storage. Frataxin has been
proposed to function as a mitochondrial
iron storage protein (95) (Fig. 3A). The
frataxin ortholog, Yfh1, was reported to be
capable of self-assembling into oligomers
and then into higher-order multimers in
the presence of increasing iron(II) (96).
The iron-dependent oligomerization of
Yfh1 is associated with the development
of ferroxidase activity that appears to exist
only in the oligomeric/multimeric struc-
ture (92). This finding was proposed to
allow Yfh1 oligomers/multimers to store
iron in a mineralized and redox-inactive
iron(III) state (92, 97). However, because
the iron-dependent oligomerization of
Yfh1 only occurs in the absence of Ca2+ and
Mg2+, which are typically abundant in the
mitochondrion (92, 93, 96), this diminishes
support for the iron-storage hypothesis.
PNAS | June 15, 2010 | vol. 107 | no. 24 | 10779
Fig. 3. Schematic of the possible functions of
frataxin. (A) Frataxin may perform a mitochondrial
iron-storage function similar to ferritin. (B) Fra-
taxin may function as an iron chaperone by binding
iron and then delivering it. (C) The frataxin or-
tholog, CyaY may function as an “iron-sensing”-
negative regulator that inhibits the rate of ISC as-
sembly under conditions of high mitochondrial
iron and low ISC apo-acceptor availabilities. (D)
Frataxin may also function as a “metabolic switch”
that allows the mitochondrion to favor heme or
ISC/heme syntheses, depending on frataxin and
protoporphyrin IX levels (91, 99) (see text).
Spontaneously oligomerized recombi-
nant human frataxin appears capable of
binding and storing iron (97, 98). However,
recombinant human frataxin monomers,
unlike those of Yfh1, cannot be induced to
oligomerize in vitro by iron (98). More-
over, the spontaneous oligomerization of
human frataxin appears to be dependent
on heterologous overexpression (98). Such
considerations argue against an in vivo
iron-storage function for human frataxin.
The observation that the manipulation
of mitochondrial iron levels does not affect
frataxin expression levels is not consistent
with a significant role in mitochondrial
iron storage (99). Theoretical support for
the generality of the “iron-storage” hy-
pothesis was also lessened upon the dis-
covery of Ftmt in higher organisms (66,
100). The existence of Ftmt appears to
make redundant any significant iron-
storage role for human frataxin. However,
yeast cells do not express ferritins (100),
and it is possible that Yfh1 may possess
an additional iron-storage capacity not
shared by frataxin orthologs in higher
organisms.
Frataxin as an iron chaperone. Perhaps the
most promising emerging role for frataxin
is as an iron chaperone (Fig. 3B) for ISC and
heme biosyntheses. Frataxin has been ob-
served to interact with, and presumably
donate iron to, iron-dependent proteins
involved in ISC and heme biosyntheses (90,
91). For example, Yfh1 appears capable of
interacting with the central ISC assembly
complex comprising the scaffold protein,
Isu, and the cysteine desulfurase, Nfs1, in
a manner enhanced by iron(II) (90, 101).
Recently, it has been suggested that
frataxin interacts with Isu1 via a highly
conserved tryptophan residue (W131a) in
its conserved β-sheet region (102). Anal-
ogously, human frataxin demonstrates
iron-enhanced interactions with Isu and
ferrochelatase (91, 103). Importantly, the
interaction of frataxin with either Isu or
ferrochelatase appears to increase the rate
of ISC synthesis (90) or the ferrochelatase-
catalyzed insertion of iron(II) into PPIX
(91), respectively. Such observations sug-
gest frataxin may function as an iron-
donor to Isu and ferrochelatase. Emerging
data indicate that the nature of frataxin’s
facilitatory interactions with the ISC
and heme synthesis machinery may be
more complex than just simple iron-
donation. As discussed in the next sec-
tion, a possible primary function of fra-
taxin’s interactions may be to additionally
negatively regulate the respective bio-
synthetic interactions.
Frataxin as an iron-sensing negative-regulator.
A recent study with CyaY suggests that
the protein may act as an “iron-sensor”
(Fig. 3C) that negatively regulates the rate
of ISC biosynthesis under conditions of
high iron and low ISC apo-acceptor
availabilities (89). If we extend this model
to eukaryotic systems, a deficiency in fra-
taxin expression is presumably deleteri-
ous because the rate of ISC biosynthesis
may exceed the availability of ISC apo-
acceptors, resulting in the overproduction
of ISCs that are unstable in an unbound
form (89). Essentially, this model suggests
that over and above functioning as an iron
donor in ISC biosynthesis, frataxin may
exert “kinetic control” over the rate of
ISC biosynthesis, depending on the rela-
tive availabilities of iron and ISC apo-
acceptors. This possible iron-sensing role
is consistent with the relatively low (i.e.,
micromolar) affinities of iron-binding by
bacterial, yeast and human frataxin or-
thologs (92). The applicability of this
model remains to be examined in mam-
mals. It also needs to be assessed whether
any such kinetic control is elicited by fra-
taxin’s interaction with ferrochelatase
during heme biosynthesis.
Frataxin as an expression-regulated “metabolic
switch.” An extension of the notion of fra-
taxin as a negative regulator to frataxin’s
functional role in heme biosynthesis may
help to explain the decline in frataxin
levels during erythroid differentiation (99)
(Fig. 3D). That is, because frataxin ex-
pression is markedly decreased during
Friend cell hemoglobinization (99), it
is possible that frataxin may be down-
regulated during erythroid differentiation
to allow higher rates of heme synthesis,
potentially at the expense of decreased
levels of ISC synthesis (99). This hypoth-
10780 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107
esis is supported by the observation that
the immediate precursor for heme syn-
thesis, PPIX, down-regulates frataxin ex-
pression (99). Hence, increased PPIX
levels, which indicate a requirement for
heme synthesis, lead to decreased frataxin
expression and a diversion of iron from
other mitochondrial pathways (i.e., ISC
synthesis or iron storage) to heme bio-
genesis (99).
This latter hypothesis is supported by the
observation that an increase in frataxin
levels relative to ferrochelatase (i.e., above
a molar ratio of 1:1 frataxin:ferrochelatase
dimer) results in decreased rates of
heme synthesis in vitro (91). It has also
been observed that iron-bound human
frataxin has a higher putative binding af-
finity for ferrochelatase (17 nM) than
Isu (480 nM) (91). These observations
provide a basic mechanism that supports
the hypothesis that frataxin may allow
metabolic switching between ISC and
heme synthesis pathways depending on
expression levels relative to those of Isu
and ferrochelatase (91, 99).
A frataxin metabolon? Frataxin’s putative
ability to modulate iron-dependent bio-
chemical reactions through protein-
protein interactions is suggestive of the
possibility that it may form one or more
metabolons, or protein complexes, with
proteins involved in ISC and heme bio-
syntheses (94). The conserved tryptophan
residue-131 in frataxin, which is respon-
sible for its interaction with Isu1, suggests
the association is crucial for its function,
which is underlined by the fact that mu-
tating this residue results in a loss of mi-
tochondrial aconitase activity (102). This
hypothesis suggests that the loss of the
interaction with Isu1 results in an im-
pairment of ISC synthesis and supports
the notion of a functional protein com-
plex involving frataxin. In general, me-
tabolons are dynamic protein complexes
that greatly enhance the efficiency of
metabolic reactions through processes,
such as substrate channeling. On the basis
of the possibility that frataxin may act
as an iron-donor, it might be expected
that frataxin could be part of a mitochon-
drial metabolon consisting of ISC as-
sembly components, such as Iscu, and
heme biosynthesis enzymes, such as fer-
rochelatase (Fig. 3).
Sideroblastic Anemias. The characteristic
feature of all sideroblastic anemias is the
ring sideroblast. This is a pathological
erythroid precursor containing excessive
deposits of nonheme iron in mitochondria
with peri-nuclear distribution responsi-
ble for the ring appearance. With consid-
erable simplification, and from the point of
view of pathogenesis, sideroblastic ane-
mias can be divided into those with or
without a heme synthesis defect in ery-
throblasts.
It can be proposed that the combination
of several factors plays a role in the path-
ogenesis of mitochondrial iron accumula-
tion in sideroblastic anemias associated
with a heme synthesis defect: (i) iron is
specifically targeted toward erythroid mi-
tochondria; (ii) iron cannot be used for
heme synthesis because of the lack of
PPIX; (iii) there is a lack of heme, the
negative regulator of iron uptake from Tf;
and (iv) iron can leave erythroid mito-
chondria only after being inserted into
PPIX. A key example of this scenario is X-
linked sideroblastic anemia, which is
caused by mutations in erythroid-specific
ALAS2 (104). As already discussed, a dis-
tinct form of X-linked sideroblastic ane-
mia is XLSA/A. This condition is caused
by mutations in ABCB7 (79), whose
product is thought to transfer an ISC
precursor from mitochondria to the cyto-
sol. In XLSA/A, as is the case in ALAS2-
associated sideroblastic anemia, decreased
levels of heme are likely to contribute to
the pathogenesis of ring sideroblast for-
mation. In refractory anemia with ring
sideroblasts (RARS), there is no evidence
for a defect in PPIX formation in patients’
erythroblasts. In some patients with
RARS, acquired mutations in subunits
of cytochrome oxidase encoded by mito-
chondrial DNA have been described
(105). It has been proposed that this defect
could lead to impaired iron reduction
that is needed for heme and ISC synthesis,
and without this, mitochondrial iron de-
posits occur.
It can also be hypothesized that ery-
throid progenitors of patients with RARS,
characterized by genomic instability and
premature apoptosis, exhibit anomalous
induction of mitochondrial ferritin that
would lead to a shift of iron into their
mitochondria (50, 69). This would prevent
the use of iron for hemoglobin synthesis
and cause a ring-sideroblast phenotype. In
fact, any metabolic abnormality that
markedly affects the synthesis of ISCs or
heme is likely to result in mitochondrial
iron-loading. Actually, it has recently been
shown that mutations in the putative gly-
cine transporter, SLC25A38, lead to
a rare form of sideroblastic anemia (106).
This iron-loading probably occurs be-
cause ALAS catalyzes the reaction of
glycine with succinyl CoA to generate 5-
aminolevulinate. Without glycine, PPIX
synthesis would be inhibited which pre-
vents heme generation. Defects in other
metabolic pathways, which affect mito-
chondrial iron metabolism, can also lead
to sideroblastic anemia. An example of
this would be patients with mutations in
pseudouridine synthase-1, which is in-
volved in the processing of mitochondrial
tRNAs (107).
Richardson et al.
Summary
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point for iron utilization in heme and ISC
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that the mitochondrion can play an im-
portant role in orchestrating whole-cell
iron metabolism. Indeed, analysis of dis-
ease states has enabled understanding of
the role of the mitochondrion in regu-
lating cellular iron metabolism. There is
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ACKNOWLEDGMENTS. D.R.R.’s laboratory
was supported by grants from the National Health
and Medical Research Council of Australia, the
US Muscular Dystrophy Association (MDA),
MDA New South Wales, Friedreich’s Ataxia Re-
search Alliance (FARA) Australia, and FARA USA.
P.P and A.D.S. were supported by grants from
the Canadian Institutes of Health Research.
Y.S.R. was a grateful recipient of a Cancer Institute
New South Wales Early Career Development
Fellowship.
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