Structure 14, 1535–1546, October 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.str.2006.08.010
The Structures of Frataxin Oligomers
Reveal the Mechanism for the Delivery
and Detoxification of Iron
Tobias Karlberg,1Ulrika Schagerlo ¨f,1
Oleksandr Gakh,3Sungjo Park,3Ulf Ryde,2
Martin Lindahl,1Kirstin Leath,4Elspeth Garman,4
Grazia Isaya,3,* and Salam Al-Karadaghi1,*
1Department of Molecular Biophysics
2Department of Theoretical Chemistry
Center for Chemistry and Chemical Engineering
P.O. Box 124
SE-221 00 Lund
3Departments of Pediatric & Adolescent Medicine
and Biochemistry & Molecular Biology
Mayo Clinic College of Medicine
Rochester, Minnesota 55905
4Department of Biochemistry
University of Oxford
South Parks Road
Oxford, OX1 3QU
Defects in the mitochondrial protein frataxin are re-
sponsible for Friedreich ataxia, a neurodegenerative
and cardiac disease that affects 1:40,000 children.
Here, we present the crystal structures of the iron-
free and iron-loaded frataxin trimers, and a single-
particle electron microscopy reconstruction of a 24
subunit oligomer. The structures reveal fundamental
aspects of the frataxin mechanism. The trimer has a
central channel in which one atom of iron binds. Two
conformations of the channel with different metal-
binding affinities suggest that a gating mechanism
controls whether the bound iron is delivered to other
proteins or transferred to detoxification sites. The tri-
mer constitutes the basic structural unit of the 24 sub-
unit oligomer. The architecture of this oligomer and
several features of the trimer structure demonstrate
striking similarities to the iron-storage protein ferritin.
The data reveal how stepwise assembly provides fra-
taxin with the structural flexibility to perform two func-
tions: metal delivery and detoxification.
Theabilitytoincorporate ironintoprostheticgroups and
proteins is essential for the biogenesis of many vital en-
zymes in all organisms (Al-Karadaghi et al., 2006; Lill
et al., 1999). However, in the presence of atmospheric
oxygen at neutral pH, ferrous iron (Fe2+) is readily
oxidized to the ferric form (Fe3+) with a decrease in sol-
ubility from %10210M to %10218M (Williams, 1982). In
addition, Fe2+can interact with hydrogen peroxide,
a byproduct of oxygen metabolism, and catalyze the
generation of highly toxic hydroxyl radicals, which ulti-
mately results in oxidative damage to proteins, mem-
branes, and DNA (Halliwell, 1978). Thus, to maintain an
adequate supply of iron, cells require molecular mecha-
nisms to overcome both the limited bioavailability and
potential toxicity of this transition metal. A well-charac-
terized mechanism is the one utilized by the ferritins,
a conserved family ofproteins that assemble into hollow
particles able to accommodate large amounts of iron,
which play a critical role in iron detoxification and stor-
age in the cytoplasm and other cellular compartments
(Chasteen and Harrison, 1999). In mitochondria of
has been recently shown to perform the dual function of
making Fe2+bioavailable and detoxifying surplus iron
(reviewed in Al-Karadaghi et al., 2006).
The function of frataxin has been the subject of in-
tense investigation ever since defects in this protein
were linked to Friedreich ataxia, a progressive disease
of children and adolescents characterized by neurolog-
ical impairment, cardiomyopathy, and diabetes mellitus
(Campuzano et al., 1996). In vivo frataxin promotes the
biosynthesis of heme (Schoenfeld et al., 2005; Zhang
et al., 2005) as well as the assembly and repair of iron-
sulfur clusters (Bulteau et al., 2004; Duby et al., 2002;
Gerber et al., 2003). This correlates well with the ability
of frataxin to deliver Fe2+to ferrochelatase, the enzyme
that catalyzesthe last stepof heme biosynthesis (O’Neill
et al., 2005a; Park et al., 2003; Yoon and Cowan, 2004);
to the iron-sulfur cluster scaffold protein IscU, which ini-
tiates the assembly of iron-sulfur clusters (Layer et al.,
2006; Yoon and Cowan, 2003); and to the [3Fe-4S]+
form of mitochondrial aconitase, which yields the active
[4Fe-4S]2+enzyme (Bulteau et al., 2004). In vivo frataxin
also plays a primary role in the protection against oxida-
tive stress (Gakh et al., 2006; Schultz et al., 2000; Thier-
bach et al., 2005; Vazquez-Manrique et al., 2006). This is
consistent with the ability of frataxin to catalyze the
oxidation of Fe2+to Fe3+, by using O2or H2O2as the
oxidant, and to promote the conversion of Fe3+to a
stable protein-bound mineral (Bou-Abdallah et al.,
2004; Nichol et al., 2003; O’Neill et al., 2005a; Park
et al., 2002).
Biochemicalstudies haveshown thatself-assembly is
a central part of the mechanism of iron detoxification by
frataxin. Thus, the bacterial frataxin homolog, CyaY,
forms a tetramer when Fe2+is added anaerobically,
while larger oligomers are formed in the presence of
Fe2+and atmospheric O2(Bou-Abdallah et al., 2004;
Layer et al., 2006). Using this mechanism, CyaY seques-
ters w25 atoms of iron per subunit in a polynuclear Fe3+
hydroxo(oxo) mineral. Assembly of yeast frataxin also
depends on the presence of iron, and it proceeds
according to the progression a / a3/ a6/ a12/
a24/ a48(Adamec et al., 2000; Gakh et al., 2002). The
48 subunit oligomer can store w50–75 iron atoms per
subunit in 1–2 nm cores, which are structurally similar
to ferrihydrite, the main biomineral formed by vertebrate
ferritins (Park et al., 2003; Nichol et al., 2003). Inter-
actions leading to stepwise assembly of yeast frataxin
*Correspondence: firstname.lastname@example.org (G.I.), salam.al-karadaghi@
oligomers have been suggested to be mediated by
alignment and complexation of ferrihydrite crystallites,
formed at separate mineralization sites (Park et al.,
2003). Indeed, yeast frataxin oligomers readily disas-
semble into monomers upon reduction of their ferric
iron cores (Park et al., 2003). Unlike CyaY and yeast fra-
taxin, human frataxin assembles in an iron-independent
manner via stable subunit-subunit interactions medi-
ated by the nonconserved N-terminal region of the pro-
tein (O’Neill et al., 2005b). Like yeast frataxin, the human
protein has ferroxidase activity and forms iron cores
structurally similar to ferrihydrite (Nichol et al., 2003).
Although the three-dimensional structures of the
monomers of CyaY, yeast, and human frataxin have
been determined (Adinolfi et al., 2002; Cho et al., 2000;
Cook et al., 2006; Dhe-Paganon et al., 2000; He et al.,
2004), the molecular mechanism that enables frataxin
to function in both iron delivery and detoxification re-
mains elusive. In light of the high degree of amino acid
sequence conservation among frataxins from different
property of the protein, we screened for point mutations
that would enable yeast frataxin to form stable oligo-
mers in an iron-independent manner, similar to human
frataxin. Here, iron-free and iron-loaded trimers and
a 24 subunit oligomer formed by an Y73A variant of
yeast frataxin were characterized by X-ray crystallogra-
struction. The data suggest a conserved mechanism in
while higher-order oligomers provide a basic structure
suitable for iron detoxification and storage.
Results and Discussion
Preparation of Stable Oligomers of Yeast Frataxin
The yeast frataxin monomer assembles stepwise in an
iron-dependent manner (Adamec et al., 2000). Trimer
was suggested to represent the functional unit of yeast
frataxin, but the instability of this species hampered
structural studies (Park et al., 2002; unpublished data).
In contrast, the human frataxin monomer assembles in
an iron-independent manner via stable protein-protein
interactions (O’Neill et al., 2005b). However, human fra-
taxin particles are not suitable for structural studies due
to their tendency to polymerize (Cavadini et al., 2002).
We therefore screened for point mutations that would
enable the yeast frataxin monomer to oligomerize in an
iron-independent manner. The mature form of the pro-
tide) (Branda et al., 1999) was subjected to site-directed
mutagenesis whereby residues conserved between
yeast and human frataxin were replaced by alanine res-
idues; then, mutant proteins were expressed in E. coli
and bacterial cell extracts analyzed by size-exclusion
chromatography as described (Gakh et al., 2006; O’Neill
et al., 2005b). Mutations in two regions (N terminus and
strands b2–b3) resulted in proteins that oligomerized
during expression in E. coli, yielding trimer and lower
levels of larger oligomers. The trimer and a 24 subunit
oligomer formed by one of these proteins (Y73A) were
further characterized, as described below.
Crystal Structure of the Trimer
The Y73A yeast frataxin crystallized in space group I213.
The structure was solved by molecular replacement by
using the structures of CyaY (Cho et al., 2000) and hu-
man frataxin (Dhe-Paganon et al., 2000) monomers as
search models. The NMR structure of the yeast frataxin
monomer available at that time (He et al., 2004) did not
give a satisfactory molecular-replacement solution.
The trimer structure was refined to the crystallographic
tein used in this study consisted of 123 amino acid res-
idues (52–174). Of these, the first 9 and the last 2 were
disordered in the electron density map and could not
be modeled. The final refinement statistics are pre-
sented in Table 1.
The overall arrangement of monomers in the trimer
structure is almost flat, with dimensions of 75 A˚ 3
75 A˚3 30 A˚(Figures 1A and 1B). Each subunit is folded
into an a/b sandwich with two a helices (a2, a3) packed
against a five-stranded (b1–b5), antiparallel, twisted
b sheet. Two additional b strands (b6, b7) build up
lix at the protein N terminus (a1), together with a 7 resi-
due extended coil region connected to helix a2, build
up an N-terminal extension that interacts with the neigh-
boring monomer within the trimer (Figures 1A and 1B).
earlier for different monomeric forms of the protein, al-
though with some important deviations. Helix a2 is
about two turns shorter in the trimer structure, suggest-
ing that oligomerization involves partial unfolding of this
element. Moreover, the N-terminal extension, which is
highly flexible in the yeast frataxin monomer (Cook
et al., 2006), is stabilized within the trimer by the interac-
tions between neighboring subunits (Figure 1B).
Table 1. Crystallographic Data Collection and Refinement
a, b, c (A˚)
Resolution range (A˚)
I/s(I) > 3 (%)
Solvent content (%)
aRmerge= SjIi2 <I>j/SI, where Iiis an individual intensity measure-
ment and <I> is the average intensity for this reflection.
bRcryst= SjFo2 Fcj/SFo, where Foand Fcare the observed and
calculated structure factor amplitudes, respectively. Rfree is the
same as Rcryst, but it is calculated on 5% of the data excluded
The plane of the b sheet in each subunit is inclined
with an angle of about 30?relative to the plane of the tri-
mer. Solvent-accessible parts of helix a3 and strand b7,
between strands b5 and b6 create one surface of the tri-
mer (Figure 1A). The opposite surface is created by ele-
ments from two different subunits, with the N-terminal
face of the next, which, in turn, contributes helix a2,
strands b1–b3, and the loop between b3 and b4 to the
surface (Figure 1B).
The Role of the N-Terminal Region
The N-terminal region of each subunit appears to play
a crucial role in the stabilization of the trimer. In each
monomer, the N-terminal region is anchored to the
core structure at the base (Figure 2A) and through helix
a1, which interacts with the b sheet of another subunit
(Figure 2B). Figure 2C shows a 3Fo2 2Fcelectron den-
sity map superimposed on the N-terminal region. At
the base, the conformation of the N-terminal extension
is stabilized by an extensive network of interactions be-
tween the main chain carbonyls of A72, H74, and E75
and the amide groups of H74, E76, and D78, respec-
tively, and between the side chains of H74, E76, and
of another subunit include residues P62, E64, V65, and
L68 from helix a1 and residues T110, E112, T118, and
P62, V65, and L68 in the N-terminal extension are di-
rectly packed against residues T110 and T118 (Fig-
ure 2B). Only the side chain of T118 and the carbonyl
oxygen of E64 are at hydrogen-bonding distance from
Interestingly, the N-terminal extension also plays
a major role in the packing of trimers within the crystals,
where two monomers from neighboring trimers pack
with their b sheets facing each other. The N-terminal ex-
tension is responsible for the interactions within the
crystal lattice and is sandwiched between the structural
elements (not shown). Similar interactions may be re-
oligomer (see below).
The Channel at the 3-Fold Axis of the Trimer
The trimer is further stabilized by interactions between
the loops around a central channel at the 3-fold axis
(Figures 1A and 1B). On one surface of the trimer, the
b hairpin loop between strands b6 and b7 (residues
S151–G155), the C terminus of strand b5, and the loop
between strands b5 and b6 (residues L144–W149) build
up a unique helical structural element (shown in red in
Figures 1A and 3A). Three such elements are arranged
around the 3-fold axis of the trimer, with the loop be-
tween strands b5 and b6 of one subunit packed against
the hairpin loop of the next subunit (Figure 3A). Two pu-
tative hydrogen bonds between subunits, one between
the carbonyl oxygen of L152 and the amide group of
G147 and the other between the side chain of N146
and the carbonyl oxygen of Q154, contribute to the
stabilization of the loops (Figure 3A). The side chains
of L145, V150, and L152 from the three subunits are ex-
posed to the solvent and together create a hydrophobic
edge around the entrance to the channel (Figure 3A). In
the sequence of human frataxin, L145, which is closer to
the surface, is replaced by a threonine, L152 is replaced
by a serine, while V150 is conserved (Figure 4). Each
subunit contributes an invariant aspartate residue
(D143, at the end of strand b5); the three D143 residues
are located approximately in the middle of the channel,
with their side chains directed toward the opening at
the opposite end (Figure 3A).
is built up by residues between G117 and D143, which
belong to strands b3, b4, and b5 (Figures 1B and 3B).
Figure 1. Overall Architecture of the Yeast Frataxin Trimer in Ribbon
(A) Suggested outer surface of the trimer with the exposed helices
a2 and a3 (shown in yellow). Loops and b strands are shown in
brown or red.
(B) Suggested inner surface of the trimer. The N-terminal exten-
sion, which contributes to the stabilization of the trimer, is shown
The Structures of Frataxin Oligomers
This is the most highly conserved stretch of amino acids
within eukaryotic frataxin sequences (Figure 4). Resi-
dues K123–I130 build up part of strands b3 and b4 and
the loop between them. The strands are sharply bent
by w80?relative to the plane of the central b sheet, while
the loop between them (residues P125–K128) extends
into the putative exit of the channel. Several interactions
may contribute to the stabilization of this loop. Among
them is a putative hydrogen bond between the side
chain of D143 and the amide of Q129 of the same sub-
unit. Additional interactions involve putative hydrogen
bonds between the side chain of Q129 and the carbonyl
oxygen of P126 of the next subunit, and between the
side chains of N127 of one subunit and E75 of another
(Figure 3B). Residues P126 and Q129 are invariant in
all frataxin sequences (Figure 4).
same crystallographic structure, loop P125–K128 may
exist in two alternative conformations, which approxi-
mately have the same occupancy (w0.5) (Figure 3C). In
the secondconformation, the tip ofthe loop isdisplaced
by about 8 A˚. This brings residues N127 and Q129 in
actions are formed between N127 and Q129, and be-
tween D143 and the side chains of N127 and Q129 of
the same subunit. The interactions between N127 and
E75 and between Q129 and P126 are not present in
this conformation. The peptide unit of P126 is flipped,
resulting in the carbonyl oxygen pointing toward the
interior of the channel, a direction opposite to that ob-
served in the first conformation (Figure 3C). A shift of
the side chain of K128 creates a positively charged lid
at the channel opening (Figure 3C).
The channel is funnel shaped, with openings of w15 A˚
ing acidity toward the latter (Figures 5A–5D). In analogy
with the channels at the 3-fold axis of ferritin shells
(Grant et al., 1998; Hempstead et al., 1994; Stillman
et al., 2001), the larger and smaller openings may serve
as an entrance and exit, respectively, for iron ions. How-
ever, while the entrance to the ferritin channel is lined by
basic residues, the putative entrance to the frataxin
channel is lined by hydrophobic residues (Figures 3A
and 5C), which may serve not only to guide ions into
the channel, but also to provide a docking surface for
the proteins that interact with frataxin (Bulteau et al.,
2004; Park et al., 2003; Yoon and Cowan, 2003).
The Crystal Structure of the Iron-Loaded Trimer
Iron loading of the frataxin trimer was achieved by aero-
bic incubation of the purified oligomer with Fe2+at a
molar ratio of two iron atoms per monomer, followed
by isolation of the iron-loaded trimer by size-exclusion
chromatography. A stoichiometric ratio of 0.9 (60.1)
Figure 2. Stabilization of the Trimer by the N-Terminal Extension
(A) Interactions at the base of the N-terminal extension.
(B) Interactions of helix a1 of one subunit of the trimer with the b sheet of another subunit.
(C) Stereoview showing a 3Fo2 2Fcdensity map (blue) contoured at the 1.3s level superimposed on helix a1.
iron atoms per iron-loaded trimer was measured by
using particle-induced X-ray emission (microPIXE)
(Garman and Grime, 2005), whereas there was no mea-
surable iron in the unloaded control. The iron-loaded
trimer was crystallized at conditions similar to those of
the iron-free complex, and diffraction data were col-
lected to a resolution of 3.6 A˚. Despite the low resolution
a high sigma peak (electron density is visible up to the
level of 5s; Figure 5C) at the center of the channel at
the 3-fold axis of the trimer. The presence of one iron
atom per trimer is in agreement with the microPIXE
results. Three additional peaks, which most probably
correspond to solvent molecules, are positioned at
w2 A˚from the center of the density (Figures 5C and
5D). This distance is within the range expected for
metal-solvent bond distances. Interestingly, in the pres-
ence of iron, the P125–K128 loop is present only in the
first of the two conformations described above. The
metal binds close to the suggested entrance at w4 A˚
from the side chains of the three D143 residues (Figures
molecules, not visible at the present resolution of the
data, bridge the interactions between the iron and pro-
tein groups. Owing to the hydrophobic lid formed by
V150, L152, and especially L145, the iron atom is fully
enclosed within the channel (Figure 5C).
Calculations of the electrostatic potential and corre-
sponding metal-binding energies in the central pore of
the frataxin trimer indicate that the change in the confor-
mation of the P125–K128 loop may affect the iron-
binding properties of the channel, with the second con-
formation demonstrating higher metal-binding affinity
(Figure 6; also see Experimental Procedures). This is in
apparent contrast with the fact that in the structure of
the iron-loaded trimer the P125–K128 loop is present
in the first, low-affinity binding conformation. A possible
explanation is that upon iron binding the interaction
between D143 and the metal prevents D143 from con-
tributing to the stabilization of the second, high-affinity
binding conformation, favoring a shift to the low-affinity
conformation. This may facilitate iron delivery to pro-
teins docked at the channel entrance, while conditions
that stabilize the second conformation, which remain
to be identified, may promote iron passage through
the channel. The suggested channel exit appears to be
Figure 3. Stereoview of the Structure of the
(A) Putative channel entrance. The helical
structural motif from each monomer is shown
(B) The first conformation of the proposed
channel exit. The residues that contribute to
the structure and stabilization of the trimer
are shown as sticks. In (A) and (B), the elec-
tron density contoured at the 1.3s level is
shown superimposed on the model.
(C)The second conformation ofthe proposed
channel exit. The teal ribbon model with
amino acid side chains (shown as sticks) rep-
nel exit. The structure of the first conforma-
tion shown as a brown ribbon model is
superimposed to demonstrate the two posi-
tions of the P125–K128 loop.
The Structures of Frataxin Oligomers
connected to negatively charged patches on the side of
each subunit (Figure 5B). The residues forming the
patches, D78, D79, D82, D86, E89, E90, and E93 from
helix a2, and D101 from strand b1, are conserved in eu-
nomeric frataxins have implicated these residues in iron
binding (Cook et al., 2006; Nair et al., 2004). In addition,
site-directed mutagenesis has shown that they com-
prise functionally distinct sites that are involved in iron
oxidation and mineralization, and that they are impor-
tant for iron detoxification in vivo (Gakh et al., 2006).
Each patch is lined by hydrophobic and positively
charged residues (H74, H106, K123), which together
with the side chain of K128 (in the high-affinity binding
ification sites. The putative ferroxidation site is in close
vicinity of the channel exit and entails an arrangement
of residues (H74, D78, D79, D82, and H83) reminiscent
of the di-iron site of ribonucleotide reductase (Nordlund
and Eklund, 1995) (Figure 5E). The residues constituting
this site have a different arrangement in the trimer com-
pared to the monomer structure of yeast frataxin (Cook
et al., 2006), due to the partial unfolding of helix a2, im-
plying that trimer formation is needed for the formation
of a functional ferroxidation site.
Position of Pathogenic Frataxin Point Mutations
Defects in frataxin are linked to the neurodegenerative
disease Friedreich ataxia (Campuzano et al., 1996).
Most patients carry large GAA trinucleotide repeat ex-
pansions in the first intron of the frataxin gene that ham-
per transcriptional elongation, causing a severe reduc-
tion in the levels of frataxin. About 4% of the patients
carry an expansion in one allele and a point mutation
in the other allele (Campuzano et al., 1996). While the
majority of the pathogenic point mutations affect the
hydrophobic core of the structure (L84S, I130F, L132P,
W149G, L158H, and L158F, yeast numbering), four mu-
tations are likely to destabilize formation of the trimeric
complex (Figure 7). These include G107V, W131R,
R141C (G130V, W155R, and R165C, human numbering),
and D122Y at a position that corresponds to P100 in
yeast. P100 is located in the loop between helix a2 and
strand b1, at the tip of the subunit close to the surface
against which helix a1 is packed. G107 is located in
a loop between strands b1 and b2. Its carbonyl oxygen
makes a putative hydrogen bond with the amide group
of K123, thus contributing to the stabilization of the con-
formation of the regions between K123 and I130. K123 is
also involved in interactions with the N-terminal ex-
tension through a putative hydrogen bond with D78.
Moreover, W131, which is exposed to the surface of
the central b sheet, is involved in interactions with the
N-terminal extension. Another mutation, R141C, may
disrupt a putative hydrogen bond to the carbonyl of
P125, which may affect the conformation of the loop at
the channel exit. Solution studies of the effect of these
mutations, in combination with a rational design of com-
pounds that stabilize the frataxin trimer, might offer
a new opportunity for the treatment of Friedreich ataxia.
EM Single-Particle Reconstruction
of the 24 Subunit Oligomer
The 24 subunit oligomer formed by yeast frataxin Y73A
was reconstructed from 4,000 particles at a resolution
of 19 A˚(Figures 8A and 8B). The reconstruction shows
a cubic shape in which the trimer represents the basic
structural unit, with the center of each trimer positioned
at the 3-fold axis of the cube (Figure 8C). Docking of the
trimer into the reconstruction (Figure 8D) shows that the
particle is primarily stabilized by interactions between
Figure 4. Alignment of Frataxin Sequences
The amino acid sequences of frataxin from yeast, human, mouse, and Drosophila are aligned with the bacterial CyaY frataxin homologs from
S. typhimurium, E. coli, and P. aeruginosa. Residues conserved only in eukaryotic or bacterial sequences are marked in green and blue, respec-
tively; invariant residues are marked in red. The positions of secondary structural elements are shown along the sequence. The overall sequence
identity is 40% between the yeast and human sequences, and it is 28% between the 106 overlapping residues (68–174) of the yeast and E. coli
sequences. This figure was generated with Alscript (Barton, 1993). The sequences are identified by their Swiss-Prot entry names.
the N termini of subunits from neighboring trimers
(Figure 8E). The best fit is obtained with the N termini
of the N termini places the suggested entrance to the
channel on the outside of the particle (Figure 8F), while
the exit and the oxidation and mineralization sites are
located in the interior. Ongoing reconstruction of iron-
loaded oligomers shows that the packing of trimers de-
limits a cavity for iron deposition (unpublished data).
This architecture is in agreement with the iron-storage
function of frataxin and is remarkably reminiscent of
the iron-storage protein ferritin (Grant et al., 1998;
Hempstead et al., 1994; Stillman et al., 2001) (Figure 8F).
A reduction in the levels of frataxin is responsible for
Friedreich ataxia, a fatal neurodegenerative and cardiac
disease (Campuzano et al., 1996). Frataxin is an iron-
binding protein required for the maintenance of mito-
chondrial iron balance in humans and other eukaryotes,
owing to its roles in the delivery of iron to the heme and
iron-sulfur cluster biosynthetic pathways and the detox-
ification of surplus iron (reviewed in Al-Karadaghi et al.,
2006). The mechanism that enables frataxin to perform
such essential functions has been eagerly pursued but
has remained elusive even after different structures of
omeric frataxin species described here suggest that
self-assembly provides the protein with the means to
bind iron and either transfer it to other proteins or detox-
ify and store it.
Whereas the assembly of yeast frataxin and CyaY is
driven by the binding and oxidation of iron (Layer
et al., 2006; Park et al., 2003), assembly of human fra-
taxin occurs in an iron-independent manner via sub-
unit-subunit interactions mediated by the protein N
Figure 5. Potential Functional Sites and
Metal Binding to the Central Channel of the
(A and B) Electrostatic surface-potential dis-
tribution for the suggested outer (A) and inner
(B) surfaces of the frataxin trimer. Red and
blue denote negative and positive potentials,
respectively. The residues constituting one
of the three proposed ferroxidation sites are
shown as sticks in (B).
(C) Top view of the channel with bound iron.
The Fo2 Fcdifference electron density map
contoured at the 4s level (magenta) is shown
superimposed on the 3Fo2 2Fcdensity map
(blue). The bound iron (shown as a blue
sphere) is located w6 A˚from the entrance
to the channel, as defined by the side chain
of L145. The red spheres correspond to sol-
(D)Cross-sectionthrough the central channel
showing the electrostatic-potential distribu-
tion within the channel. The bound iron and
surrounding solvent molecules are shown
(E) The proposed ferroxidation site of frataxin
viewed with an electrostatic-potential sur-
face. Distances (A˚) between the side chains
are indicated in the figure.
The Structures of Frataxin Oligomers
terminus (O’Neill et al., 2005b). The Y73A variant used in
the present work was identified by screening for point
mutations that would enable yeast frataxin to form sta-
bleoligomers inan iron-independent mannerlike human
frataxin. Similar variants found in the screen included
single substitutions at the base of the N-terminal exten-
sion, H74A and D78A, and double substitutions in the
b sheet, V108A/T110A and T118A/V120A. These muta-
tions probably induce conformational changes that fa-
vor the interaction of the N-terminal extension of one
subunit with the b sheet of another, thereby facilitating
is 18 residues longer than that of the yeast protein and
has a significantly different amino acid sequence,
whereas CyaY only has a short stretch of amino acids
upstream of helix a2 (Figure 4). Thus, we speculate
that different N-terminal regions reflect different modes
by which frataxin assembly can be induced and regu-
lated in different organisms.
nel at the 3-fold axis of the trimer. The two possible con-
formations of the channel suggest that a gated mecha-
nism controls whether the iron stays in the channel or
is transferred to the detoxification sites at the channel
exit. Because a single iron atom is delivered to ferroche-
latase during heme synthesis, IscU during iron-sulfur
repair, the trimer could be a suitable source of metal in
all of these processes in which frataxin has been impli-
cated as the iron donor. In addition, the relatively large
hydrophobic surface that lines the entrance to the chan-
nel may enable the trimer to interact with structurally
different proteins. The reconstruction of a 24 subunit
oligomer further shows that the trimer is the building
similarities in the structural organization of frataxin olig-
omers and the iron-storage protein ferritin, despite the
lack of any apparent evolutionary relationship between
these two protein families. It can also be expected that
the organization of the iron core of frataxin will be re-
miniscent of the iron core of ferritin (Nichol et al., 2003).
Together these data suggest that self-assembly pro-
vides frataxin with the structural features required to
perform both iron delivery and iron detoxification. The
insights gained from this work provide a framework to
identify natural factors or develop compounds that
enhance frataxin activity by modulating its oligomeriza-
Protein Preparation and Crystallization
The mutant Y73A frataxin from S. cerevisiae (residues 52–174 of the
yeast frataxin sequence, corresponding to the mature form of the
protein) (Branda et al., 1999) was recombinantly expressed in
cedure (Cavadini et al., 2002). Briefly, bacterial lysate (w25 ml at
26 mg/ml total protein) was applied to a Macro-Prep DEAE column
(16 mm 3 50 cm) (Bio-Rad), and protein was eluted with a 1 l linear
gradient, from 50 to 525 mM NaCl, in 20 mM Tris-HCl (pH 8.0) at
a flow rate of 10 ml/min. Most Y73A frataxin was eluted in two sep-
arate pools, a low-salt pool (300–380 mM NaCl) containing a3, and
a high-salt pool (440–510 mM) containing a24. Each DEAE pool
was diluted to 600 ml and loaded onto a Macro-Prep High Q column
(Bioscale MT20) (Bio-Rad), and the protein was eluted with a 500 ml
linear NaCl gradient as described above at a flow rate of 5 ml/min.
Two High Q pools were obtained, a24(w410–510 mM NaCl) and a3
(w200–290 mM NaCl). Each pool was concentrated to 1 ml, loaded
onto a Sephacryl 300 column (16 mm 3 60 cm) (Amersham-Biosci-
ences), and eluted with 120 ml of 10 mM HEPES-KOH (pH 7.3),
100 mM NaCl at a flow rate of 0.4 ml/min. Fractions containing trimer
(a3)ora24subunit oligomer(a24)werepooled andapplied toaMono
Q HR5/5 column (Amersham-Biosciences) and eluted with a linear
try Axis of the Trimer
Electrostatic interaction energies with a +2-charged ion were calcu-
lated for each of the two conformations of the P125–K128 loop as
described in Experimental Procedures. The red and blue plots
show the metal-binding energies for the first and second configura-
tions, respectively, along the 3-fold-symmetry axis of the trimer. The
position of the two plots along the 3-fold axis is defined by the Mol-
Script y coordinate (y coord, in angstroms from the unit cell origin).
The position of the metal-binding energy in the first configuration of
the channel (red plot) is in agreement with the position of the metal
ion in the iron-loaded structure (w6 A˚from the side chain of L145).
Figure 7. Position of Pathogenic FRDA Mutations
Residues that are mutated in some Friedreich ataxia patients are
mapped on one subunit of the trimer. See Results for details.
containing a3(w200–250 mM NaCl) or a24(w280–310 mM NaCl)
were concentrated and buffer exchanged to 10 mM HEPES-KOH
Trimer crystals were grown by vapor diffusion in a hanging drop
at 15?C. Crystallization drops were made by mixing 3 ml of a protein
solution at a concentration of 7 mg/ml in 10 mM KOH-HEPES
(pH 7.3) with 3 ml of a reservoir solution containing 0.1 M Bis-Tris
(pH 5.5), 2.0 M ammonium sulfate, and 4% (v/v) g-butyrolactone.
Crystals appeared after 5 days. The best crystals had dimensions
of w0.15 mm 3 0.15 mm 3 0.15mm. The crystalsbelongedto space
the purified oligomer with ferrous iron at a molar ratio of two iron
atoms per monomer for 2 hr at 30?C, followed by isolation of the
iron-loaded trimer by size-exclusion chromatography. Crystals in
space group I213 were obtained in the same manner as for the
Data Collection and Structure Determination
For data collection, 25% glycerol was used as cryoprotectant, and
crystals were mounted on a rayon loop and flash frozen directly in
Figure 8. Electron Microscopic Model of a 24
Subunit Frataxin Oligomer
(A) EM images of the 24 subunit oligomer in
1% uranyl acetate negative stain on the re-
(B) A comparison between projection images
of the reconstructed model (to the left) and
the class averages (to the right). See Experi-
mental Procedures for details.
(C) View approximately along the 2-fold axis,
which relates two trimers. A total of eight tri-
mers make the oligomer.
(D) Packing of trimers docked into the EM re-
construction. The trimers (green and purple)
are shown in surface representation.
(E) A stereoview showing interactions be-
tween the N termini of two subunits from
neighboring trimers packed within the 24
(F) Surface representation of frataxin (left)
and horse-spleen ferritin (right) 24-meric olig-
omers viewed along the 3-fold axis, repre-
sented with the same scale. The width of
the particles is 115 A˚and 130 A˚, respectively.
Trimers are colored according to their elec-
trostatic surface potentials.
The Structures of Frataxin Oligomers
a stream of boiled-off nitrogen gas held at 100 K. A native data set to
3.0 A˚resolution (I213) was collected with a MAR Research CCD de-
tector at MAX II synchrotron laboratory stations I711 and I911-2 in
Lund, Sweden (Cerenius et al., 2000; Mammen et al., 2004). The res-
olution of the data could not beimproved atthe higher-energy Swiss
Light Source synchrotron. A data set of the iron-loaded trimer was
collected with an ADSC CCD detector at beamline ID23-1, ESRF
synchrotron facility in Grenoble, France, to a resolution of 3.6 A˚
(I213). All data sets were processed with the XDS package (Kabsch,
1993). The structure was solved by molecular replacement with the
program Phaser (McCoy et al., 2005) by using a combination of the
solved crystal structures of human frataxin and the E. coli homolog,
CyaY (Cho et al.,2000; Dhe-Paganon et al.,2000), as a search probe.
The model was built by using the graphical program O (Jones et al.,
1991) and was refined with CNS (Brunger et al., 1998). The progress
of refinement was followed by decreasing R and Rfreevalues. Data
collection and refinement statistics are shown in Table 1.
Particle-Induced X-Ray Emission Measurements
The particle-induced X-ray emission (microPIXE) measurements
were carried out at the National Ion Beam Centre, University of Sur-
rey, UK on a beamline arranged as described by Grime et al. (1991).
Small volumes (0.2 ml) of the unloaded and iron-loaded protein con-
centrated to 20 mg/ml in Tris-HCl buffer (pH 7.3) were each pipetted
onto two separate 2 mm thick mylar films stretched over three alumi-
num target holders and dried, as described (Garman, 1999). A
2.5 MeV proton beam of 3 mm diameter was used to induce charac-
teristic X-ray emission from the dried liquid droplet under vacuum.
The X-rays were detected in a solid-state lithium-drifted silicon de-
tector with high energy resolution. The proton beam was then
scanned spatially in the x and y dimensions. Using the data collec-
tion software, spatial maps of all elements heavier than sodium
that were present in the sample were obtained. Quantitative infor-
mation was obtained by collecting spectra at four separate selected
points on the drop and also at a point on the backing foil. These
spectra were analyzed with GUPIX (Johansson et al., 1995) to ex-
tract the areal density of each element of interest in the sample
with respect to the sulfur peak from the methionines and cysteines
in the protein. The sulfur signal provides a very convenient internal
calibration, allowing the number of iron atoms per protein monomer,
and hence per trimer, to be obtained.
Calculation of Metal Interaction Energies in the Channel
along the 3-Fold Axis
Calculations of the electrostatic potential in the central pore of the
frataxin trimer were performed by solving the Poisson-Boltzmann
equation with the program MEAD (Macroscopic Electrostatics with
Atomic Detail) (Bashford, 1997; Bashford and Gerwert, 1992). The
electrostatic potential was calculated for each of the two different
conformations of the P125–K128 loop present in the iron-free struc-
ture of the trimer. The protein was described as a volume with a di-
electric constant of 4, dissolved in waterwith a dielectric constant of
80. All atoms in the protein were assigned a partial charge, taken
from the AMBER 2003 force field (Case et al., 2004; Cornell et al.,
1995). Aspartate and glutamic acid residues were assumed to be
negatively charged, and lysine and arginine residues were assumed
to be positively charged. Based on the presumed hydrogen bond
pattern, the surroundings, and the solvent exposure, we let H74,
H83, and H106 be doubly protonated and therefore positively
charged, whereas H95 was protonated on the N32atom. Several ad-
ditional calculations were made with different assignments of the
histidine residues, e.g., all residues protonated on the N32atom or
all residues doubly protonated, as well as different ionic strength
values. These variations had some influence on the absolute value
of the potential, but the shapes and positions of the two curves
shown in Figure 6 were not affected. Hydrogen atoms were added
and optimized by AMBER 8 (Case et al., 2004) before the MEAD
calculations. All atoms were assigned PARSE radii (Sitkoff et al.,
1994), and the solvent probe radius was 1.4 A˚(water). The electro-
static potential was calculated along the 3-fold symmetry axis by
using a 0.5 A˚grid with at least twice the size of the protein (2513
grid points). The calculations were run at 300 K. The electrostatic
potential was used to calculate the electrostatic interaction energies
obtained by multiplying the electrostatic potential at any given posi-
in Figure 6 exclude the solvation energy of the unbound metal ion,
which will reduce the binding energies by a constant factor for
both curves. In Figure 6, the position of the two curves along the
used to calculate the electrostatic potential.
EM Data Collection and Image Processing
Protein solution (2 ml of 0.2 mg/ml) was applied to carbon-coated,
glow-discharged copper grids (400-mesh, Electron Microscopy Sci-
ences, USA) and was allowed to adsorb for 1 min. Excess solution
was blotted, and the grids were subsequently stained with either
1% uranyl acetate in water (2 ml for 10 s) or 1% phosphor tungsten
acid in 20 mM phosphate buffer (pH 6.9) (2 ml for 1 min). Images
were acquired with a Philips CM120 equipped with a Gatan GIF
100 energy filter and Gatan 791 CCD camera (1024 3 1024 pixels)
at a magnification of 52,400-fold (sampling distance 4.67 A˚). The im-
age processing was performed with the Eman software package
(Ludtke et al., 1999), applying octahedral point group symmetry.
The iterative classification procedure converged after 8 cycles of
reclassifications, and 4000 particles out of the 5400 were used for
calculating the final reconstruction. The resolution of the frataxin
oligomer was determined to 19 A˚according to the 0.5 Fourier shell
correlation criteria. Strict damping of the FSC curve in high-
resolution shells was observed, which, together with the compari-
son between class averages and projections, is an indication that
the envelope of the classification process is not limiting. The dock-
ing of the X-ray structure into the EM reconstruction was done in
Chimera (Pettersen et al., 2004). The modification of the trimeric
X-ray structure was done in O (Jones et al., 1991). The final recon-
struction of the 24 subunit oligomer was evaluated by comparing
class averages with projection images in the same angular orienta-
tion of the interpolated model (Figure 8B). The image pairs revealed
the same structural features, an indication of the reconstruction be-
ing in a global minimum, rather than trapped in a local one. An initial
reconstruction was performed without any symmetry. The result
revealsacubic shape ofthe molecule, vindicating ouruse of octahe-
dral symmetry (result not shown). Several independent reconstruc-
tions aligning on either point group symmetry or varying reference
models gave similar results. To induce a better fit to the EM model,
the monomer had to be tilted by an angle of about 12?compared to
its position in the X-ray model. The tilt did not introduce any notable
changes in the internal interactions within the trimeric structure, and
the size of the channel was conserved.
We acknowledge MAX-lab and the European Synchrotron Radiation
Facility for provision of synchrotron radiation facilities, and we thank
the National Center for High-Resolution Electron Microscopy elec-
tron biomicroscopy unit. This work was supported by grants from
the Swedish ResearchCounciltoS.A.-K.and fromtheNationalInsti-
tutes of Health/National Institute on Aging (AG15709) and the Frie-
dreich Ataxia Research Alliance (FARA) to G.I.
Received: March 21, 2006
Revised: August 21, 2006
Accepted: August 28, 2006
Published: October 10, 2006
Adamec,J.,Rusnak, F., Owen,W.G.,Naylor, S.,Benson, L.M., Gacy,
A.M., and Isaya, G. (2000). Iron-dependent self-assembly of
recombinant yeast frataxin: implications for Friedreich ataxia. Am.
J. Hum. Genet. 67, 549–562.
Adinolfi, S., Trifuoggi, M., Politou, A.S., Martin, S., and Pastore, A.
(2002). A structural approach to understanding the iron-binding
properties of phylogenetically different frataxins. Hum. Mol. Genet.
Al-Karadaghi, S., Franco, R., Hansson, M., Shelnutt, J.A., Isaya, G.,
and Ferreira, G.C. (2006). Chelatases: distort to select? Trends Bio-
chem. Sci. 31, 135–142.
Barton, G.J. (1993). An efficient algorithm to locate all locally optimal
alignments between two sequences allowing for gaps. Comput.
Appl. Biosci. 9, 729–734.
Bashford, D. (1997). An object-oriented programming suite for elec-
trostatic effects in biological molecules. In Scientific Computing in
Object-Oriented Parallel Environments, Y. Ishikawa, R.R. Oldehoeft,
J.V.W. Reynders, and M. Tholburn, eds. (Berlin: Springer), pp. 233–
pKa values of ionizable groups in bacteriorhodopsin. J. Mol. Biol.
Bou-Abdallah,F., Adinolfi, S.,Pastore, A.,Laue,T.M., andChasteen,
N.D. (2004). Iron binding and oxidation kinetics in frataxin CyaY of
Escherichia coli. J. Mol. Biol. 341, 605–615.
Branda, S.S., Cavadini, P., Adamec, J., Kalousek, F., Taroni, F., and
Isaya, G. (1999). Yeast and human frataxin are processed to mature
form in two sequential steps by the mitochondrial processing pepti-
dase. J. Biol. Chem. 274, 22763–22769.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P.,
Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,
Pannu, N.S., et al. (1998). Crystallography & NMR system: a new
software suite for macromolecular structure determination. Acta
Crystallogr. D Biol. Crystallogr. 54, 905–921.
Bulteau, A.L., O’Neill, H.A., Kennedy, M.C., Ikeda-Saito, M., Isaya,
G., and Szweda, L.I. (2004). Frataxin acts as an iron chaperone pro-
tein to modulate mitochondrial aconitase activity. Science 305, 242–
Campuzano, V., Montermini, L., Molto, M.D., Pianese, L., Cossee,
M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A.,
et al. (1996). Friedreich’s ataxia: autosomal recessive disease
caused by an intronic GAA triplet repeat expansion. Science 271,
Case, D.A., Darden, T.A., Cheatham, T.E., Simmerling, C.L., Wang,
J., Duke, R.E., Luo, R., Merz, K.M., Wang, B., Pearlman, D.A., et al.
(2004). AMBER 8 (www.amber.scripps.edu).
Cavadini, P., O’Neill, H.A., Benada, O., and Isaya, G. (2002). Assem-
bly and iron binding properties of human frataxin, the protein defi-
cient in Friedreich ataxia. Hum. Mol. Genet. 33, 217–227.
Cerenius, Y., Sta ˚hl, K., Svensson, L.A., Ursby, T., Oskarsson, A˚.,
Albertsson, J., and Liljas, A. (2000). The crystallography beamline
I711 at MAX II. J. Synchrotron Radiat. 7, 203–208.
Chasteen, N.D., and Harrison, P.M. (1999). Mineralization in ferritin:
an efficient means of iron storage. J. Struct. Biol. 126, 182–194.
Cho, S.J., Lee, M.G., Yang, J.K., Lee, J.Y., Song, H.K., and Suh, S.W.
(2000). Crystal structure of Escherichia coli CyaY protein reveals
a previously unidentified fold for the evolutionarily conserved fra-
taxin family. Proc. Natl. Acad. Sci. USA 97, 8932–8937.
Cook, J.D., Bencze, K.Z., Jankovic, A.D., Crater, A.K., Busch, C.N.,
Bradley, P.B., Stemmler, A.J., Spaller, M.R., and Stemmler, T.L.
(2006). Monomeric yeast frataxin is an iron-binding protein. Bio-
chemistry 45, 7767–7777.
Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, K.M., Fergu-
son, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., and Kollman,
P.A. (1995). A second generation force field for the simulation of pro-
teins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117,
Dhe-Paganon, S., Shigeta, R., Chi, Y.I., Ristow, M., and Shoelson,
S.E. (2000). Crystal structure of human frataxin. J. Biol. Chem. 275,
Duby, G.,Foury, F.,Ramazzotti,A., Herrmann, J.,andLutz,T.(2002).
A non-essential function for yeast frataxin in iron-sulfur cluster as-
sembly. Hum. Mol. Genet. 11, 2635–2643.
Gakh, O., Adamec, J., Gacy, M.A., Twesten, R.D., Owen, W.G., and
Isaya, G. (2002). Physical evidence that yeast frataxin is an iron stor-
age protein. Biochemistry 41, 6798–6804.
Gakh, O., Park, S., Liu, G., Macomber, L., Imlay, J.A., Ferreira, G.C.,
and Isaya, G. (2006). Mitochondrial iron detoxification is a primary
function of frataxin that limits oxidative damage and preserves cell
longevity. Hum. Mol. Genet. 15, 467–479.
Garman, E. (1999). Leaving no element of doubt: analysis of proteins
using microPIXE. Structure 7, R291–R299.
by microPIXE. Prog. Biophys. Mol. Biol. 89, 173–205.
Gerber, J., Muhlenhoff, U., and Lill, R. (2003).An interaction between
frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on
Isu1. EMBO Rep. 4, 906–911.
Grant, R.A., Filman, D.J., Finkel, S.E., Kolter, R., and Hogle, J.M.
(1998). The crystal structure of Dps, a ferritin homolog that binds
and protects DNA. Nat. Struct. Biol. 5, 294–303.
Grime, G.W., Dawson, M., Marsh, M., McArthur, I.C., and Watt, F.
(1991). The Oxford submicron nuclear facility. Nucleic Instrum.
Meth. B 54, 52–53.
Halliwell, B. (1978). Superoxide-dependent formation of hydroxyl
radicals in the presence of iron salts. Its role in degradation of hya-
luronic acid bya superoxide-generatingsystem.FEBS Lett. 96, 238–
He, Y., Alam, S.L., Proteasa, S.V., Zhang, Y., Lesuisse, E., Dancis, A.,
and Stemmler, T.L. (2004). Yeast frataxin solution structure, iron
binding, and ferrochelatase interaction. Biochemistry 43, 16254–
Hempstead, P.D., Hudson, A.J., Artymiuk, P.J., Andrews, S.C., Ban-
field, M.J., Guest, J.R., and Harrison, P.M. (1994). Direct observation
of the iron binding sites in a ferritin. FEBS Lett. 350, 258–262.
Johansson, S.A.E., Campbell, J.L., and Malqvist, K.G. (1995).
Particle induced X-ray emission spectrometry (PIXE). In Chemical
Analysis: A Series of Monographs on Analytical Chemistry and Its
Applications, S.A.E. Johansson, J.L. Campbell, and K.G. Malmqvist,
eds. (New York: John Wiley and Sons, Inc.), p. 167.
Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard. (1991). Im-
proved methods for building protein models in electron density
maps and the location of errors in these models. Acta Crystallogr.
A 47(Pt. 2), 110–119.
Kabsch, W. (1993). Automatic processing of rotation diffraction data
from crystals of initially unknown symmetry and cell constants.
J. Appl. Crystallogr. 26, 795–800.
(2006). Iron-sulfur cluster biosynthesis: characterization of Escheri-
chia coli CyaY as an iron donor for the assembly of [2Fe-2S] clusters
in the scaffold IscU. J. Biol. Chem. 281, 16256–16263.
Lill, R., Diekert, K., Kaut, A., Lange, H., Pelzer, W., Prohl, C., and
Kispal, G. (1999). The essential role of mitochondria in the biogene-
sis of cellular iron-sulfur proteins. Biol. Chem. 380, 1157–1166.
Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiauto-
mated software for high-resolution single-particle reconstructions.
J. Struct. Biol. 128, 82–97.
Bent Diamond Crystals and Multilayer Based Optics at the new
MAX-lab. AIP Conference Proceedings 705, 808–811.
McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C., and Read, R.J.
(2005).Likelihood-enhanced fast translation functions. Acta Crystal-
logr. D Biol. Crystallogr. 61, 458–464.
Nair, M., Adinolfi, S., Pastore, C., Kelly, G., Temussi, P., and Pastore,
A. (2004). Solution structure of the bacterial frataxin ortholog, CyaY:
mapping the iron binding sites. Structure 12, 2037–2048.
Nichol, H., Gakh, O., O’Neill, H.A., Pickering, I.J., Isaya, G., and
George, G.N. (2003). Structure of frataxin iron cores: an X-ray ab-
sorption spectroscopic study. Biochemistry 42, 5971–5976.
Nordlund, P., and Eklund, H. (1995). Di-iron-carboxylate proteins.
Curr. Opin. Struct. Biol. 5, 758–766.
O’Neill, H.A., Gakh, O., Park, S., Cui, J., Mooney, S.M., Sampson, M.,
Ferreira, G.C., and Isaya, G. (2005a). Assembly of human frataxin is
a mechanism to detoxify redox-active iron. Biochemistry 44, 537–
O’Neill, H.A., Gakh, O., and Isaya, G. (2005b). Supramolecular
assemblies of human frataxin are formed via subunit-subunit
The Structures of Frataxin Oligomers
interactions mediated by a non-conserved amino-terminal region. Download full-text
J. Mol. Biol. 345, 433–439.
Park, S., Gakh, O., Mooney, S.M., and Isaya, G. (2002). The ferroxi-
dase activity of yeast frataxin. J. Biol. Chem. 277, 38589–38595.
Park, S., Gakh, O., O’Neill, H.A., Mangravita, A., Nichol, H., Ferreira,
G.C., and Isaya, G. (2003). Yeast frataxin sequentially chaperones
and stores iron by coupling protein assembly with iron oxidation.
J. Biol. Chem. 278, 31340–31351.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Green-
blatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera—a
visualization system for exploratory research and analysis. J. Com-
put. Chem. 25, 1605–1612.
Schoenfeld, R.A., Napoli, E., Wong, A., Zhan, S., Morin, D., Buckpitt,
A.R., Taroni, F., Lonnerdal, B., Ristow, M., Puccio, H., and Corto-
passi, G.A. (2005). Frataxin deficiency alters heme pathway tran-
scripts and decreases mitochondrial heme metabolites in mamma-
lian cells. Hum. Mol. Genet. 14, 3787–3799.
Schultz, J.B., Dehmer, T., Schols, L., Mende, H., Hardt, C., Vorgerd,
M., Burk, K., Matson, W., Dichgans, J., Beal, M.F., and Bogdanov,
M.B. (2000). Oxidative stress in patients with Friedreich’s ataxia.
Neurology 55, 1719–1721.
Sitkoff, D., Sharp, K.A., and Honig, B. (1994). Accurate calculation of
hydration free-energies using macroscopic solvent models. J. Phys.
Chem. 98, 1978–1988.
Stillman, T.J., Hempstead, P.D., Artymiuk, P.J., Andrews, S.C., Hud-
resolution X-ray crystallographic structure of the ferritin (EcFtnA) of
Escherichia coli; comparison with human H ferritin (HuHF) and the
structures of the Fe(3+) and Zn(2+) derivatives. J. Mol. Biol. 307,
Thierbach, R., Schulz, T.J., Isken, F., Voigt, A., Mietzner, B., Drewes,
G., von Kleist-Retzow, J.C., Wiesner, R.J., Magnuson, M.A., Puccio,
H., et al. (2005). Targeted disruption of hepatic frataxin expression
causes impaired mitochondrial function, decreased life span and tu-
mor growth in mice. Hum. Mol. Genet. 14, 3857–3864.
Vazquez-Manrique, R.P., Gonzalez-Cabo, P., Ros, S., Aziz, H., Bay-
lis, H.A., and Palau, F. (2006). Reduction of Caenorhabditis elegans
frataxin increases sensitivity to oxidative stress, reduces lifespan,
and causes lethality in a mitochondrial complex II mutant. FASEB
J. 20, 172–174.
Williams, R.J.(1982).Free manganese (II) and iron (II) cationscan act
as intracellular cell controls. FEBS Lett. 140, 3–10.
Yoon, T., and Cowan, J.A. (2003). Iron-sulfur cluster biosynthesis.
Characterization of frataxin as an iron donor for assembly of [2Fe-
Yoon, T., and Cowan, J.A. (2004). Frataxin-mediated iron delivery to
Ferrochelatase in the final step of Heme biosynthesis. J. Biol. Chem.
Zhang, Y., Lyver, E.R., Knight, S.A., Lesuisse, E., and Dancis, A.
(2005). Frataxin and mitochondrial carrier proteins, Mrs3p and
Mrs4p, cooperate in providing iron for heme synthesis. J. Biol.
Chem. 280, 19794–19807.
Atomic coordinates for the reported crystal structures have been
deposited in the Protein Data Bank with accession number 2FQL.