REVIEW ARTICLE
Seeking the determinants of the elusive functions of Sco
proteins
Lucia Banci
1,2
, Ivano Bertini
1,2
, Gabriele Cavallaro
1
and Simone Ciofi-Baffoni
1,2
1 Magnetic Resonance Center (CERM), University of Florence, Italy
2 Department of Chemistry, University of Florence, Italy
Introduction
The first member of the family of Sco (synthesis of
cytochrome c oxidase) proteins was identified in yeast
as a gene product essential for accumulation of the
mitochondrially synthesized subunit II (Cox2) of
cytochrome c oxidase (COX) [1]. COX is the terminal
component of the respiratory chain, located in the
inner mitochondrial membrane of eukaryotes and in
the plasma membrane of many prokaryotes. The
catalytic core of the enzyme is composed of the three
largest subunits (Cox1, Cox2 and Cox3), which are
highly conserved between prokaryotes and eukaryotes
[2]. Both Cox1 and Cox2 contain metal cofactors
which are required for COX to function, and include
one copper ion in Cox1 (termed Cu
B
) and two copper
ions forming a dinuclear centre in Cox2 (termed Cu

mitochondrial cytochrome c oxidase; however their precise role in this pro-
cess has not yet been elucidated at the molecular level. In particular, some
but not all eukaryotes including humans possess two Sco proteins whose
individual functions remain unclear. There is evidence that eukaryotic Sco
proteins are also implicated in other cellular processes such as redox signal-
ling and regulation of copper homeostasis. The range of physiological
functions of Sco proteins appears to be even wider in prokaryotes, where
Sco-encoding genes have been duplicated many times during evolution.
While some prokaryotic Sco proteins are required for the biosynthesis of
cytochrome c oxidase, others are most likely to take part in different
processes such as copper delivery to other enzymes and protection against
oxidative stress. The detailed understanding of the multiplicity of roles
ascribed to Sco proteins requires the identification of the subtle determi-
nants that modulate the two properties central to their known and poten-
tial functions, i.e. copper binding and redox properties. In this review, we
provide a comprehensive summary of the current knowledge on Sco
proteins gained by genetic, structural and functional studies on both
eukaryotic and prokaryotic homologues, and propose some hints to unveil
the elusive molecular mechanisms underlying their functions.
Abbreviations
BsSco, apo-Sco from Bacillus subtilis; IMS, intermembrane space; Trx, thioredoxin.
2244 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
The two copper ions in Cu
A
are coordinated by two
bridging Cys sulfur atoms, two His nitrogen atoms,
and 2 weak ligands provided by a Met sulfur and a
backbone carbonyl oxygen [5]. The highly covalent
and rigid Cu
2

site of Cox2 [12]. The details of the mechanism
by which Sco proteins accomplish this function, how-
ever, remain a controversial issue, which is complicated
by the fact that different mechanisms appear to oper-
ate in different organisms. Long recognized evidence in
this sense comes from the observation that two Sco
proteins (Sco1 and Sco2) playing distinct roles are
required for maturation of the Cu
A
site in humans
[13,14], whereas yeast, despite having two Sco proteins
as well, needs only one of them [9,15]. Furthermore, to
make the matter more puzzling, the human proteins
have been proposed to fulfil additional functions
besides COX assembly, including mitochondrial redox
signalling [16] and regulation of copper homeostasis
[17].
Sco proteins are also found in prokaryotic organ-
isms, leading to the widespread postulation that their
function in COX assembly is conserved between
eukaryotes and prokaryotes [18]. Although this
assumption is supported by experimental data, the pre-
cise mode of action of Sco proteins in the insertion of
copper into Cox2 is as uncertain in prokaryotes as it is
in eukaryotes, and can also differ in different organ-
isms [19]. In addition, prokaryotic Sco proteins have
also been implicated in functions that are unrelated to
COX assembly, such as in regulation of gene expres-
sion [20] and in protection against oxidative stress [21].
The functional divergence of Sco proteins in prokary-

present in the last common ancestor of lineages that
diverged as early as metazoans and flowering plants,
i.e. more than 900 million years ago. Also, it showed
that the genomes of vertebrates and flowering plants
contain two Sco genes, which derive from two inde-
pendent duplication events. To complement and extend
these data, we have searched Sco genes in a total of 66
eukaryotic species (27 animals, 18 fungi, 9 plants and
12 protists) including, in addition to those examined in
[23], all species whose complete genome sequences are
available at the NCBI as of December 2010 (http://
www.ncbi.nlm.nih.gov/genomes/leuks.cgi). A summary
of our results is shown in Table 1.
Sco genes have been found in 61 of the 66 eukaryotes
analysed, with the exceptions of the microsporidia
Encephalitozoon cuniculi and Encephalitozoon intestinal-
is, the amoebae Entamoeba dispar and Entamoeba
histolytica, and the apicomplexan Cryptosporidium
parvum. The absence of Sco genes in these organisms is
not unexpected, as all of them are obligate intracellular
parasites that contain degenerated mitochondria
called mitosomes, which lack many of the functions of
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2245
Table 1. Occurrence of genes encoding Sco proteins in eukaryotic organisms, sorted by taxonomic group. Organisms that were analysed in
[23] are highlighted in grey. Gene and protein IDs reported as not available (n ⁄ a) indicate genes that were identified in [23] but not by our
search (presumably due to incomplete genome sequences). For the number of Sco genes in Pan troglodytes, see text.
Organism Group Subgroup # Sco genes NCBI gene IDs NCBI protein IDs
Xenopus tropicalis Animals Amphibians 2 100494754
100497895

Macaca mulatta Animals Mammals 2 720679
722074
XP_001116350.2
XP_001118271.1
Mus musculus Animals Mammals 2 52892
100126824
NP_001035115.1
NP_001104758.1
Pan troglodytes Animals Mammals 1 (2) 745696 XP_001164786.1
Sus scrofa Animals Mammals 2 100516804
100517855
XP_003126813.1
XP_003132044.1
Branchiostoma floridae Animals Other animals 1 7233239 XP_002613836.1
Hydra magnipapillata Animals Other animals 1 100198802 XP_002156667.1
Ixodes scapularis Animals Other animals 1 8026286 XP_002402970.1
Nematostella vectensis Animals Other animals 1 5522188 XP_001641939.1
Strongylocentrotus purpuratus Animals Other animals 1 763450 XP_001199433.1
Caenorhabditis elegans Animals Roundworms 1 173763 NP_494755.1
Ashbya gossypii Fungi Ascomycetes 1 4620854 NP_984670.2
Aspergillus nidulans Fungi Ascomycetes 1 2872639 XP_662446.1
Aspergillus oryzae Fungi Ascomycetes 1 5996623 XP_001824537.2
Candida dubliniensis Fungi Ascomycetes 1 8049436 XP_002422402.1
Candida glabrata Fungi Ascomycetes 2 2886568
2889365
XP_445160.1
XP_447458.1
Debaryomyces hansenii Fungi Ascomycetes 1 2899722 XP_002769958.1
Kluyveromyces lactis Fungi Ascomycetes 1 2893043 XP_453226.1
Lachancea thermotolerans Fungi Ascomycetes 1 8290333 XP_002551538.1

In addition to plants and vertebrates, multiple Sco
genes also occur in the fungi Saccharomyces cerevisiae
and Candida glabrata, which have two such genes, and
in kinetoplast protozoa, which have three (apart from
Leishmania braziliensis, which has two). A neighbour-
joining tree built from the multiple alignment of all the
Sco proteins identified (Fig. 1) indicates that indepen-
dent duplications occurred (a) in a common ancestor
of vertebrates, (b) in a common ancestor of land
plants, (c) in a common ancestor of S. cerevisiae and
C. glabrata, and possibly of other fungi, and (d) in a
common ancestor of kinetoplasts, where two duplica-
tions occurred. This scenario implies that in eukaryotes
containing two or three Sco proteins these proteins
have distinct physiological functions, which are not nec-
essarily the same in organisms belonging to different
Table 1. (Continued).
Organism Group Subgroup # Sco genes NCBI gene IDs NCBI protein IDs
Encephalitozoon intestinalis Fungi Other fungi 0 – –
Micromonas sp. RCC299 Plants Green algae 1 8246970 XP_002508419.1
Ostreococcus lucimarinus Plants Green algae 1 5006467 XP_001422358.1
Ostreococcus tauri Plants Green algae 1 9838624 XP_003084388.1
Arabidopsis thaliana Plants Land plants 2 820046
830129
NP_566339.1
NP_568068.1
Oryza sativa Plants Land plants 2 4328372
4346889
NP_001045964.1
NP_001063017.1

Leishmania major Protists Kinetoplasts 3 3684900
5651436
5653126
XP_888624.1
XP_001682836.1
XP_001684203.1
Trypanosoma brucei Protists Kinetoplasts 3 3660260
3660582
4357233
XP_803555.1
XP_827193.1
XP_001218860.1
Trypanosoma cruzi Protists Kinetoplasts 3 3535712
3537405
3540368
XP_805842.1
XP_807216.1
XP_809712.1
Entamoeba dispar Protists Other protists 0 – –
Entamoeba histolytica Protists Other protists 0 – –
Monosiga brevicollis Protists Other protists 1 5887529 XP_001742585.1
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2247
0.1
Homo sapiens|NP 004580.1
Pan troglodytes|XP 001164786.1
1000
Macaca mulatta|XP 001118271.1
1000
Bos taurus|NP 001073712.1

418
Hydra magnipapillata|XP 002156667.1
317
347
253
Homo sapiens|NP 005129.2
Macaca mulatta|XP 001116350.2
1000
Bos taurus|NP 001098963.1
Sus scrofa|XP 003126813.1
898
866
Mus musculus|NP 001104758.1
1000
Xenopus tropicalis|XP 002935088.1
1000
394
Caenorhabditis elegans|NP 494755.1
841
Sorghum bicolor|XP 002453341.1
Zea mays|NP 001130056.1
1000
Oryza sativa|NP 001045964.1
1000
Vitis vinifera|XP 002263427.1
644
Arabidopsis thaliana|NP 566339.1
771
Populus trichocarpa|XP 002323592.1
1000

1000
1000
410
Leishmania infantum|XP 001462953.1
Leishmania major|XP 888624.1
1000
Leishmania braziliensis|XP 001561796.1
1000
Trypanosoma brucei|XP 827193.1
Trypanosoma cruzi|XP 805842.1
946
1000
Leishmania infantum|XP 001465217.1
Leishmania major|XP 001682836.1
1000
Trypanosoma cruzi|XP 809712.1
806
Trypanosoma brucei|XP 001218860.1
1000
413
535
240
Ashbya gossypii|NP 984670.2
Lachancea thermotolerans|XP 002551538.1
752
Kluyveromyces lactis|XP 453226.1
544
Zygosaccharomyces rouxii|XP 002494544.1
483
Candida glabrata|XP 447458.1

Kinetoplasts
Animals
Sco1 land plants
Green algae
Apicomplexans
Sco1 vertebrates
Fungi
Sco1 fungi
Sco2 fungi
Determinants of the elusive functions of Sco proteins L. Banci et al.
2248 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
kingdoms, however. As mentioned in the Introduction,
it is indeed well known that the physiological roles of
Sco2 in humans and yeast must be diverse. It would
then be useful to assess experimentally the roles of the
duplicated proteins in kinetoplasts and land plants as
well. In particular, determining the function of the
duplicated plant proteins would be especially interest-
ing, as in all plants one of the two proteins (indicated
as ‘Sco2 land plants’ in Fig. 1) lacks the characteristic
CXXXC motif present in all the other Sco proteins
and is thus presumably unable to bind copper (a
CXXXG motif is found in the proteins from Oryza sa-
tiva, Sorghum bicolor and Zea mays, and a SXXXG
motif in those from Arabidopsis thaliana, Popu-
lus trichocarpa and Vitis vinifera).
The distribution of Sco proteins across prokaryotic
species is far more variable than in eukaryotes. A bioin-
formatics analysis of 311 prokaryotic genomes (285
from Bacteria and 26 from Archaea) revealed that Sco

share most of their major features: all the residues that
are highly conserved in prokaryotes, including copper
ligands and two aspartates in a DXXXD motif, are
present and highly conserved in eukaryotes as well, and
the additional highly conserved residues in eukaryotes
are generally those found most frequently (though
being more variable) in the corresponding positions in
prokaryotic sequences. In this respect, the most remark-
able differences are the presence in eukaryotes of a
DEXXK motif downstream of the CXXXC motif
which has no counterpart in prokaryotes, and two other
changes also involving the occurrence of charged resi-
dues in eukaryotes in the place of non-polar residues in
prokaryotes (Glu and Arg for Ala and Gly, respec-
tively; see Fig. 2).
Fig. 2. Profile–profile comparison of eukaryotic and prokaryotic Sco protein sequences obtained using the program HHSEARCH [94]. The profile
of eukaryotic sequences was constructed from their multiple alignment using the program
HMMER [95], while that of prokaryotic sequences
was taken from [22]. Highly conserved residues (i.e. residues occurring at a given position with probability > 0.5) are shown in bold. Copper-
binding residues are highlighted in yellow. Positions where the two profiles differ most are highlighted in red.
Fig. 1. Neighbour-joining tree built (using the program CLUSTALW [91]) from the multiple alignment of eukaryotic Sco proteins (constructed
using the program
MUSCLE [92]). Relevant subgroups are shown. Numbers on branches are bootstrap values based on 1000 replicates. The
tree was visualized with the program
TREEVIEW [93].
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2249
Structural studies on eukaryotic and
prokaryotic Sco proteins: hints for
function

solution, multiple local conformational states exchang-
ing with each other on the intermediate or slow NMR
timescale (ls to ms) [27] (Fig. 4). This effect is particu-
larly observed in human apo-Sco2 which indeed, at
variance with human apo-Sco1, displays a conforma-
tional heterogeneity involving, in addition to the
metal-binding site region, also the b sheet and the sur-
rounding a helices which constitute the protein core of
Sco2 [28]. Cu(I) binding, however, is in both Sco pro-
teins able to ‘freeze’ the above regions in an ordered,
more rigid conformation (Fig. 4). This behaviour can
be rationalized taking into account the spatial location
of metal ligands. Cu(I) ion is in fact coordinated by
the two Cys residues of the CXXXC conserved motif,
located in loop 3 and helix a1, and by a conserved His
which is far in the sequence from the CXXXC motif,
i.e. in the b-hairpin present in the extended, solvent-
exposed loop (Fig. 4). Therefore, the involvement in
the metal-binding site of residues from two different
regions of the protein contributes to produce a com-
pact structure of the metal-loaded protein state with
respect to the apo form. The large conformational var-
iability of the His-containing loop observed in the apo-
Sco1 solution structure [27] indicates that backbone
structural changes are necessary to locate the metal
ligand His260 in the vicinity of the other two ligands,
Cys169 and Cys173. This behaviour is also confirmed
by the crystal structures [16,27,31]. Even if the loop
segments of apo-Sco1 have a continuous electron den-
sity with similar backbone conformations in all three

Sco proteins (inserted at the N-terminus and between strand b4
and helix a3) are shown in red. A specific property of the eukaryotic
Sco fold is the presence of an extended, solvent-exposed loop con-
taining a b-hairpin (shown in green) connecting helix a3 and strand
b6.
+Cu(I)
Fig. 4. Illustration of how metal binding ‘freezes’ the conforma-
tional heterogeneity of the metal-binding region in Sco proteins.
From an apo state characterized by conformational disorder in the
CXXXC motif and the loop containing the histidine ligand, one com-
pact conformer with the appropriate metal-ligand distances is
selected upon metal addition. The cysteine ligands are shown in
yellow, the histidine ligand in blue, and the Cu(I) ion in light blue.
Determinants of the elusive functions of Sco proteins L. Banci et al.
2250 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
acquires a more ordered conformation as a conse-
quence of Ni(II) binding [27]. This higher order is rec-
ognized by a definition of the electron density map in
that region for both molecules of the asymmetric unit
of Ni(II)-Sco1 higher than that in the apo-Sco1 crystal
structure. A further confirmation comes from the sig-
nificantly lower temperature factors of the atoms
belonging to the His-containing loop in the structure
of Ni(II)-Sco1 with respect to those in the apo-Sco1
structure. Crystallization therefore most likely selects,
in apo-Sco1, the lowest-energy conformers between the
multiple ones present in solution. Backbone conforma-
tional changes to allow the formation of a Cu(I)-bind-
ing site appear to be necessary also in the crystallized
apo-Sco1 state, in agreement with the demand of a

and Cys216, two cysteine residues present in yeast Sco1
but not conserved in human Sco1 and Sco2 and not
belonging to the conserved CXXXC motif. A possible
explanation of this result is that the soaking solution
contained Cu(II) rather than Cu(I) ions, and the Cu(II)
ions could then have catalysed oxidation of the
conserved cysteines, which therefore cannot bind cop-
per. The copper ion was then bound at an adventitious
site formed by the non-conserved Cys181 and Cys216
plus the conserved His239 in the flexible long loop.
These structural data on eukaryotic Sco proteins
indicate that, despite the full conservation of the three
metal-binding ligands, the metal-binding site has an
intrinsic structural flexibility, indicating the absence of
a binding site structurally well organized to receive the
metal. The latter property can thus explain the efficient
formation of a disulfide bond between the Cys ligands
and the movement of the His ligand towards a copper-
binding site located in a different position with respect
to the typical metal-binding site of the Sco proteins.
The His-ligand-containing loop indeed displays the
largest backbone fluctuations from the apo- to the
Cu(I)-bound state, positioning the imidazole ring of
His260 about 10 A
˚
from the sulfur atoms of the metal-
binding Cys residues, in apo-Sco1. However, from an
open apo conformation with local disorder, the struc-
ture converts, upon metal binding, into a well defined
compact state. In particular, the His ligand coordina-

including at least one histidine, and possibly a weakly
bound water molecule.
Both NMR and crystallographic data on BsSco show
structural properties very similar to those found for
eukaryotic apo-Sco proteins. Backbone conformational
exchange processes have been detected in solution
for the CXXXC metal binding motif and the
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2251
His-containing loop of BsSco. Accordingly, the RMSD
between the His-containing loop of the two crystallo-
graphically independent molecules A and B is quite
high (4.66 A
˚
compared with an overall value of
0.14 A
˚
). Also, the average temperature B-factor of this
loop is 53.56, compared with the average B-factor of
30.81 for molecule A and 31.23 for molecule B, and the
loop has a weak electron density map. The CXXXC-
containing loop also exhibits differences between
molecule A and molecule B (RMSD of 1.56 A
˚
),
although much less than the His-containing loop, with
the average B-factor (49.40) also higher than the protein
average. In some structures of apo-BsSco obtained in
the presence of copper, a disulfide bridge is observed
between the Cys of the CXXXC motif, similarly to

which a rapidly formed intermediate state of Cu(II)-
BsSco, with low-micromolar metal affinity, is then
slowly converted into the stable final Cu(II)-bound
form [38]. However, high ionic strength can induce
destabilization of the Cu(II)-BsSco complex and metal
release, indicating that structural flexibility of the
metal-binding site can be easily promoted also in this
case [36]. In a physiological context, it could be possible
that, for BsSco as well as for human Sco proteins, the
interactions with a specific protein partner can induce
conformational changes of the metal-binding site, thus
promoting the metal release to the Cu
A
site.
Eukaryotic Sco proteins in the
assembly of the Cu
A
site of COX
In eukaryotes, a large number of nuclear genes are
required for the proper assembly and function of COX
[39]. The most thoroughly characterized aspect of
COX assembly is that of mitochondrial copper delivery
to the nascent holoenzyme complex, and in particular
delivery of copper to the Cu
A
site. Such process
involves Sco proteins, specifically two highly homolo-
gous members of the family, Sco1 and Sco2, and
Cox17. Solution structure of the latter protein shows
that a highly conserved twin Cx

Cox17 [50], indicating that Sco1 functions downstream
of Cox17 in copper delivery to COX. Copper-binding
properties [51], mutational analysis of the metal-bind-
ing CXXXC motif [52] and physical interactions with
Cox2 [53] suggested that Sco1 specifically delivers cop-
per to the Cu
A
site in the Cox2 subunit [52,53]. A ser-
ies of conserved residues on the leading edge of the
Determinants of the elusive functions of Sco proteins L. Banci et al.
2252 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
His-containing loop have been suggested to be impli-
cated in Cox2 interaction, but not in the interaction
with Cox17, thus indicating different surfaces on Sco1
for the interaction with Cox17 and Cox2 [54]. The
copper transfer from Sco1 to Cox2 has never been
directly observed in vitro as all the attempts to stabilize
eukaryotic Cox2 domains have been unsuccessful so
far [55]. At variance with Sco1 mutants, yeast Sco2
mutants lack an obvious phenotype associated with
respiration, even if, similarly to Sco1, Sco2 interacts
with the C-terminal portion of Cox2 [56]. Although
Sco2, like Sco1, can restore respiratory growth in the
Cox17 null mutant, rescue in this case requires addi-
tion of copper to the growth medium [9]. Sco2 does
not suppress a Sco1 null mutant, although it is able to
partially rescue a Sco1 point mutant [9]. The ability of
Sco2 to restore respiration in Cox17 but not Sco1
mutants is taken as an indication that Sco1 and Sco2
have overlapping but not identical functions. Most

3S-S
2GSH
GSSG
Cu, 2e
–
Cu(I)Sco1
2e
–
, Cu(I)
apoCox2
Cu(I)Sco2
Cu(I)Cox17
Cu(I)
apoCox17
2S-S
Cu(I)Cox17
Cu(I)
Cu(I), 2e
–
Cu, 2e
–
apoCox17
2S-S
Cytoplasm
IMS
Matrix
Fig. 5. Pathway of copper insertion into the Cu
A
site of COX in humans. The structures of Cox2 and of Cox17, Sco1 and Sco2 in their differ-
ent metal or redox states are shown. Cysteine residues involved in copper binding or disulfide bond formation are shown as yellow sticks.

trol to specifically transfer the metal to the correct pro-
tein. The same reaction of copper-electron-coupled
transfer does not occur with human Sco2 (Fig. 5), for
kinetic reasons that may be ascribed to the lack of a
specific metal-bridged protein–protein complex, which
is instead observed in the Cu(I)-Cox17 ⁄ Sco1 interaction
[60]. The different Sco1 ⁄ Sco2 metallation properties
seem to be conserved also in the cell environment, as
the metallation of human Sco1, but not of Sco2, when
expressed in the yeast cytoplasm, is dependent on the
co-expression of human Cox17 [58]. Cu(II) binding has
also been suggested to be crucial for normal Sco1 func-
tion, but how the Cu(II) site in Sco protein is generated
still remains an open question, since only Cu(I) can be
bound to the physiological copper donor Cox17.
Pathogenic missense mutations in both Sco1 and
Sco2 genes produce respiratory chain deficiency associ-
ated with COX assembly defects [64–66] that result in
early onset diseases with fatal clinical outcomes. How-
ever, human Sco2 patients present neonatal encephalo-
cardiomyopathy [67,68], whereas Sco1 patients exhibit
neonatal hepatic failure and ketoacidotic coma [69] or
a fatal hypertrophic cardiomyopathy [70]. These dis-
tinctive clinical presentations are not a result of tissue-
specific expression of the two genes, as Sco1 and Sco2
are ubiquitously expressed and exhibit a similar expres-
sion pattern in different human tissues [65]. The mis-
sense mutation in human Sco1 of a conserved proline,
adjacent to the CXXXC motif, into a leucine (P174L)
is associated with a fatal neonatal hepatopathy. This

the Sco1 protein [59].
Besides being Cu(I) chaperones, human Sco proteins
have been proposed to perform other functional roles
in the final step of Cu
A
biogenesis, which are strictly
linked to the complete understanding of the functional
role of their CXXXC motif. Structural studies [27,28]
have indeed suggested that the CXXXC motif of
human Sco proteins confers a redox activity to the
proteins, resulting in a potential thioredoxin function.
It has been suggested that this thioredoxin activity
could be implicated in a disulfide exchange reaction
from Sco2 to Sco1 (Fig. 5) [14] and toward an oxidized
state of Cox2, i.e. with Cox2 metal-binding cysteines
forming a disulfide bond so as to allow copper incor-
poration into the Cu
A
site (Fig. 5) [14,27]. Recent find-
ings also implicate Sco2, but not Sco1, in the
stabilization of newly synthesized Cox2 molecules,
indicating that Sco2 also plays a role in Cu
A
biogenesis
upstream of Sco1 and that it is indispensable for Cox2
polypeptide synthesis [14]. It is important to stress that
the suggested redox-dependent processes represent a
working model of Cu
A
site biogenesis. In fact, much

minal segment (containing the residues protruding into
the mitochondrial matrix, the transmembrane helix
and the following  20 residues) is crucial to determin-
ing the aggregation state of these proteins. The data
therefore support the hypothesis that this N-terminal
region is important for modulating the aggregation
state of the proteins.
In conclusion, Cu
A
biogenesis in humans is a com-
plex mechanism involving both Cu(I) and disulfide
exchange reactions from Cox17 to the apo-Cu
A
site
passing through Sco1 and Sco2 proteins (Fig. 5), but
the molecular details of the role of Sco proteins in the
copper insertion into the Cu
A
site needs to be further
investigated.
Other functions of eukaryotic Sco
proteins
Genetic and biochemical studies identified the existence
of a bioactive copper pool within the mitochondrial
matrix that is used to metallate both COX and super-
oxide dismutase [73,74]. Copper is delivered to the
organelle by a small non-proteinaceous ligand, not yet
identified, that probably translocates from the cytosol
to the matrix upon metal ion binding [75]. It is
thought that the copper-loaded anionic ligand diffuses

catalyses copper efflux from the cell in fibroblasts) are
established yet. However, it has been proposed, only
on the basis of genetic data [17], that the molecular
basis for the mitochondrial signal might be generated
by Sco2-dependent modulation of the redox state of
the cysteines within the CXXXC motif of Sco1. Specif-
ically, this proposal was based on the fact that signifi-
cant perturbations were detected in the redox state of
the cysteines of Sco1 in both Sco1 and Sco2 patient
backgrounds, and these correlate well with the severity
of the observed cellular copper deficiency [17]. The
thiol-disulfide oxidoreductase function of the CXXXC
motif of human Sco proteins could therefore be impli-
cated not only in the maturation of the Cu
A
site of
Cox2 but also in the maintenance of cellular copper
homeostasis.
The involvement of human Sco1 in mitochondrial
signalling pathways has also been evoked for another
process. Indeed, on the basis of its structural similarity
to peroxiredoxins, which have been implicated in a
number of signalling pathways, and of the high perox-
ide sensitivity of yeast DSco1 cells, it has been sug-
gested that Sco1 deficiency may disturb the ability of
the mitochondrion to sense its redox state or to react
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2255
to peroxide signals, thus suggesting a role for Sco1 in
mitochondrial redox signalling pathways [16]. Human

A
cysteine ligands [19]. Copper insertion into
Cox2, in the form of Cu(I) ions, is carried out by a
periplasmic protein called PCu
A
C which is able to
selectively and sequentially deliver two Cu(I) ions to
apo-Cu
A
giving rise to the native Cu(I)
2
-CuA site [19].
PCu
A
C has been proposed to be a periplasmic copper
chaperone, thus taking the role of Cox17 in bacteria.
The solution structure and extended X-ray absorption
fine structure data of the PCu
A
C homologue from Dei-
nococcus radiodurans revealed that the protein binds
Cu(I) through histidine and methionine ligands in a
solvent-exposed copper-binding site, which is thus well
poised for metal transfer chemistry [78]. In summary,
the proposed mechanism of Cu
A
assembly consists of
the sequential insertion of two Cu(I) ions donated by a
metallochaperone into the Cu
A

COX assembly in this organism under microaerobic
conditions, which B. japonicum encounters in the sym-
biotic bacteroid form [81]. However, the significance of
this observation is unclear, as the main COX enzyme
expressed by B. japonicum under microaerobic condi-
tions is a Cu
A
-lacking cbb
3
oxidase [81]. Indeed,
another study reported that B. japonicum Sco does not
affect the assembly of cbb
3
oxidase, but rather is
required for the maturation of the Cu
A
-containing
COX which is predominant for aerobic growth, thus
leaving open the question as to the identity of the oxi-
dase enzyme whose assembly is affected by PCu
A
C
and Sco in the symbiotic state [82]. A similar uncer-
tainty exists with the Sco protein (called SenC) from
the metabolically versatile bacterium Pseudomo-
nas aeruginosa, which was reported to function in cop-
per delivery to cbb
3
oxidase and possibly to other
types of COX [83]. On the other hand, copper delivery

observed for the prokaryotic Sco protein from the pur-
ple photosynthetic bacteria Rhodobacter sphaeroides
(called PrrC), which indeed has been reported to bind
Cu(I) not specifically, to be able to reduce Cu(II) to
Determinants of the elusive functions of Sco proteins L. Banci et al.
2256 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
Cu(I), and to possess thiol-disulfide oxidoreductase
activity [86,87]. The disulfide reductase activity of
these two prokaryotic Sco proteins indicates that pro-
karyotic Sco proteins can generally act as periplasmic
thiol-disulfide oxidoreductases. The structural charac-
terization of a Sco protein that has redox but not cop-
per-binding properties, like Sco from P. putida, was
important as it provided a useful basis to find the
structural factors that determine the formation of a
tight-affinity or a weak-affinity copper-binding site in
Sco proteins. Specifically, a model was proposed where
the histidine ligand may or may not adopt conforma-
tions suitable for copper coordination in dependence
on the occurrence of hydrophobic interactions in the
surroundings of the metal-binding site (Fig. 6), and
only once histidine coordination is lost can Sco pro-
teins acquire redox activity [85]. The histidine ligand
coordination is therefore the discriminating factor for
introducing a high-affinity copper-binding site in Sco
proteins. Such a general model would mainly delineate
prokaryotic Sco proteins as redox-active proteins
whose activity is modulated by copper (Cu(I) or
Cu(II)) binding, rather than proteins serving a copper
chaperone function. On this basis, it can be argued

role [88]. However, at variance with eukaryotic Sco
proteins, a copper-bound form of BsSco has never
been structurally solved, despite its documented ability
to bind Cu(I) and Cu(II) ion through the CXXXC
motif [34–36]. Also, currently available information
from in vitro experiments does not support a copper
chaperone function [89]. The two cysteines in the
CXXXC motif were indeed found to easily intercon-
vert between the oxidized disulfide and the reduced
dithiol states, supporting the idea that BsSco would
fulfil a redox role in Cu
A
assembly [33]. In particular,
at high ionic strength and in the presence of excess
copper, the Cu(II)-bound protein undergoes oxidation
forming a metal-free, disulfide-bonded state with con-
comitant formation of Cu(I) [36], and mutation of the
copper-binding histidine to alanine increases the redox
sensitivity of Cu(II)-BsSco by three orders of magni-
tude at normal ionic strength [89]. This mutation
therefore does not result in elimination of the copper-
binding capability, but rather produces a variant with
altered redox chemistry. This led to the suggestion that
the Cu(II)–histidine bond may act as a switch for the
Ser
Lys
Thr
Ser
Asn
Ser

considering that copper-loaded BsSco was capable of
< 20% reconstitution of Cox2 in vitro [89] and that
the effect of the histidine-to-methionine mutation on
the redox potential of the CXXXC motif has not been
investigated, all the available data could be reconciled
considering that BsSco works in the Cu
A
maturation
process as a thioredoxin to maintain the correct oxida-
tion state of the Cu
A
cysteine ligands to allow copper
binding. The Cu(II)-bound form may stabilize the
reduced state of the cysteines, being activated toward
the disulfide exchange reaction only upon Cox2-specific
recognition.
A role for Sco proteins unrelated to copper trans-
port, and specifically in protection against oxidative
stress, was also proposed earlier for the homologues
found in the pathogens Neisseria meningitidis and
Neisseria gonorrhoeae, two organisms which do not
have a Cu
A
-containing COX [21]. In Neisseria species,
Sco is not required for the assembly of cbb
3
oxidase,
and its inactivation results in a much increased sensi-
tivity to oxidative killing by paraquat [21]. These find-
ings suggested that Sco could act in the protection of

Cox2, the histidine ligand is released from
Cu(I), thereby activating copper chaperone
and thioredoxin activities of Sco1 ⁄ Sco2
toward its Cox2 partner. The van der Waals
contact surface of the hydrophobic residues
in the metal-binding region of human Cu(I)-
Sco1 ⁄ Sco2 is shown in blue. The cysteines
in the CXXXC motif and the histidine ligand
are shown in yellow and green, respectively.
The Cu(I) ion is shown as an orange sphere.
Determinants of the elusive functions of Sco proteins L. Banci et al.
2258 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
occurred in the surroundings of the CXXXC catalytic
site to acquire copper-binding properties at different
levels from bacteria to eukaryotes, thus introducing a
copper chaperone function in a protein with thioredox-
in properties. Specifically, a histidine ligand in a
flexible loop region close to the CXXXC motif is able
to modulate the metal affinity of the protein. This
combination of redox and metal-binding properties
would determine the functional versatility of Sco pro-
teins, and their capability to act in copper trafficking
as well as in redox-related processes, possibly including
signalling pathways depending on either or both of
these processes.
Subtle structural and chemical features determine
the specific copper-binding properties of individual Sco
proteins, and consequently their ability to engage in
redox reactions and in turn their molecular functions.
As suggested by the recent data obtained from the

˚
resolution of cytochrome c oxidase
from Paracoccus denitrificans. Nature 376, 660–669.
3 Richter OM & Ludwig B (2003) Cytochrome c oxidase
– structure, function, and physiology of a redox-driven
molecular machine. Rev Physiol Biochem Pharmacol
147, 47–74.
4 Hill BC (1993) The sequence of electron carriers in the
reaction of cytochrome c oxidase with oxygen. J Bioen-
erg Biomembr 25, 115–120.
5 Tsukihara T, Aoyama H, Yamashita E, Tomizaki T,
Yamaguchi H, Shinzawa-Itoh K, Nakashima R, Yaono
R & Yoshikawa S (1995) Structures of metal sites of
oxidized bovine heart cytochrome c oxidase at 2.8 A
˚
.
Science 269, 1069–1074.
6 Farver O, Lu Y, Ang MC & Pecht I (1999) Enhanced
rate of intramolecular electron transfer in an engineered
purple CuA azurin. Proc Natl Acad Sci USA 96, 899–
902.
7 Kroneck PM, Antholine WA, Riester J & Zumft WG
(1988) The cupric site in nitrous oxide reductase con-
tains a mixed-valence [Cu(II),Cu(I)] binuclear center: a
multifrequency electron paramagnetic resonance investi-
gation. FEBS Lett 242, 70–74.
8 Solomon EI, Xie X & Dey A (2008) Mixed valent sites in
biological electron transfer. Chem Soc Rev 37, 623–638.
9 Glerum DM, Shtanko A & Tzagoloff A (1996) SCO1
and SCO2 Act as high copy suppressors of a mitochon-

structure of human SCO1: implications for redox
signaling by a mitochondrial cytochrome c oxidase
‘assembly’ protein. J Biol Chem 280, 15202–15211.
17 Leary SC, Cobine PA, Kaufman BA, Guercin GH,
Mattman A, Palaty J, Lockitch G, Winge DR, Rustin
L. Banci et al. Determinants of the elusive functions of Sco proteins
FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS 2259
P, Horvath R et al. (2007) The human cytochrome c
oxidase assembly factors SCO1 and SCO2 have regula-
tory roles in the maintenance of cellular copper homeo-
stasis. Cell Metab 5, 9–20.
18 Chinenov YV (2000) Cytochrome c oxidase assembly
factors with a thioredoxin fold are conserved among
prokaryotes and eukaryotes. J Mol Med 78, 239–242.
19 Abriata LA, Banci L, Bertini I, Ciofi-Baffoni S, Gkazo-
nis P, Spyroulias GA, Vila AJ & Wang S (2008) Mecha-
nism of Cu(A) assembly. Nat Chem Biol 4, 599–601.
20 Eraso JM & Kaplan S (2000) From redox flow to gene
regulation: role of the PrrC protein of Rhodobacter sph-
aeroides 2.4.1. Biochemistry 39, 2052–2062.
21 Seib KL, Jennings MP & McEwan AG (2003) A Sco
homologue plays a role in defence against oxidative
stress in pathogenic Neisseria. FEBS Lett 546, 411–415.
22 Banci L, Bertini I, Cavallaro G & Rosato A (2007) The
functions of Sco proteins from genome-based analysis.
J Proteome Res 6, 1568–1579.
23 Porcelli D, Oliva M, Duchi S, Latorre D, Cavaliere V,
Barsanti P, Villani G, Gargiulo G & Caggese C (2010)
Genetic, functional and evolutionary characterization of
scox, the Drosophila melanogaster ortholog of the

33 Ye Q, Imriskova-Sosova I, Hill BC & Jia Z (2005) Iden-
tification of a disulfide switch in BsSco, a member of
the Sco family of cytochrome c oxidase assembly pro-
teins. Biochemistry
44, 2934–2942.
34 Andruzzi L, Nakano M, Nilges MJ & Blackburn NJ
(2005) Spectroscopic studies of metal binding and metal
selectivity in Bacillus subtilis BSco, a homologue of the
yeast mitochondrial protein Sco1p. J Am Chem Soc
127, 16548–16558.
35 Imriskova-Sosova I, Andrews D, Yam K, Davidson D,
Yachnin Y & Hill BC (2006) Characterization of the
redox and metal binding activity of BsSco, a protein
implicated in the assembly of cytochrome c oxidase.
Biochemistry 44, 16949–16956.
36 Davidson DE & Hill BC (2009) Stability of oxidized,
reduced and copper bound forms of Bacillus subtilis
Sco. Biochim Biophys Acta 1794, 275–281.
37 Siluvai GS, Mayfield M, Nilges MJ, Debeer GS &
Blackburn NJ (2010) Anatomy of a red copper center:
spectroscopic identification and reactivity of the copper
centers of Bacillus subtilis Sco and its Cys-to-Ala
variants. J Am Chem Soc 132, 5215–5226.
38 Cawthorn TW, Poulsen BE, Davidson DE, Andrews
D & Hill BC (2009) Probing the kinetics and thermo-
dynamics of copper(II) binding to Bacillus subtilis
Sco, a protein involved in the assembly of the Cu(A)
center of cytochrome c oxidase. Biochemistry 48,
4448–4454.
39 Carr HS & Winge DR (2003) Assembly of cytochrome

Determinants of the elusive functions of Sco proteins L. Banci et al.
2260 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
46 Maxfield AB, Heaton DN & Winge DR (2004) Cox17
is functional when tethered to the mitochondrial inner
membrane. J Biol Chem 279, 5072–5080.
47 Punter FA & Glerum DM (2003) Mutagenesis reveals a
specific role for Cox17p in copper transport to cyto-
chrome oxidase. J Biol Chem 278, 30875–30880.
48 Neupert W & Herrmann JM (2007) Translocation of
proteins into mitochondria. Annu Rev Biochem 76,
723–749.
49 Krummeck G & Rodel G (1990) Yeast SCO1 protein is
required for a post-translational step in the accumula-
tion of mitochondrial cytochrome c oxidase subunits I
and II. Curr Genet 18, 13–15.
50 Horng YC, Cobine PA, Maxfield AB, Carr HS & Win-
ge DR (2004) Specific copper transfer from the Cox17
metallochaperone to both Sco1 and Cox11 in the
assembly of yeast cytochrome c oxidase. J Biol Chem
279, 35334–35340.
51 Nittis T, George GN & Winge DR (2001) Yeast Sco1,
a protein essential for cytochrome c oxidase function is
a Cu(I)-binding protein. J Biol Chem 276, 42520–42526.
52 Rentzsch A, Krummeck-Weiss G, Hofer A, Bartuschka
A, Ostermann K & Rodel G (1999) Mitochondrial cop-
per metabolism in yeast: mutational analysis of Sco1p
involved in the biogenesis of cytochrome c oxidase.
Curr Genet 35, 103–108.
53 Lode A, Kuschel M, Paret C & Rodel G (2000) Mito-
chondrial copper metabolism in yeast: interaction

J, Horvath R, Trepesch N, Stecker G, Freisinger P,
Thirion C et al.
(2001) Cytochrome c oxidase deficiency
due to mutations in SCO2 , encoding a mitochondrial
copper-binding protein, is rescued by copper in human
myoblasts. Hum Mol Genet 10, 3025–3035.
62 Salviati L, Hernandez-Rosa E, Walker WF, Sacconi S,
DiMauro S, Schon EA & Davidson MM (2002) Copper
supplementation restores cytochrome c oxidase activity
in cultured cells from patients with SCO2 mutations.
Biochem J 363, 321–327.
63 Hu J, Dong L & Outten CE (2008) The redox environ-
ment in the mitochondrial intermembrane space is
maintained separately from the cytosol and matrix.
J Biol Chem 283, 29126–29134.
64 Shoubridge EA (2001) Cytochrome c oxidase deficiency.
Am J Med Genet 106, 46–52.
65 Papadopoulou LC, Sue CM, Davidson MM, Tanji K,
Nishino I, Sadlock JE, Krishna S, Walker W, Selby J,
Glerum DM et al. (1999) Fatal infantile cardioencephal-
omyopathy with COX deficiency and mutations in
SCO2, a COX assembly gene. Nature Genet 23, 333–337.
66 Paret C, Lode A, Krause-Buchholz U & Rodel G
(2000) The P(174)L mutation in the human hSCO1 gene
affects the assembly of cytochrome c oxidase. Biochem
Biophys Res Commun 279, 341–347.
67 Salviati L, Sacconi S, Rasalan MM, Kronn DF, Braun
A, Canoll P, Davidson M, Shanske S, Bonilla E, Hays
AP et al. (2002) Cytochrome c oxidase deficiency due to
a novel SCO2 mutation mimics Werdnig–Hoffmann dis-

74 Cobine PA, Pierrel F, Bestwick ML & Winge DR
(2006) Mitochondrial matrix copper complex used in
metallation of cytochrome oxidase and superoxide
dismutase. J Biol Chem 281, 36552–36559.
75 Leary SC, Winge DR & Cobine PA (2009) ‘Pulling the
plug’ on cellular copper: the role of mitochondria in
copper export. Biochim Biophys Acta 1793, 146–153.
76 Leary SC (2010) Redox regulation of SCO protein func-
tion: controlling copper at a mitochondrial crossroad.
Antioxid Redox Signal 13, 1403–1416.
77 Matoba S, Kang JG, Patino WD, Wragg A, Boehm M,
Gavrilova O, Hurley PJ, Bunz F & Hwang PM (2006)
p53 regulates mitochondrial respiration. Science 312,
1650–1653.
78 Banci L, Bertini I, Ciofi-Baffoni S, Katsari E, Katsaros
N, Kubicek K & Mangani S (2005) A copper(I) protein
possibly involved in the assembly of CuA center of bac-
terial cytochrome c oxidase. Proc Natl Acad Sci USA
102, 3994–3999.
79 Arnesano F, Banci L, Bertini I & Martinelli M (2005)
Ortholog search of proteins involved in copper delivery
to cytochrome c oxidase and functional analysis of par-
alogs and gene neighbors by genomic context. J Prote-
ome Res 4, 63–70.
80 Andreini C, Bertini I, Cavallaro G, Decaria L & Rosato
A (2011) A simple protocol for the comparative analysis
of the structure and occurrence of biochemical path-
ways across superkingdoms. J Chem Inf Model 51, 730–
738.
81 Arunothayanan H, Nomura M, Hamaguchi R, Itakura

sesses thiol-disulfide oxidoreductase activity. FEBS Lett
581, 4663–4667.
88 Mattatall NR, Jazairi J & Hill BC (2000) Characteriza-
tion of YpmQ, an accessory protein required for the
expression of cytochrome c oxidase in Bacillus subtilis.
J Biol Chem 275, 28802–28809.
89 Siluvai GS, Nakano MM, Mayfield M, Nilges MJ &
Blackburn NJ (2009) H135A controls the redox activity
of the Sco copper center. Kinetic and spectroscopic
studies of the His135Ala variant of Bacillus subtilis Sco.
Biochemistry 48, 12133–12144.
90 Siluvai GS, Nakano M, Mayfield M & Blackburn NJ
(2011) The essential role of the Cu(II) state of Sco in
the maturation of the Cu(A) center of cytochrome oxi-
dase: evidence from H135Met and H135SeM variants of
the Bacillus subtilis Sco. J Biol Inorg Chem 16, 285–297.
91 Thompson JD, Higgins DG & Gibson TJ (1994)
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weight-
ing, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22, 4673–4680.
92 Edgar RC (2004) MUSCLE: multiple sequence align-
ment with high accuracy and high throughput. Nucleic
Acids Res 32, 1792–1797.
93 Page RD (1996) TreeView: an application to display
phylogenetic trees on personal computers. Comput Appl
Biosci 12 , 357–358.
94 Soding J (2005) Protein homology detection by HMM–
HMM comparison. Bioinformatics 21, 951–960.
95 Eddy SR (1998) Profile hidden Markov models.