Tài liệu Báo cáo khoa học: Seeking the determinants of the elusive functions of Sco proteins pptx

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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

A
)
[3]. The Cu
A
centre acts as the primary acceptor of
electrons coming from cytochrome c, which are then
transferred, via a low-spin heme a moiety, to the
catalytic site formed by Cu
B
and a high-spin heme a
3
where oxygen binding and reduction take place [4].
Keywords
copper; cytochrome c oxidase; redox; Sco;
thiol-disulﬁde
Correspondence
I. Bertini, Magnetic Resonance Center,
University of Florence, Via Luigi Sacconi 6,
50019 Sesto Fiorentino, Italy
Fax: +39 055 457 4271
Tel: +39 055 457 4272
E-mail: ﬁ.it
(Received 15 February 2011, revised 12
April 2011, accepted 18 April 2011)
doi:10.1111/j.1742-4658.2011.08141.x
Sco proteins are present in all types of organisms, including the vast major-
ity of eukaryotes and many prokaryotes. It is well established that Sco pro-
teins in eukaryotes are involved in the assembly of the Cu
A
cofactor of

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

S
2
core of the Cu
A
centre is considered
an important feature in determining its efﬁciency in
long-range electron transfer by virtue of a low reorga-
nization energy [6]. Another important factor in this
respect is the electronic structure of Cu
A
, which cycles
between a reduced Cu(I)–Cu(I) state and an oxidized
species consisting of a fully delocalized mixed-valence
pair with two equivalent Cu
1.5+
ions [7,8].
The proposal that Sco proteins could play a role in
copper delivery to COX within the process of COX
assembly was ﬁrst formulated based on the observation
that their overexpression could rescue respiratory deﬁ-
ciency in yeast mutants lacking the copper chaperone
Cox17 [9], a low molecular weight protein that is local-
ized within the cytoplasm and the mitochondrial inter-
membrane space (IMS) [10,11]. Many subsequent
studies in eukaryotic organisms were performed along
the lines of this hypothesis, and contributed to drawing
a picture where Sco proteins function in COX assem-
bly by mediating copper transfer from Cox17 to the
Cu
A

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-

otes is apparent from the analysis of their genomes,
some of which contain genes coding for Sco proteins
without having any genes coding for Cox2 [22].
By bringing together the available data on eukary-
otic and prokaryotic Sco proteins, a complex scenario
therefore emerges in which major questions arise as to
which is the ancestral function of Sco proteins, how
(and how many) other functions have evolved from
that, and to what extent the mechanisms operating in
prokaryotes are related to, and thus can be used to
understand, those active in the more complex eukary-
otes. The answers to these questions involve the
description of the molecular determinants that underlie
the speciﬁc functional mechanisms of these proteins. In
this work, we review the current knowledge on pro-
karyotic and eukaryotic Sco proteins, with the aim of
providing a framework to rationalize the various func-
tions of these proteins and the elusive factors that
determine these functions.
Occurrence and sequence features
of Sco proteins in eukaryotes and
prokaryotes
To date, no systematic analysis of eukaryotic genomes
has been carried out to identify genes that encode Sco
proteins. In the most comprehensive survey available,
Sco-encoding genes (Sco genes hereafter) were identi-
ﬁed in 39 eukaryotic species and their exon–intron
structure was examined to reconstruct their evolution-
ary history [23]. This analysis showed that eukaryotic
Sco genes all descend from an ancestral gene already

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

XP_002937049.1
XP_002935088.1
Danio rerio Animals Fishes 2 606683
n ⁄ a
NP_001038697.1
n ⁄ a
Takifugu rubripes Animals Fishes 2 n ⁄ a
n ⁄ a
n ⁄ a
n ⁄ a
Acyrthosiphon pisum Animals Insects 1 100160965 NP_001156100.1
Aedes aegypti Animals Insects 1 n ⁄ an⁄ a
Anopheles gambiae Animals Insects 1 1275633 XP_314900.3
Apis mellifera Animals Insects 1 726314 XP_001122061.1
Bombyx mori Animals Insects 1 n ⁄ an⁄ a
Culex pipiens Animals Insects 1 n ⁄ an⁄ a
Drosophila melanogaster Animals Insects 1 33711 NP_608884.1
Drosophila simulans Animals Insects 1 6731025 XP_002078186.1
Drosophila virilis Animals Insects 1 6628793 XP_002051936.1
Nasonia vitripennis Animals Insects 1 100122150 XP_001605752.1
Pediculus humanus corporis Animals Insects 1 8236514 XP_002424862.1
Tribolium castaneum Animals Insects 1 657827 XP_969355.1
Bos taurus Animals Mammals 2 508586
100125923
NP_001073712.1
NP_001098963.1
Homo sapiens Animals Mammals 2 6341
9997
NP_004580.1
NP_005129.2

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

Magnaporthe oryzae Fungi Ascomycetes 1 2682559 XP_366930.1
Pichia pastoris Fungi Ascomycetes 1 8200898 XP_002493635.1
Pichia stipitis Fungi Ascomycetes 1 4838144 XP_001383869.2
Saccharomyces cerevisiae Fungi Ascomycetes 2 852312
852325
NP_009580.1
NP_009593.1
Schizosaccharomyces pombe Fungi Ascomycetes 1 2539826 NP_595287.1
Yarrowia lipolytica Fungi Ascomycetes 1 2912923 XP_504291.1
Zygosaccharomyces rouxii Fungi Ascomycetes 1 8202094 XP_002494544.1
Cryptococcus neoformans Fungi Basidiomycetes 1 3259327 XP_572441.1
Encephalitozoon cuniculi Fungi Other fungi 0 – –
Determinants of the elusive functions of Sco proteins L. Banci et al.
2246 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
canonical mitochondria including oxidative phosphory-
lation [24–26]. This observation is thus in full agreement
with the notion that the primary function of eukaryotic
Sco proteins is in mitochondrial COX assembly. Our
results also conﬁrm that plants and vertebrates have
two Sco genes with the conspicuous exception of
Pan troglodytes, for which only a Sco1 homologue has
been found. However, a tblastn search in the P. troglo-
dytes genome using human Sco2 as the query sequence
reveals a close match in a region of chromosome 22
(NCBI locus NW_001231014, between nucleotides
49882300 and 49882500) where no genes are thought to
reside. It is therefore most likely that P. troglodytes also
has a Sco2 homologue, whose recognition has been
hindered by an error in the current genome assembly
(Build 2.1).

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

Populus trichocarpa Plants Land plants 2 7466084
7471514
XP_002323592.1
XP_002306313.1
Sorghum bicolor Plants Land plants 2 8065373
8074665
XP_002462290.1
XP_002453341.1
Vitis vinifera Plants Land plants 2 100246281
100247202
XP_002266556.1
XP_002263427.1
Zea mays Plants Land plants 2 100191148
100282683
NP_001130056.1
NP_001149062.1
Cryptosporidium parvum Protists Apicomplexans 0 – –
Plasmodium falciparum Protists Apicomplexans 1 2655070 XP_001349003.1
Plasmodium knowlesi Protists Apicomplexans 1 7318424 XP_002257566.1
Theileria annulata Protists Apicomplexans 1 3862043 XP_952475.1
Leishmania braziliensis Protists Kinetoplasts 2 5412667
5416541
XP_001561796.1
XP_001562419.1
Leishmania infantum Protists Kinetoplasts 3 5066371
5068632
5069910
XP_001462953.1
XP_001465217.1
XP_001470536.1

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

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

proteins are present in a large variety of species from
both Bacteria and Archaea, which in most cases (65 out
of 128, i.e. about 51%) have more than one Sco gene,
and can have up to seven [22]. On the other hand, 183
of the 311 organisms analysed (i.e. about 59%) were
found to contain no Sco homologues, which appear to
be lacking altogether in some prokaryotic groups such
as cyanobacteria [22]. In particular, by searching for
the co-occurrence in genomes of Sco and Cox2 genes, it
was pointed out that about 12% of prokaryotes have
Cox2 but not Sco genes, and about 6% have Sco but
not Cox2 genes. These observations imply that some
prokaryotes (including for example cyanobacteria)
evolved a process of COX maturation where Sco is not
required and, on the other hand, that Sco proteins in
prokaryotes can also function outside COX assembly.
The supposedly broader functional range of prokary-
otic Sco proteins with respect to their eukaryotic coun-
terparts, which is most likely to be found in those
prokaryotes where multiple duplications of Sco genes
have occurred, is reﬂected in the higher variability of
their amino acid sequences (21 ± 9% average pairwise
identity in prokaryotes versus 38 ± 9% in eukaryotes).
A comparison of the sequence proﬁles based on the
multiple alignments of eukaryotic and prokaryotic Sco
proteins, respectively, in fact shows that highly con-
served residues are more numerous in eukaryotes than
in prokaryotes, and are especially concentrated in the
regions forming the copper-binding site (Fig. 2). How-
ever, prokaryotic and eukaryotic sequences appear to

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

To date the three-dimensional structures of human
Sco1 and Sco2 have been determined in their apo- and
metal-loaded states. Speciﬁcally, apo-, Cu(I)- and
Ni(II)-Sco1 [16,27] and Cu(I)-Sco2 [28] structures are
available. From all these data it emerges that the over-
all structure contains a typical thioredoxin (Trx) fold
[29] with the insertion of further secondary structure
elements. The Trx fold is constituted by a central four-
stranded b sheet (b3, b4, b6, b7) and three ﬂanking a
helices (a1, a3, a4) (Fig. 3). On this scaffold, a b-hair-
pin structure (b1 and b2) followed by a 3
10
-helix (h1)
is inserted at the N terminus and one helix (a2)
followed by a strand (b5), the latter forming a parallel
b sheet with strand b4, are inserted between strand b4
and helix a3 (Fig. 3). This fold topology belongs to a
subset of the Trx superfamily, present in peroxiredox-
ins and glutathione peroxidases [30]. A speciﬁc
property of the eukaryotic Sco fold, absent in Trx and
Trx-like family members, is the presence of a b-hairpin
in the extended, solvent-exposed loop connecting helix
a3 and strand b6 (Fig. 3). All of these points of inser-
tion are those typically tolerated in a Trx fold without
disruption of the overall structure [29].
A comparison of the structural and dynamic proper-
ties of apo- versus metal-loaded states of human Sco1
and Sco2 provides a detailed molecular view of the
metal-binding process. In the apo forms, a large num-
ber of residues in the metal-binding area sample, in

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

independent molecules of the crystal [16], the loop
N
β
2
C
θ
1
β
3
β
4
β
7
α
2
β
5
α
1
α
4
α
3
β
6
β
1
Fig. 3. Schematic picture of the fold topology of Sco proteins. The
secondary structure elements of a typical thioredoxin fold are
shown in grey. Additional secondary structure elements present in

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

conformational sampling of Sco1 to bind the metal
ion.
The coordination sphere of Ni(II) in the crystal
structure of human Ni(II)-Sco1 [27] is quite peculiar. In
this structure, the two metal-binding Cys residues are
oxidized and form a disulﬁde bond and therefore are
not capable of binding the Ni(II) ion as thiolates. Still,
the metal ion remains in contact with the S—S bond
with an Ni—S distance of 2.0–2.2 A
˚
, suggesting the for-
mation of bonds with the lone pairs of the sulfur atoms.
The coordination sphere of Ni(II) is completed by
His260 (Ne2—Ni, 2.03–2.45 A
˚
), in agreement with the
solution structure of Ni(II)-Sco1 and a water molecule,
or more likely an anion such as chloride, arranged in a
distorted square planar geometry. The redox state of
the Cys ligands differs from that found in Ni(II)-Sco1,
Cu(I)-Sco1 and Cu(I)-Sco2 solution structures, which
have both cysteines in the reduced state [27]. This differ-
ent behaviour is due to the different experimental con-
ditions, i.e. aerobic versus anaerobic, and the presence
of 1 mm dithiothreitol disulﬁde-reductant. The only
other available crystal structure of a metal-loaded Sco1
form (yeast Sco1) [31] also shows a quite unexpected
metal coordination. The crystal has been obtained by
soaking apo-Sco1 crystals with copper ions and its
structure reveals a copper-binding site involving Cys181

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-

tion is the key event which modulates the ordered⁄ dis-
ordered state of the whole metal-binding region
(Fig. 4). Taking into account that disordered regions
in protein structures are often engaged in protein–pro-
tein interactions, one may speculate that this loop
modulates association–dissociation of Sco1 with its
partners. For example, it is possible that, once the
mitochondrial copper chaperone Cu(I)-Cox17 interacts
transiently with apo-Sco1 and donates its copper cargo
to Sco1, the His-containing loop structurally rear-
ranges, thus allowing His binding and concomitant
formation of the compact Cu(I)-Sco1 structure. The
formation of the stable compact Cu(I)-Sco1 state could
thus constitute the important driving force of the cop-
per transfer from Cox17 to Sco1.
Solution and crystal structures are also available for
prokaryotic Sco homologues. Speciﬁcally, the solution
structure of apo-Sco from Bacillus subtilis (BsSco),
solved in 2003, was the ﬁrst for this class of proteins
[32] and, later, its crystal structure [33] as well as the
solution structure of the Sco1 homologue from Ther-
mus thermophilus [19] were solved. Despite numerous
efforts, solution or crystal structures of the copper
forms of these prokaryotic Sco proteins were not
obtained. However, a combination of spectroscopic
techniques was used to ﬁnd that BsSco employs a sin-
gle metal site to bind both Cu(I) and Cu(II) [34–37],
the former via two cysteines plus a weakly bound,
unidentiﬁed ligand, and the latter via two cysteines
with unequal bond strengths, two O ⁄ N donor ligands

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

what occurs for yeast Sco1. There seems to be only a
small energy barrier separating the disulﬁde-bonded
and non-disulﬁde-bonded conformations. Taken
together, these observations indicate that (as in eukary-
otic apo-Sco proteins) both metal-ligand-containing
loops implicated in copper binding exhibit conforma-
tional plasticity in the structure. However, an important
structural difference of the metal-binding region of apo-
BsSco with respect to the corresponding region of
eukaryotic apo-Sco proteins is that, in the latter, one
cysteine is located at one end of an a helix and the other
is in the preceding short loop region, and the thiolate
groups of the cysteines are only partially exposed. In
contrast, the two Cys residues in BsSco are located in a
protruding loop that is exposed to the solvent. The
backbone conformation of the His-containing loop,
which does not have the b-hairpin present in eukaryotic
Sco proteins, is also such that it largely exposes the His
ligand to the solvent in BsSco only. These differences
suggest that apo-BsSco has a greater structural ﬂexibil-
ity than human Sco proteins, which can account for the
impossibility of getting a compact Cu(I) state even upon
Cu(I) addition. This indicates that speciﬁc amino acid
substitutions in critical points of the fold can largely
affect the structural ﬂexibility of the metal-binding
region of Sco proteins, and as a consequence their cop-
per afﬁnity. Cu(II) binding, however, is able to deter-
mine in BsSco the formation of a complex with extreme
kinetic stability and picomolar afﬁnity [35,36,38]. A
two-step model for Cu(II) binding has been proposed in

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

9
C motif forms two
disulﬁde bonds which are essential for the formation of
an a-hairpin fold [40–42]. The oxidoreductase Mia40 is
responsible in the IMS for promoting both the forma-
tion of the two disulﬁdes and the folding of Cox17
[43–45]. A ﬂexible and completely unstructured N-ter-
minal tail of Cox17 contains a CC motif which coordi-
nates one Cu(I) ion [40]. It was shown that Cu(I)
binding is essential to Cox17 function and that, in spite
of its dual localization, the proposed functional role of
Cox17 in mitochondrial copper delivery to COX is
restricted to the IMS [46,47]. A high copy suppressor
screen of a yeast Cox17 null strain led to the identiﬁca-
tion of the two highly homologous proteins Sco1 and
Sco2 [9]. Both proteins are imported in the IMS
through the TOM translocase which recognizes a
typical mitochondrial targeting sequence present at the
N-terminus of Sco1 and Sco2 [48]. Then, they are
arrested in the mitochondrial inner membrane through
a stop-transfer mechanism, in which a transmembrane
helix, subsequent to the mitochondrial targeting
sequence in both proteins, functions as a critical sort-
ing signal that causes the arrest of the precursor during
the import reaction at the level of the inner membrane
as well as in its lateral insertion into the lipid bilayer,
both processes being mediated by the TIM23 translo-
case [48].
Yeast Sco1 is absolutely required in the activation
of COX [1,49] and in vitro it can receive copper from

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

parts of yeast Sco1 (N-terminal portion amino acids
1–75 and C-terminal portion amino acids 106–295) can
indeed be replaced by the corresponding parts of yeast
Sco2 without loss of function, but a short stretch of 13
amino acids, immediately adjacent to the transmem-
brane region, is crucial for Sco1 function and cannot
be replaced by its Sco2 counterpart [52,56]. In
summary, the Sco2 function in yeast still remains
elusive.
In contrast to yeast, both Sco1 and Sco2 are
required in humans for cellular respiration and Cu
A
biogenesis [13,57]. They have non-overlapping, cooper-
ative functions in copper delivery to the Cu
A
site [13].
Both human Sco1 and Sco2 are copper-binding pro-
teins [58] and have an afﬁnity for Cu(I) higher than
that of human Cox17 [55]; accordingly, Cu(I) is quanti-
tatively transferred from Cox17 to Sco1 [59] and Sco2
[60] (Fig. 5). They also have a similar afﬁnity for Cu(I)
[55] and can rapidly exchange it with each other [60]
(Fig. 5). These data therefore strongly indicate that
both human Sco proteins can receive Cu(I) from
Cox17 to donate it to the apo-Cu
A
site, thus determin-
ing the formation of the ﬁnal active centre (Fig. 5). In
Cu(I)Cox17
apoCox17

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.

Histidine Cu(I) ligands in Sco1 and Sco2 are shown as blue sticks. Copper ions are shown as magenta spheres. Cox17 can simultaneously
transfer Cu(I) ion and two electrons to metallate oxidized apo-Sco1. Sco1 and Sco2 can act as copper chaperones and ⁄ or thioredoxins, being
implicated in copper transfer to apo-Cox2 and in a disulﬁde exchange reaction from Sco2 to Sco1 and toward apo-Cox2. Cys-reduced states
of Sco1 and Sco2 are also able to exchange Cu(I) between each other.
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 2253
agreement with this model, it was demonstrated that
the ability to bind Cu(I) is crucial to the function of
both human Sco proteins [58]. Accordingly, supplemen-
tation of the growth media with copper salts results in
either a partial or complete rescue of the observed
COX deﬁciency in Sco1 and Sco2 patient cell lines
[13,61,62]. Cu(I) can be transferred from Cox17 to
Sco1 also when the latter protein is oxidized, i.e. with
its metal-binding cysteines forming a disulﬁde bond
[60] (Fig. 5). It has indeed been shown in vitro that
human Cu(I)-Cox17 can simultaneously transfer Cu(I)
and two electrons to oxidized human apo-Sco1 [60].
The result is Cu(I)-Sco1 and the fully oxidized apo-
Cox17, which can be reduced by glutathione to the
reduced state, then being again able to bind Cu(I)
(Fig. 5). The Sco1 ⁄ Cox17 redox reaction is thermody-
namically driven by copper transfer. This reaction may
also occur in vivo because, in the IMS, the cysteines in
the CXXXC motif in Sco1 exist as a mixed population
comprising oxidized disulﬁdes and reduced thiols [14],
consistent with the redox potential of CXXXC in
human Sco1 ()277 mV) [59] compared with that of the
IMS ()255 mV) [63]. The electron-transfer-coupled
metallation of human Sco1 can be a mechanism of con-

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

pathology is caused by a loss of normal Sco1 function
rather than gain of some aberrant action of the mutant
protein [69]. Structural studies show that P174L
mutation alters hydrophobic interactions around the
metal-binding site [59], thus affecting both the copper-
binding afﬁnity and the redox properties of human
Sco1 [59] and therefore severely compromising copper
transfer from Cox17 to Sco1 [59,71].
Mapping of the known pathogenic mutations for
Sco2 onto its three-dimensional structure reveals that a
signiﬁcant number of them (six out of eight, i.e. 75%)
cluster closely around the Cu(I) ligands. The structural
data on human Sco2 [28] can help to rationalize at the
molecular level the mutant mis-functions. In particular,
all reported Sco2 patients save one [72] carry a Glu-to-
Lys (E140K) missense mutation on one allele. Glu140
is located in helix a1 and essentially not solvent
exposed, and is involved in a salt bridge with Lys143
that is disrupted in the E140K mutant. The possible
structural rearrangements induced by the mutation
could therefore affect both the copper-binding proper-
ties and the protein stability, factors which inﬂuence
protein function. Similarly, the mutation of Ser225
to Phe could affect the copper-binding properties of
human Sco2, destabilizing the Cu(I) coordination by
the proximal His224 ligand. As Cu(I) binding induces
a transition from an open apo-Sco2 state to a compact
Cu(I)-Sco2 state, the mutation could alter this confor-
mational transition determining a weaker metal afﬁn-
ity, similar to that found for the P174L mutation in

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

Determinants of the elusive functions of Sco proteins L. Banci et al.
2254 FEBS Journal 278 (2011) 2244–2262 ª 2011 The Authors Journal compilation ª 2011 FEBS
remains to be learned about the molecular events that
govern copper transfer from Sco proteins to Cox2 dur-
ing its maturation, and about the relative contributions
of Sco1 and Sco2 to this process. Our understanding
of these and other events awaits the development of
in vitro systems in which stabilized Cox2 can be
expressed and studied.
The Cu
A
assembly process also needs to take into
account other important factors which relate to the
aggregation state of human Sco1 and Sco2 and of their
complex with apo-Cox2. It has been proposed that the
maturation of Cox2 is contingent upon the formation
of a complex that includes both Sco proteins, where
ﬁrst Sco2 interacts with newly synthesized Cox2 and
then the physical interaction between Sco2 and Cox2
triggers the recruitment of Sco1 to the Sco2–Cox2
complex, as well as metallation of Sco1 by Cox17 [14].
Size exclusion chromatography data also suggest that
both human Sco1 and Sco2 proteins function as
homodimers in vivo [13]. In vitro studies on the soluble
C-terminal functional domain show that the soluble
domain of apo-Sco2 behaves as a monomer in solution
[28] and that the dimeric state of Sco1 depends on a
highly charged region close to the N-terminal trans-
membrane helix anchoring the protein to the inner
membrane of mitochondria [27]. Therefore, the N-ter-

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

across the outer membrane into the IMS through
either porin or the TOM complex; however, the highly
impermeable nature of the inner membrane needs its
protein-mediated transport to and from the matrix. At
present, it is not clear how copper is moved across the
inner membrane. Its translocation could be achieved
by either a single bidirectional transporter or two uni-
directional transporters [75]. Once copper is stored in
the matrix, integral membrane proteins with soluble
domains that localize to both the matrix and the IMS
are required for dynamic communication between
organellar and cellular copper handling pathways in
order to satisfy cellular copper requests [76]. Molecular
genetic analyses established that Sco1 and Sco2 pro-
teins are the candidate molecules to perform the above
function [17]. They have indeed been found to play
regulatory roles in the maintenance of cellular copper
homeostasis [17]. In particular, it has been established
that the copper deﬁciency in Sco patient ﬁbroblasts
was caused by the inappropriate stimulation of copper
efﬂux from the cell rather than a defect in its uptake.
The genetic data available so far [17] led to the pro-
posal of a model in which Sco2 modiﬁes an aspect of
Sco1 function that is crucial to the generation and
transduction of a mitochondrial signal that regulates
copper efﬂux from the cell. Neither the players nor the
molecular mechanisms of the signalling pathway con-
necting Sco proteins to the cytoplasmic proteins
involved in the regulation of cellular copper concentra-
tion (ATP7A is the most obvious candidate since it

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

Sco2 protein has also been identiﬁed as a mediator of
the balance between the utilization of respiratory and
glycolytic pathways in mice and human cancer cell
lines, through its involvement in the p53-linked path-
way [77]. The high structural plasticity of human Sco2,
especially observed in its apo form, can be considered
an essential requirement for its involvement in various
protein recognition pathways, including Cox2 assem-
bly, cellular copper homeostasis and the p53-linked
pathway. Indeed, local conformational disorder is
commonly associated with protein interaction sites in
conformational disorder. However, the molecular
grounds of both signalling and p53-linked pathways
involving Sco1 and Sco2, respectively, are still obscure.
Functions of Sco proteins in
prokaryotes: indications for a primary
redox role
The only prokaryotic organism for which a molecular
level description of the mechanism of Cu
A
assembly is
available is the Gram-negative thermophile T. thermo-
philus. In an NMR study, it was shown that T. thermo-
philus Sco is unable to deliver either Cu(I) or Cu(II) to
the Cu
A
site, thereby precluding its role as a copper
chaperone, and instead functions as a thiol-disulﬁde
oxidoreductase to maintain the correct oxidation state
of the Cu

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

centre, whose cysteines
are kept reduced by a Sco protein working as thiore-
doxin [19]. The afﬁnities for copper of the three pro-
teins involved are completely in agreement with the
observed Cu
A
reconstitution process, the Cox2 site
having an afﬁnity higher than that of PCu
A
C, and the
PCu
A
C site higher than that of Sco1 [19]. Genomic
context analysis indicated that Sco and PCu
A
C homo-
logues are often encoded in the same operon, suggest-
ing that the process described for T. thermophilus is
likely to occur in other Gram-negative bacteria as well
[22,79]. In fact, a recent bioinformatics analysis per-
formed on over a thousand prokaryotic genomes
found that about 70% of the organisms that possess a
Cox2 and a Sco homologue also have a PCu
A
C homo-
logue [80]. A recent study on the symbiotic nitrogen-
ﬁxing bacterium Bradyrhizobium japonicum supports
the model that Sco and PCu
A
C are both required for

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

to cbb
3
oxidase was proposed to be the unique func-
tion of the Sco protein (also called SenC) from the
purple photosynthetic bacterium Rhodobacter capsula-
tus, which does not have a Cu
A
-containing COX [84].
A role for Sco proteins in cbb
3
oxidase assembly could
in part, but not entirely, account for their occurrence
in COX-lacking organisms, as two-thirds of the ge-
nomes that contain Sco but not COX2 genes also
encode cbb
3
oxidase [22].
A recent study performed on Pseudomonas putida
also supported an exclusive redox role for prokaryotic
Sco proteins. It was focused on the structural and
functional characterization of an intriguing two-
domain protein consisting of a Sco domain and a cyto-
chrome c domain [85]. It was shown that the Sco
domain does not bind Cu(II), and binds Cu(I) with
low, not physiologically relevant, afﬁnity only through
the cysteines in the CXXXC motif. In addition, it has
thioredoxin-like activity as well as the capability,
through the CXXXC motif, of reducing Cu(II) to
Cu(I) as well as Fe(III) to Fe(II) in the cytochrome c
domain [85]. Similar redox properties have been

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

that in eukaryotic Sco proteins the removal of the his-
tidine ligand switches on the thioredoxin function in
order to maintain the correct oxidation state of the
Cu
A
cysteine ligands, so that copper can be inserted by
Sco proteins which also work as copper chaperones in
the Cu
A
maturation.
The above-mentioned model is in agreement with
the large amount of data available for the Sco protein
from the Gram-positive B. subtilis, called YpmQ or
BsSco. The ﬁrst characterization of this protein
showed that deletion of its encoding gene disrupts the
expression of COX but not of menaquinol oxidase,
which does not contain a Cu
A
centre [88]. In addition,
it was found that levels of COX recover after addition
of copper to the growth medium or when BsSco is
expressed from a plasmid, whereas mutant forms of
BsSco with substitutions in the potential copper-bind-
ing residues are ineffective [88]. It was thus proposed
that BsSco is involved in the assembly of the Cu
A
site
of COX, and based on the above data it was initially
suggested that it is functionally equivalent to its
eukaryotic homologues playing a copper chaperone

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

Fig. 6. Comparison between P. putida (left)
and human Sco1 (right) highlighting that
hydrophilic residues in the region around the
metal-binding site of P. putida Sco (shown
in red) are replaced by hydrophobic residues
in eukaryotic Sco proteins (shown in blue).
The van der Waals contact surface of the
hydrophobic residues in human Cu(I)-Sco1 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.
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 2257
redox activity of BsSco, which would pass from an
inactive Cu(II)-bound state to an active Cu-free state
upon interaction with partner proteins that destabilize
the Cu(II)–histidine bond [89]. As the histidine to ala-
nine mutated BsSco is not functional in COX assembly
and yet Cu(I) binding is not affected by the loss of the
histidine ligand, it was argued that the Cu(II)-bound
state has a major role in the mechanism of action of
BsSco, ruling out the simple model of direct Cu(I)
insertion into the Cu
A
site [89]. Further support for
this view came from the observation that a BsSco vari-
ant designed to destabilize the Cu(II)-bound state by
substitution of the histidine ligand with a methionine
is also not functional in COX assembly [90]. However,

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

periplasmic proteins from oxidative damage, and led
to the more general proposal that in species possessing
COX the failure in COX assembly caused by Sco inac-
tivation could actually be due to oxidative damage of
the Cu
A
site rather than to impairment of copper
insertion [21].
Concluding remarks: towards a uniﬁed
model for the functions of Sco proteins
Sco proteins have been implicated in several functional
processes, ranging from COX assembly to regulation
of gene expression, protection against oxidative stress,
mitochondrial redox signalling, and regulation of cop-
per homeostasis. It appears that the involvement of
Sco proteins in each of these processes depends on the
organism, resulting from evolutionary processes that
produced a puzzling variety of possible functions.
Given that the fold of Sco proteins resembles that of
proteins involved in thiol-disulﬁde redox chemistry, it
is reasonable to assume that the primary role of Sco
proteins should be linked to a thiol-disulﬁde oxidore-
ductase activity associated with the conserved CXXXC
motif. During evolution, speciﬁc amino acid variations
Fig. 7. Potential molecular mechanism for
copper incorporation into the Cu
A
site of
COX in eukaryotes. Upon speciﬁc protein–
protein recognition between Sco1 ⁄ Sco2 and

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

characterization of the atypical Sco protein from
P. putida, an important (though possibly not unique)
factor affecting these properties is the hydrophobic or
hydrophilic character of the protein regions in the
proximity of the potential copper ligands. This ﬁnding
allowed us to propose a molecular model for copper
incorporation into the Cu
A
site of eukaryotes in which
Sco proteins can work upon removal of His ligand,
induced by a speciﬁc protein–protein recognition, as
copper chaperone and thioredoxin towards the Cox2
partner (Fig. 7). Elucidation of this and other similar
determinants is crucial to achieving a complete under-
standing of this critically important and functionally
divergent class of proteins.
Acknowledgements
This work was supported by Ministero Italiano
dell’Universita
`
e della Ricerca (MIUR) through the
FIRB project RBRN07BMCT and the FIRB project
RBFR08WGXT.
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