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: ivanobertini@cerm.unifi.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 Sus scrofa|XP 003132044.1 1000 979 Mus musculus|NP 001035115.1 1000 Xenopus tropicalis|XP 002937049.1 855 Branchiostoma floridae|XP 002613836.1 678 Strongylocentrotus purpuratus|XP 001199433.1 458 Ixodes scapularis|XP 002402970.1 Nematostella vectensis|XP 001641939.1 164 319 Drosophila melanogaster|NP 608884.1 Drosophila simulans|XP 002078186.1 1000 Drosophila virilis|XP 002051936.1 1000 Anopheles gambiae|XP 314900.3 979 Tribolium castaneum|XP 969355.1 830 Apis mellifera|XP 001122061.1 Nasonia vitripennis|XP 001605752.1 997 420 Pediculus humanus corporis|XP 002424862.1 Acyrthosiphon pisum|NP 001156100.1 418 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 Ostreococcus lucimarinus|XP 001422358.1 Ostreococcus tauri|XP 003084388.1 1000 Micromonas RCC299|XP 002508419.1 932 865 299 Plasmodium falciparum|XP 001349003.1 Plasmodium knowlesi|XP 002257566.1 1000 Theileria annulata|XP 952475.1 959 Sorghum bicolor|XP 002462290.1 Zea mays|NP 001149062.1 1000 Oryza sativa|NP 001063017.1 1000 Arabidopsis thaliana|NP 568068.1 Populus trichocarpa|XP 002306313.1 770 Vitis vinifera|XP 002266556.1 998 1000 Leishmania infantum|XP 001470536.1 Leishmania major|XP 001684203.1 1000 Leishmania braziliensis|XP 001562419.1 1000 Trypanosoma brucei|XP 803555.1 Trypanosoma cruzi|XP 807216.1 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 Saccharomyces cerevisiae|NP 009593.1 569 986 Pichia pastoris|XP 002493635.1 558 Debaryomyces hansenii|XP 002769958.1 Pichia stipitis|XP 001383869.2 990 Candida dubliniensis|XP 002422402.1 950 752 Candida glabrata|XP 445160.1 Saccharomyces cerevisiae|NP 009580.1 962 879 Yarrowia lipolytica|XP 504291.1 500 Aspergillus nidulans|XP 662446.1 Aspergillus oryzae|XP 001824537.2 1000 Magnaporthe oryzae|XP 366930.1 990 628 Schizosaccharomyces pombe|NP 595287.1 833 Cryptococcus neoformans|XP 572441.1 771 Monosiga brevicollis|XP 001742585.1 Sco2 vertebrates Sco2 land plants 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 [...]... 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... 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 Specifically, this proposal was based on the fact that significant perturbations were detected in the redox state of the cysteines of Sco1 in both Sco1 and Sco2 patient backgrounds, and these correlate... aggregation state of the proteins In conclusion, CuA biogenesis in humans is a complex mechanism involving both Cu(I) and disulfide exchange reactions from Cox17 to the apo-CuA 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 CuA site needs to be further investigated Other functions of eukaryotic Sco proteins Genetic.. .Determinants of the elusive functions of Sco proteins L Banci et al 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, supplementation 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... 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 implicated not only in the maturation of the CuA 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... 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 oxidase: evidence from H135Met and H135SeM variants of the Bacillus subtilis Sco J Biol Inorg... anchoring the protein to the inner membrane of mitochondria [27] Therefore, the N-terminal segment (containing the residues protruding into the mitochondrial matrix, the transmembrane helix and the following  20 residues) is crucial to determining 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. .. in eukaryotic Sco proteins the removal of the histidine ligand switches on the thioredoxin function in order to maintain the correct oxidation state of the CuA cysteine ligands, so that copper can be inserted by Sco proteins which also work as copper chaperones in the CuA maturation The above-mentioned model is in agreement with the large amount of data available for the Sco protein from the Gram-positive... across the outer membrane into the IMS through Determinants of the elusive functions of Sco proteins 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... to regulation of gene expression, protection against oxidative stress, mitochondrial redox signalling, and regulation of copper 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 . 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. 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