Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein

Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.     

Mapping the functional interaction of Sco1 and Cox2 in cytochrome oxidase biogenesis

Sco1 is implicated in the copper metallation of the Cu(A) site in Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated and apo-conformers revealed structural dynamics primarily in an exposed region designated loop 8. The structural dynamics of loop 8 in Sco1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif (217)KKYRVYF(223) on the leading edge of this loop are shown presently to be important for yeast Sco1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant Sco1 proteins failed to restore respiratory growth or cytochrome oxidase activity in sco1Delta cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-Sco1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient Sco1-Cox2 interaction, the mutant Sco1 molecules appear compromised in a function with Cox2. The mutants failed to suppress the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of sco1Delta cells. These studies implicate different surfaces on Sco1 for interaction or function with Cox17 and Cox2.     

Crystal structure of yeast Sco1

The Sco family of proteins are involved in the assembly of the dinuclear Cu_A site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-Å resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function.     

Sco proteins are involved in electron transfer processes

Sco proteins are widespread in eukaryotic and in many prokaryotic organisms. They have a thioredoxin-like fold and bind a single copper(I) or copper(II) ion through a CXXXC motif and a conserved His ligand, with both tight and weak affinities. They have been implicated in the assembly of the Cu_A site of cytochrome c oxidase as copper chaperones and/or thioredoxins. In this work we have structurally characterized a Sco domain which is naturally fused with a typical electron transfer molecule, i.e., cytochrome c, in Pseudomonas putida. The thioredoxin-like Sco domain does not bind copper(II), binds copper(I) with weak affinity without involving the conserved His, and has redox properties consisting of a thioredoxin activity and of the ability of reducing copper(II) to copper(I), and iron(III) to iron(II) of the cytochrome c domain. These findings indicate that the His ligand coordination is the discriminating factor for introducing a metallochaperone function in a thioredoxin-like fold, typically responsible for electron transfer processes. A comparative structural analysis of the Sco domain from P. putida versus eukaryotic Sco proteins revealed structural determinants affecting the formation of a tight-affinity versus a weak-affinity copper binding site in Sco proteins.     

Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.     

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.     

The antioxidant function of Sco proteins depends on a critical surface-exposed residue

BackgroundBesides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity.MethodsNative and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays.ResultsA functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways.ConclusionThis study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology.General significanceThese findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.     

Characterization of the redox and metal binding activity of BsSco, a protein implicated in the assembly of cytochrome c oxidase

Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans. The soluble domain of Sco has a thioredoxin fold that is suggestive of redox activity for this protein. We have characterized the soluble domain of the Sco protein from Bacillus subtilis (i.e., sBsSco) for its redox reactivity and metal binding capacity. In oxidized sBsSco, the cysteines are present as an intramolecular disulfide. Oxidized sBsSco does not bind metal, but can be reduced in vitro to a metal-binding form. Reduction of the disulfide in sBsSco is accompanied by increased intrinsic fluorescence. The reducibility of the cystine is unchanged when the conserved histidine is mutated to alanine. Tight binding by reduced sBsSco is observed for Cu(II) by electronic absorption, intrinsic fluorescence, and EPR spectroscopies, and isothermal titration calorimetry with an observed stoichiometry of one Cu(II) ion per sBsSco and a KD of approximately 50 nM. Tight binding of Cu(I) and Ag(I) is observed by quenching of intrinsic tryptophan fluorescence. Cobalt(II) exhibits weak binding, whereas Ni(II) and Zn(II) do not appear to bind. The high-affinity binding of metals by BsSco is triggered by its redox state, and this property could be important for its function in vivo.     

Let's Sco1, Oxidase! Let's Sco!

The solution structure of Sco1 from Bacillus subtilis is the first structure of a protein important in the assembly of cytochrome c oxidase (CcO). The assembly of CcO requires the insertion of multiple cofactors. Sco1 is a conserved protein implicated in formation of the binuclear Cu(A) center.     

The functions of Sco proteins from genome-based analysis

Sco proteins are widespread proteins found in eukaryotic as well as in many prokaryotic organisms. The 3D structure of representatives from human, yeast, and Bacillus subtilis has been determined, showing a thioredoxin-like fold. Sco proteins have been implicated mainly as copper transporters involved in the assembly of the CuA cofactor in cytochrome c oxidase. Some mutations have been identified in humans that lead to defective cytochrome c oxidase formation and thus to fatal illnesses. However, it appears that the physiological function of Sco proteins goes beyond assembly of the CuA cofactor. Extensive analysis of completely sequenced prokaryotic genomes reveals that 18% of them contain either Sco proteins but not CuA-containing proteins or vice versa. In addition, in several cases, multiple Sco-encoding genes occur even if only a single potential Sco target is encoded in the genome. Genomic context analysis indeed points to a more general role for Sco proteins in copper transport, also to copper enzymes lacking a CuA cofactor. To obtain further insight into the possible role of Sco in the assembly of other cofactors, a search for Cox11 proteins, which are important for CuB biosynthesis, was also performed. A general framework for the action of Sco proteins is proposed, based on the hypothesis that they can couple metal transport and thiol/disulfide-based oxidoreductase activity, as well as select between either of these two cellular functions. This model reconciles the variety of experimental observations made on these proteins over the years, and can constitute a basis for further studies.     

The essential role of the Cu(II) state of Sco in the maturation of the Cu<Subscript>A</Subscript> center of cytochrome oxidase: evidence from H135Met and H135SeM variants of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Sco

Sco is a red copper protein that plays an essential yet poorly understood role in the metalation of the Cu_A center of cytochrome oxidase, and is stable in both the Cu(I) and Cu(II) forms. To determine which oxidation state is important for function, we constructed His135 to Met or selenomethionine (SeM) variants that were designed to stabilize the Cu(I) over the Cu(II) state. H135M was unable to complement a scoΔ strain of Bacillus subtilis, indicating that the His to Met substitution abrogated cytochrome oxidase maturation. The Cu(I) binding affinities of H135M and H135SeM were comparable to that of the WT and 100-fold tighter than that of the H135A variant. The coordination chemistry of the H135M and H135SeM variants was studied by UV/vis, EPR, and XAS spectroscopy in both the Cu(I) and the Cu(II) forms. Both oxidation states bound copper via the S atoms of C45, C49 and M135. In particular, EXAFS data collected at both the Cu and the Se edges of the H135SeM derivative provided unambiguous evidence for selenomethionine coordination. Whereas the coordination chemistry and copper binding affinity of the Cu(I) state closely resembled that of the WT protein, the Cu(II) state was unstable, undergoing autoreduction to Cu(I). H135M also reacted faster with H₂O₂ than WT Sco. These data, when coupled with the complete elimination of function in the H135M variant, imply that the Cu(I) state cannot be the sole determinant of function; the Cu(II) state must be involved in function at some stage of the reaction cycle.     

Identification of a disulfide switch in BsSco, a member of the Sco family of cytochrome c oxidase assembly proteins

BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.     

Solution structure of Sco1: a thioredoxin-like protein Involved in cytochrome c oxidase assembly

Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment. The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized. Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins. In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological. The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins. The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S. cerevisiae and to discuss the physiological role of the Sco family.     

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.     

Yeast cox17 solution structure and Copper(I) binding

Cox17 is a 69-residue cysteine-rich, copper-binding protein that has been implicated in the delivery of copper to the Cu(A) and Cu(B) centers of cytochrome c oxidase via the copper-binding proteins Sco1 and Cox11, respectively. According to isothermal titration calorimetry experiments, fully reduced Cox17 binds one Cu(I) ion with a K(a) of (6.15 +/- 5.83) x 10(6) M(-1). The solution structures of both apo and Cu(I)-loaded Cox17 reveal two alpha helices preceded by an extensive, unstructured N-terminal region. This region is reminiscent of intrinsically unfolded proteins. The two structures are very similar overall with residues in the copper-binding region becoming more ordered in Cu(I)-loaded Cox17. Based on the NMR data, the Cu(I) ion has been modeled as two-coordinate with ligation by conserved residues Cys(23) and Cys(26). This site is similar to those observed for the Atx1 family of copper chaperones and is consistent with reported mutagenesis studies. A number of conserved, positively charged residues may interact with complementary surfaces on Sco1 and Cox11, facilitating docking and copper transfer. Taken together, these data suggest that Cox17 is not only well suited to a copper chaperone function but is specifically designed to interact with two different target proteins.     

Seeking the determinants of the elusive functions of Sco proteins

Sco proteins are present in all types of organisms, including the vast majority of eukaryotes and many prokaryotes. It is well established that Sco proteins in eukaryotes are involved in the assembly of the Cu(A) cofactor of mitochondrial cytochrome c oxidase; however their precise role in this process 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 signalling 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 determinants that modulate the two properties central to their known and potential 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.     

Purification and characterization of yeast Sco1p, a mitochondrial copper protein

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains approximately 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.     

Novel insights into phenotype and mitochondrial proteome of yeast mutants lacking proteins Sco1p or Sco2p

The yeast Saccharomyces cerevisiae is a facultative anaerobe and its mitochondrial morphology is linked to its metabolic activity. The Sco proteins (Sco1p and Sco2p) were characterized as proteins required for copper delivery to cytochrome c oxidase. Our results indicated a higher fermentative capacity of the sco1-Δ mutant in comparison to the control and the sco2-Δ mutant strains. The mitochondrial proteome analysis showed that the sco1-Δ mutant down-regulated components of the respiratory chain, the TCA cycle and transport of metabolites across the mitochondrial membrane. This evidence suggests that the absence of Sco1p causes irreversible damage to the mitochondria.     

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052-2062; Oh, J.-I. and Kaplan, S. (2000) EMBO J. 19, 4237-4247). It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal-free rPrrC (apo-PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a lambda(max) of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role.     

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.