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.     

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.     

Ortholog search of proteins involved in copper delivery to cytochrome C oxidase and functional analysis of paralogs and gene neighbors by genomic context

Cytochrome c oxidase (COX) is a multi-subunit enzyme of the mitochondrial respiratory chain. Delivery of metal cofactors to COX is essential for assembly, which represents a long-standing puzzle. The proteins Cox17, Sco1/2, and Cox11 are necessary for copper insertion into CuA and CuB redox centers of COX in eukaryotes. A genome-wide search in all prokaryotic genomes combined with genomic context reveals that only Sco and Cox11 have orthologs in prokaryotes. However, while Cox11 function is confined to COX assembly, Sco acts as a multifunctional linker connecting a variety of biological processes. Multifunctionality is achieved by gene duplication and paralogs. Neighbor genes of Sco paralogs often encode cuproenzymes and cytochrome c domains and, in some cases, Sco is fused to cytochrome c. This led us to suggest that cytochrome c might be relevant to Sco function and the two proteins might jointly be involved in COX assembly. Sco is also related, in terms of gene neighborhood and phylogenetic occurrence, to a newly detected protein involved in copper trafficking in bacteria and archaea, but with no sequence similarity to the mitochondrial copper chaperone Cox17. By linking the assembly system to the copper uptake system, Sco allows COX to face alternative copper trafficking pathways.     

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.     

Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase

The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme. Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p. Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria. Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p. Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype. We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants. These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone.     

A structural-dynamical characterization of human Cox17

Human Cox17 is a key mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy transducing respiratory chain. A structural and dynamical characterization of human Cox17 in its various functional metallated and redox states is presented here. The NMR solution structure of the partially oxidized Cox17 (Cox17(2S-S)) consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds involving Cys(25)-Cys(54) and Cys(35)-Cys(44), preceded by a flexible and completely unstructured N-terminal tail. In human Cu(I)Cox17(2S-S) the copper(I) ion is coordinated by the sulfurs of Cys(22) and Cys(23), and this is the first example of a Cys-Cys binding motif in copper proteins. Copper(I) binding as well as the formation of a third disulfide involving Cys(22) and Cys(23) cause structural and dynamical changes only restricted to the metal-binding region. Redox properties of the disulfides of human Cox17, here investigated, strongly support the current hypothesis that the unstructured fully reduced Cox17 protein is present in the cytoplasm and enters the intermembrane space (IMS) where is then oxidized by Mia40 to Cox17(2S-S), thus becoming partially structured and trapped into the IMS. Cox17(2S-S) is the functional species in the IMS, it can bind only one copper(I) ion and is then ready to enter the pathway of copper delivery to cytochrome c oxidase. The copper(I) form of Cox17(2S-S) has features specific for copper chaperones.     

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.     

characterization of the cytochrome c oxidase assembly factor Cox19 of Saccharomyces cerevisiae

Cox19 is an important accessory protein in the assembly of cytochrome c oxidase in yeast. The protein is functional when tethered to the mitochondrial inner membrane, suggesting its functional role within the intermembrane space. Cox19 resembles Cox17 in having a twin CX(9)C sequence motif that adopts a helical hairpin in Cox17. The function of Cox17 appears to be a Cu(I) donor protein in the assembly of the copper centers in cytochrome c oxidase. Cox19 also resembles Cox17 in its ability to coordinate Cu(I). Recombinant Cox19 binds 1 mol eq of Cu(I) per monomer and exists as a dimeric protein. Cox19 isolated from the mitochondrial intermembrane space contains variable quantities of copper, suggesting that Cu(I) binding may be a transient property. Cysteinyl residues important for Cu(I) binding are also shown to be important for the in vivo function of Cox19. Thus, a correlation exists in the ability to bind Cu(I) and in vivo function.     

Mammalian copper chaperone Cox17p has an essential role in activation of cytochrome C oxidase and embryonic development

Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(-/-), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(-/-) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(-/-). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.     

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.     

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.     

Cox17 is functional when tethered to the mitochondrial inner membrane

Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.     

In vivo correction of COX deficiency by activation of the AMPK/PGC-1α axis

Increased mitochondrial biogenesis by activation of PPAR- or AMPK/PGC-1α-dependent homeostatic pathways has been proposed as a treatment for mitochondrial disease. We tested this hypothesis on three recombinant mouse models characterized by defective cytochrome c-oxidase (COX) activity:?a knockout (KO) mouse for Surf1, a knockout/knockin mouse for Sco2, and a muscle-restricted KO mouse for Cox15. First, we demonstrated that double-recombinant animals overexpressing PGC-1α in skeletal muscle on a Surf1 KO background showed robust induction of mitochondrial biogenesis and increase of mitochondrial respiratory chain activities, including COX. No such effect was obtained by treating both Surf1(-/-) and Cox15(-/-) mice with the pan-PPAR agonist bezafibrate, which instead showed adverse effects in either model. Contrariwise, treatment with the AMPK agonist AICAR led to partial correction of COX deficiency in all three models, and, importantly, significant motor improvement up to normal in the Sco2(KO/KI) mouse. These results open new perspectives for therapy of mitochondrial disease.     

The Cardiac Copper Chaperone Proteins Sco1 and CCS are Up-Regulated,but Cox 1 and Cox4 are Down-Regulated,by Copper Deficiency

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long–Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS–PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.     

COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae

We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies.     

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.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.     

Functional role of two interhelical disulfide bonds in human Cox17 protein from a structural perspective

Human Cox17 is the mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy-transducing respiratory chain. It consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds and binds one copper(I) ion through a Cys-Cys motif. Here, the structures and the backbone mobilities of two Cox17 mutated forms with only one interhelical disulfide bond have been analyzed. It appears that the inner disulfide bond (formed by Cys-36 and Cys-45) stabilizes interhelical hydrophobic interactions, providing a structure with essentially the same structural dynamic properties of the mature Cox17 state. On the contrary, the external disulfide bond (formed by Cys-26 and Cys-55) generates a conformationally flexible α-helical protein, indicating that it is not able to stabilize interhelical packing contacts, but is important for structurally organizing the copper-binding site region.