A Gain of Superoxide Dismutase (SOD) Activity Obtained with CCS, the Copper Metallochaperone for SOD1
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.36952
ISSN1083-351X
AutoresPaul J. Schmidt, Minerva Ramos‐Gómez, Valeria Culotta,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoThe incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp200). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS. The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp200). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS. superoxide dismutase nitro blue tetrazolium N,N,N′,N'-tetramethylethylenediamine In eukaryotic cells, copper is delivered to specific protein targets via the action of a family of copper carrier proteins termed "metallochaperones" (1Pufahl R. Singer C. Peariso K.L. Lin S.J. Schmidt P. Fahrni C. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (589) Google Scholar). These molecules are well conserved between yeast and humans and serve to guide the metal to discrete cellular locations and facilitate incorporation of the cofactor into target metalloenzymes (reviewed in Refs. 2Valentine J.S. Gralla E.B. Science. 1997; 278: 817-818Crossref PubMed Scopus (189) Google Scholar, 3Pena M.O. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (603) Google Scholar, 4Poulos T.L. Nature Struct. Biol. 1999; 6: 709-711Crossref PubMed Scopus (20) Google Scholar). One such copper chaperone, COX17, acts in the delivery of copper to mitochondrial cytochrome oxidase (5Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 6Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 7Amaravadi R. Glerum D.M. 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Labesse G. Mathews F.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 1749-1754Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Thirdly, copper delivery and incorporation into cytosolic superoxide dismutase 1 (SOD1)1 is mediated by the soluble copper carrier, CCS (copperchaperone for SOD), also known inSaccharomyces cerevisiae as LYS7 (13Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 14Gamonet F. Lauquin G.J.-M. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar). Studies with the yeast metallochaperone have shown that CCS directly incorporates copper into SOD1 despite exquisitely low levels of available free copper (15Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1357) Google Scholar). The target of the CCS metallochaperone, SOD1, is a homodimeric copper- and zinc-requiring enzyme that acts to disproportionate superoxide (O2) to hydrogen peroxide (H2O2) and oxygen in a reaction catalyzed by the redox cycling of bound copper (16McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). However, SOD1 is also capable of catalyzing deleterious reactions involving the redox active copper cofactor. The Cu(I) form of SOD1 can react with H2O2 to generate the highly toxic hydroxyl radical (OH⋅) (17Borders C.L. Fridovich I. Arch. Biochem. Biophys. 1985; 241: 472-476Crossref PubMed Scopus (55) Google Scholar, 18Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5006-5010Crossref PubMed Scopus (316) Google Scholar, 19Yim M.B. Chock P.B. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar). In fact, it has been suggested that this inherent peroxidase activity of SOD1 may be involved in cases of familial amyotrophic lateral sclerosis in which disease results from dominant mutations in SOD1 (20Yim M.B. Kang J.H. Yim H.S. Kwak H.S. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5709-5714Crossref PubMed Scopus (428) Google Scholar, 21Yim H.-S. Kang J.-H. Chock P.B. Stadtman E.R. Yim M.B. J. Biol. Chem. 1997; 272: 8861-8863Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 22Liochev S.I. Chen L.L. Hallewell R.A. Fridovich I. Arch. Biochem. Biophys. 1997; 346: 263-268Crossref PubMed Scopus (42) Google Scholar, 23Wiedau-Pazos M. Goto J. Rabizadeh S. Gralla E.B. Roe J.A. Lee M.K. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Crossref PubMed Scopus (660) Google Scholar, 24Goto J.J. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 1998; 273: 30104-30109Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It is noteworthy that the human CCS metallochaperone harbors a polypeptide region bearing striking resemblance to SOD1. This region, found in the central 16-kDa portion of CCS, is postulated to serve in target recognition of SOD1 (25Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), whereas smaller segments at the N and C terminus of CCS are thought to facilitate the binding and release of copper into SOD1 (26Schmidt P. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Homology between the central portion of CCS and SOD1 approaches 50% identity and 60% similarity (25Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Based on this curious conservation of sequence, CCS was originally postulated to be "SOD4," the fourth mammalian SOD (GenBankTM accession number 1608528). The concordance of sequence between SOD1 and CCS was the focus of current studies. CCS molecules from diverse mammals contain all of the zinc binding ligands found in SOD1 and three of four histidine copper binding ligands; the fourth histidine is always substituted by an aspartate. We demonstrate here that the presence of this single aspartate prohibits CCS from functioning as a SOD. Substituting this aspartate with a histidine results in a CCS molecule with significant superoxide scavenging activity. Furthermore, this variant of CCS appears to be a bifunctional SOD, capable of self-activation with copper. The isogenic wild type SY1699 and lys7 null SY2950 strains were previously described (27Horecka J. Kinsey P.T. Sprague G.F. Gene ( Amst. ). 1995; 162: 87-92Crossref PubMed Scopus (34) Google Scholar), as was the sod1Δ sod2Δ strain KS100 (28Culotta V.C. Joh H.D. Lin S.J. Slekar K.H. Strain J. J. Biol. Chem. 1995; 270: 29991-29997Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). VC107 and VC279 are sod1Δ::TRP1 derivatives of SY1699 and SY2950, respectively, and PS120 is a lys7Δ::URA3derivative of KS100. Stocks of yeast strains were maintained by growth on enriched YPD medium (29Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1978: 163-168Google Scholar) in anaerobic culture jars (BBL, GasPak), whereas growth tests for lysine auxotrophy utilized a synthetic minimal medium (29Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1978: 163-168Google Scholar). The vector for expression of human CCS in yeast cells is pCCS-HIS, containing the human CCS coding sequence under the control of theS. cerevisiae PGK1 (phosphoglycerol kinase) promoter. pCCS-HIS was constructed by mobilizing the PGK1-CCS fusion from pSMCCS (13Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar) by digestion with BamHI and SalI and by insertion at these same sites into pRS423 (HIS32μ; Ref. 30Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Human SOD1 was expressed in yeast by the pLC1 vector (URA3 2μ) where the SOD1 cDNA was placed under the control of the PGK1 promoter as described (31Corson L.B. Strain J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (139) Google Scholar). pSM703 is the PGK1 promoter-containing vector (URA3 2μ) that served as the parent plasmid for construction of all human SOD1 and CCS expression plasmids. Plasmids pMR002 and pPS030 for the expression of D200H CCS and H120D SOD1 were derived from pCCS-HIS and pLC1, respectively, through use of the Quick Change Site-Directed Mutagenesis Kit (Stratagene) per manufacturer's instructions. Cell lysates were prepared essentially as described (13Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). Strains transformed with the appropriate plasmids were grown approximately 18 h to an A 600 of 1.5 in 50 ml of selecting SD medium (29Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1978: 163-168Google Scholar); cells were harvested and were lysed by glass bead homogenization in 0.2–0.5 ml of a lysis buffer containing 10 mm NaPO4 (pH 7.8), 0.1 mm EDTA, 0.1% Triton, 50 mm NaCl, 20 μg/ml leupeptin, 10 μg/ml pepstatin, and 1.0 mmphenylmethylsulfonyl fluoride. Glycerol was added to the final lysates at a concentration of 5%. As needed, lysates were concentrated by Microcon-10 Microconcentrator (Amicon) columns per manufacturer's instructions. For analysis of SOD activity, extracts were applied directly without boiling to a nondenaturing 12% precast polyacrylamide gel (Novex). Following electrophoresis, the gel was subject to nitro blue tetrazolium (NBT) staining for superoxide (32Flohe L. Otting F. Packer L. Methods in Enzymology: Oxygen Radicals in Biological Systems. Academic Press, New York1984: 93-104Google Scholar) in a 75-ml solution containing one tablet of NBT (10 mg/tablet), 50 mmKPO4 (pH 7.8), 0.1 mg/ml riboflavin, and 1 μl/ml TEMED. Western blot analyses of lysates were carried out as described (33Lin S.J. Culotta V.C. Mol. Cell. Biol. 1996; 16: 6303-6312Crossref PubMed Scopus (66) Google Scholar, 34Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.39-7.52Google Scholar) using a denaturing 14% polyacrylamide gel. A polyclonal rabbit anti-human SOD1 serum (kind gift of D. Borchelt, Johns Hopkins University) and a polyclonal CCS antibody (obtained from Jeff Rothstein, Johns Hopkins University) were used at 1:5000 and 1:500 dilutions, respectively, and were detected using as secondary antibody, anti-rabbit donkey IgG (Amersham Pharmacia Biotech) conjugated to horseradish peroxidase. Visualization involved the Hybond ECL kit (Amersham Pharmacia Biotech). For immunoprecipitation of human CCS and yeast SOD1, 50–100 μg of cell lysate protein was reacted with either anti-CCS (1:100 dilution), anti-yeast SOD1 (1:500 dilution; kind gift of Dan Kosman, SUNY, Buffalo), or preimmune serum (1:100 dilution) in a buffer containing 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 150 mm NaCl, and 0.1% Nonidet P-40, as needed. Following incubation for 2 h at 4 °C, 20 μl of a 50% suspension of A-Sepharose beads (Sigma) was added, and incubation continued for an additional hour. The mixture was subjected to microcentrifugation for 1 min at 4 °C, and the resulting supernatants were concentrated on Microcon-10 Microconcentrator (Amicon) columns. The central region of human CCS encompassing amino acids 78 to 232 shares nearly 50% identity with human SOD1. All four of the zinc binding ligands of SOD1 and three of four histidine copper binding ligands are present in CCS; the fourth histidine is replaced by an aspartate residue, which is also a possible ligand for copper (35Lippard S.J. Berg J.M. Principles of Bioinorganic Chemistry. University Science Books, Mill Valley, CA1994: 45Google Scholar) (Fig. 1 A). Interestingly, this precise pattern of homology to SOD1 is observed with murine CCS (36Nishihara E. Furuyama T. Yamashita S. Mori N. Mol. Neurosci. 1998; 9: 3259-3263Google Scholar). Based on sequence analysis alone it would seem plausible that mammalian CCS should possess SOD activity. To address this question in vivo, human CCS was expressed in a yeast strain lacking SOD1 and was assayed for the ability to overcome the oxidative damage of these cells. Yeast sod1Δ mutants cannot synthesize lysine when grown in air due to oxidative damage of lysine biosynthetic component(s) (37Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 38Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (138) Google Scholar, 39Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar). This defect is readily corrected by expression of the heterologous human SOD1 (Fig. 1 C). However, expression of human CCS failed to complement the lysine auxotrophy of the sod1 mutant (Fig. 1 C) even though the protein accumulated to substantial levels (Fig. 1 B). By a NBT gel assay for SOD activity (32Flohe L. Otting F. Packer L. Methods in Enzymology: Oxygen Radicals in Biological Systems. Academic Press, New York1984: 93-104Google Scholar), CCS exhibited no detectable scavenging of superoxide (Fig. 2 A, lane 5). Therefore, despite the exquisite homology between SOD1 and human CCS, the copper chaperone appears nonfunctional for SOD activity. The most noteworthy difference between SOD1 and the SOD1 homology domain of human CCS is the substitution of a copper binding histidine with an aspartate (Fig. 1 A). To test whether this single amino acid variance precludes SOD activity, the aspartate at CCS position 200 was mutated to a histidine. By Western blot analysis, D200H CCS accumulated to wild type levels when expressed in yeast (Fig. 1 B). Surprisingly, expression of this mutant CCS molecule effectively complemented the yeast sod1 mutation (Fig. 1 C), suggesting that this metallochaperone had acquired SOD activity. D200H CCS was directly examined for superoxide scavenging activity by the NBT gel assay. These studies were conducted in the background of a sod1Δ sod2Δ strain devoid of any endogenous SOD activity (Fig. 2 A, lane 1). As seen in Fig. 2 A, a dose response of superoxide scavenging activity was detected in lysates from cells expressing D200H CCS. 2It is unclear whether D200H CCS is active as a monomer or dimer. On nondenaturing gels, the superoxide scavenging activity associated with D200H migrates to a position that lies between dimeric SOD1 and tetrameric SOD2. To establish that the mutant CCS molecule was indeed responsible for this activity, cell lysates were subjected to immunoprecipitation with either an anti-CCS antibody or preimmune control sera prior to analysis of superoxide scavenging activity. As seen in Fig. 2 B, treatment with anti-CCS depleted all activity from the cell lysates, demonstrating that D200H CCS is capable of scavenging superoxide. The gain of activity observed with D200H CCS indicated that aspartate 200 in this molecule is sufficient to prohibit SOD activity. To confirm this, we conducted the reverse mutagenesis experiment in which the fourth histidine copper ligand in human SOD1 was substituted with an aspartate (Fig. 1 A). The resultant H120D SOD1 molecule accumulated to significant levels when expressed in thesod1Δ strain and like other mutant alleles of human SOD1 (31Corson L.B. Strain J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (139) Google Scholar, 40Borchelt D.R. Guarnieri M. Wong P.C. Lee M.K. Slunt H.S. Xu Z.-S. Sisodia S.S. Price D.L. Cleveland D.W. J. Biol. Chem. 1995; 270: 3234-3238Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 41Borchelt D.R. Lee M.K. Slunt H.H. Guarnieri M. Xu Z. Wong P.C. Brown R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (524) Google Scholar), exhibited an altered mobility on denaturing gels (Fig. 3 A). As seen in Fig. 3 B, H120D SOD1 was nonfunctional in complementing the aerobic lysine auxotrophy of the sod1Δ mutant. Furthermore, when more directly visualized by the NBT gel assay, the H120D SOD1 mutant exhibited no detectable SOD activity (Fig. 3 C). The strong inhibitory effect of the H120D mutation on SOD1 activity supports the notion that the aspartate residue at CCS position 200 is sufficient to prevent this molecule from functioning as a SOD. We next tested whether amino acid Asp200 in human CCS evolved to facilitate copper transfer to SOD1. To monitor the metallochaperone activity of CCS, wild type and D200H CCS molecules were expressed in a lys7Δ null strain lacking the yeast CCS molecule (Fig. 4 A). Yeast SOD1 is still present in lys7Δ strains but is normally inactive because the enzyme is apo for copper (13Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 15Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1357) Google Scholar, 42Lyons T.J. Nerissian A. Goto J.J. Zhu H. Gralla E.B. Valentine J.S. J. Bioinorg. Chem. 1998; 3: 650-662Google Scholar). However, yeast SOD1 can be fully activated by expression of the human CCS metallochaperone (Fig. 4 B). By direct monitoring of SOD1 activity, the wild type and D200H mutant CCS molecules appeared equally capable of charging yeast SOD1 with copper (Fig. 4 B). Compared with the experiment of Fig. 2, this study utilized a low level of cell lysate to minimize interference from the superoxide scavenging activity of D200H CCS. To confirm that the activity observed in Fig. 4 B indeed reflected SOD1 and not D200H CCS, an immunodepletion experiment was conducted. As seen in Fig. 4 C, the activity observed with cells coexpressing SOD1 and D200H CCS was immunodepleted with an antibody directed against yeast SOD1 and not human CCS. Thus, D200H CCS retains full activity as a metallochaperone for SOD1. Because the D200H CCS molecule retains its function as a copper chaperone and also exhibits superoxide scavenging activity, is it possible that this SOD-like molecule can act as its own metallochaperone? To address this, the mutant CCS molecule was expressed in a strain lacking both SOD1 and the yeast copper chaperone LYS7. This strain cannot grow on medium lacking lysine; however, the D200H CCS mutant rescued oxidative damage and supported lysine independent growth (Fig. 5 A). By the NBT gel assay, it is evident that D200H CCS is capable of superoxide scavenging even in the absence of the yeast copper chaperone LYS7 (Fig. 5 B,lanes 2–4). Therefore, the D200H mutant of CCS appears to self-activate itself for superoxide scavenging activity. The mammalian CCS metallochaperone has evolved with a domain exhibiting remarkable homology to its target of copper delivery, SOD1. Based on this high degree of sequence identity, SOD activity would not have been an unreasonable assumption. In fact, a number of copper-containing complexes have been shown to scavenge superoxide (43Lengfelder E. Weser U. Bull. Eur. Physiopathol. Respir. 1981; 17: 73-80PubMed Google Scholar, 44deAlvare L.R. Goda K. Kimura T. Biochem. Biophys. Res. Commun. 1976; 69: 687-694Crossref PubMed Scopus (100) Google Scholar, 45Batinic-Haberle I. Liochev S.I. Spasojevic I. Fridovich I. Arch. Biochem. Biophys. 1997; 343: 225-233Crossref PubMed Scopus (119) Google Scholar). However, CCS exhibits no detectable superoxide scavenging activity, and prohibition of SOD activity is in part accomplished by the presence of a single aspartate residue (Asp200) at the putative copper site. Substitution of this aspartate with histidine helps to reconstruct the four-histidine copper site of SOD1 and is sufficient to unlock the superoxide scavenging capacity of CCS. It is curious that mammalian CCS has evolved to look so much like SOD1 without retaining SOD activity. Presumably, this striking homology is required for CCS recognition of SOD1. SOD1 normally exists as a homodimer (46Tainer J.A. Getzoff E.D. Beem K.M. Richardson J.S. Richardson D.C. J. Mol. Biol. 1982; 160: 181-217Crossref PubMed Scopus (898) Google Scholar), and the formation of a transient heterodimer (or multimer) between enzyme and CCS metallochaperone may initiate metal transfer. Physical interaction between SOD1 and the SOD homology domain of CCS has in fact been demonstrated by Gitlin and colleagues (25Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Because metal binding can affect the conformation of SOD1 (42Lyons T.J. Nerissian A. Goto J.J. Zhu H. Gralla E.B. Valentine J.S. J. Bioinorg. Chem. 1998; 3: 650-662Google Scholar), the presence of a SOD1-like copper site in CCS may contribute to the overall domain structure needed for target recognition. It is not known whether copper binds at this site, yet if this were the case, the metal appears incapable of the redox cycling needed for superoxide scavenging. As an added advantage, abrogation of copper redox chemistry should also prohibit the deleterious peroxidase activity typical of SOD1 (18Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5006-5010Crossref PubMed Scopus (316) Google Scholar, 19Yim M.B. Chock P.B. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar). Based on homology to SOD1, there are four possible copper ligands in the central domain of CCS, and of these, placement of an aspartate at position 200 seems best suited to preclude redox damage to the protein. Uchida and Kawakishi (47Uchida K. Kawakishi S. J. Biol. Chem. 1994; 269: 2405-2410Abstract Full Text PDF PubMed Google Scholar) have shown that at the analogous position in human SOD1 (His120) the histidine is highly susceptible to oxidation, whereas the remaining three histidine copper ligands are not. Hence, the presence of Asp200 in CCS appears perfectly designed to prohibit self-oxidation of the copper chaperone or oxidation of its intimate partner, SOD1. Although mammalian CCS exhibits great homology to SOD1, far less homology is evident when one compares the central domain of yeast CCS to that of yeast SOD1 (approximately 25% identity at the amino acid level). Furthermore, as revealed through x-ray crystallographic studies by Rosenzweig and co-workers (48Lamb A.L. Wernimont A.K. Pufahl R.A. Culotta V.C. O'Halloran T.V. Rosenzweig A.C. Nature Struct. Biol. 1999; 6: 724-729Crossref PubMed Scopus (175) Google Scholar), the central domain of yeast CCS is devoid of a metal binding cavity, even though the overall structure is remarkably similar to SOD1. The loss of this metal binding site in yeast CCS may reflect a unique requirement for activation of yeast SOD1. Valentine and co-workers (42Lyons T.J. Nerissian A. Goto J.J. Zhu H. Gralla E.B. Valentine J.S. J. Bioinorg. Chem. 1998; 3: 650-662Google Scholar) have demonstrated that unlike mammalian SOD1, which forms a symmetrical homodimer, the yeast enzyme adopts an asymmetrical conformation in which there is unequal metallation of the two subunits (42Lyons T.J. Nerissian A. Goto J.J. Zhu H. Gralla E.B. Valentine J.S. J. Bioinorg. Chem. 1998; 3: 650-662Google Scholar). As fungal SOD1 diverged from the human enzyme, it is likely that their respective metallochaperones evolved concomitantly to conform to specific metallation requirements. Why do separate molecules exist for SOD1 and its metallochaperone? Our studies here with human D200H CCS indicate that it is possible to manufacture a self-sufficient SOD molecule that can charge itself with copper. Although more energy-consuming, the synthesis of separate molecules for superoxide scavenging and metal incorporation may allow for control of SOD1 activity at the post-translational level. In general, the SOD1 polypeptide is ubiquitously expressed, but the fraction of apo- to holoSOD1 can vary greatly among cell types and tissues (49Petrovic N. Comi A. Ettinger M.J. J. Biol. Chem. 1996; 271: 28331-28334Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 50Rossi L. Marchese E. DeMartino A. Rotilio G. Ciriolo M.R. 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