Human Sco1 and Sco2 Function as Copper-binding Proteins
2005; Elsevier BV; Volume: 280; Issue: 40 Linguagem: Inglês
10.1074/jbc.m506801200
ISSN1083-351X
AutoresYih‐Chern Horng, Scot C. Leary, Paul A. Cobine, Fiona B. Young, Graham N. George, Eric A. Shoubridge, Dennis R. Winge,
Tópico(s)RNA Research and Splicing
ResumoThe function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function. The function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function. Cytochrome c oxidase (CcO) 5The abbreviations used are: CcO, cytochrome c oxidase; IMS, intermembrane space; NTA, nitrilotriacetic acid; DTT, dithiothreitol; WT, wild type. is the terminal enzyme of the energy-transducing, electron transfer chain within the mitochondrial inner membrane. Mammalian CcO consists of 13 polypeptide subunits, three of which (CoxI-CoxIII) are encoded by the mitochondrial genome and the remaining 10 of which are encoded by the nuclear genome (1Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (520) Google Scholar, 2Poyton R.O. Goehring B. Droste M. Sevarion K.A. Allen L.A. Zhao X.J. Methods Enzymol. 1995; 260: 97-116Crossref PubMed Scopus (51) Google Scholar). An additional 30 proteins are believed to be required for the assembly of the CcO complex (3Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). A number of these accessory proteins are important in the processing and translation of COXI-COXIII mRNA transcripts, in membrane insertion of subunits, and in either the synthesis or delivery of cofactors. The cofactors in CcO include two copper sites (CuA and CuB), two heme A moieties, and a magnesium and zinc ion (4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1292) Google Scholar). The CuA site is a binuclear, mixed valent copper center localized in the CoxII subunit, whereas the CuB site consists of a single copper ion within a Cu-heme A binuclear center in the CoxI subunit (4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1292) Google Scholar). The CoxI and CoxII subunits are synthesized within the mitochondrion, so copper site insertion must occur during insertion of the nascent polypeptides into the inner membrane. Several proteins, including Cox11, Cox17, Cox19, Cox23, and Sco1, have been implicated in the assembly of the copper centers in CcO in yeast and all have human homologs (5Carr H.S. Winge D.R. Acc. Chem. Res. 2003; 36: 309-316Crossref PubMed Scopus (196) Google Scholar). Cox17 is a soluble copper metallochaperone within the mitochondrial intermembrane space (IMS). Sco1 and Cox11 are inner membrane proteins tethered by a single transmembrane helix and are implicated in the assembly of the CuA and CuB centers, respectively (6Hiser L. Di Valentin M. Hamer A.G. Hosler J.P. J. Biol. Chem. 2000; 275: 619-623Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 7Dickinson E.K. Adams D.L. Schon E.A. Glerum D.M. J. Biol. Chem. 2000; 275: 26780-26785Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Both proteins are copper-binding proteins, and mutations that abrogate Cu(I) binding attenuate in vivo assembly of CcO (8Rentzsch N. Krummeck-WeiB G. Hofer A. Bartuschka A. Ostermann K. Rodel G. Curr. Genet. 1999; 35: 103-108Crossref PubMed Scopus (80) Google Scholar, 9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 2002; 277: 22185-22190Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Recently, we demonstrated that Cox17 is capable of donating Cu(I) to both Sco1 and Cox11 (12Horng Y.C. Cobine P.A. Maxfield A.B. Carr H.S. Winge D.R. J. Biol. Chem. 2004; 279: 35334-35340Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The prediction is that copper ions are only transiently bound to Sco1 and Cox11 in yeast prior to donation to CoxII and CoxI, respectively. Yeast Sco1 was shown to form a transient complex with CoxII (13Lode A. Kuschel M. Paret C. Rodel G. FEBS Lett. 2000; 448: 1-6Google Scholar). Assembly of CcO in mammalian cells has additional components, since two distinct Sco-like molecules are involved in the assembly process. Mutations in both human genes, SCO1 and SCO2, have been identified, and these result in respiratory chain deficiency associated with CcO assembly defects (14Papadopolou L.C. Sue C.M. Davidson M.M. Tanji K. Nishion I. Sadlock J.E. Selby J. Glerum D.M. Van Coster R. Lyon G. Scalais E. Lebel R. Kaplan P. Shanske S. De Vivo D.C. Bonilla E. Hirano M. DiMauro S. Schon E.A. Nat. Genet. 1999; 23: 333-337Crossref PubMed Scopus (490) Google Scholar, 15Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.-P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar, 16Jaksch M. Ogilvie I. Yao J. Kortenhaus G. Bresser H.-G. Gerbitz K.-D. Shoubridge E.A. Hum. Mol. Genet. 2000; 9: 795-801Crossref PubMed Scopus (195) Google Scholar). Although yeast also has a second Sco protein, designated Sco2, it has no function in CcO assembly (17Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The human Sco1 and Sco2 molecules share the Cu(I) binding motif of yeast Sco1; however, nothing is currently known about the copper binding by human Sco1, whereas limited data exist on copper binding by human Sco2 (18Jaksch M. Paret C. Stucka R. Horn N. Muller-Hocker J. Horvath R. Trepesch N. Stecker G. Freisinger P. Thirion C. Muller J. Lunkwitz R. Rodel G. Shoubridge E.A. Lochmuller H. Hum. Mol. Genet. 2001; 10: 3025-3035Crossref PubMed Scopus (109) Google Scholar, 19Foltopoulou P.F. Zachariadis G.A. Politou A.S. Tsiftsoglou A.S. Papadopoulou L.C. Mol. Genet. Metab. 2004; 81: 225-236Crossref PubMed Scopus (26) Google Scholar). Neither human Sco1 nor Sco2 is able to rescue the respiratory defect of yeast sco1 null cells (20Paret C. Ostermann K. Krause-Buchholz U. Rentzsch A. Rodel G. FEBS Lett. 1999; 447: 65-70Crossref PubMed Scopus (40) Google Scholar). However, a chimera containing the N-terminal segment of yeast Sco1 fused to the C-terminal segment of human Sco1 is functional in sco1 null cells (20Paret C. Ostermann K. Krause-Buchholz U. Rentzsch A. Rodel G. FEBS Lett. 1999; 447: 65-70Crossref PubMed Scopus (40) Google Scholar). Two lines of evidence suggest that both human Sco1 and Sco2 proteins probably function in copper metallation of CcO. First, overexpression of COX17 partially rescues the CcO deficiency of SCO2, but not SCO1, patient cells (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). Second, the CcO deficiency in SCO1 and SCO2 patient cell lines is partially suppressed by the addition of exogenous copper to the culture medium (18Jaksch M. Paret C. Stucka R. Horn N. Muller-Hocker J. Horvath R. Trepesch N. Stecker G. Freisinger P. Thirion C. Muller J. Lunkwitz R. Rodel G. Shoubridge E.A. Lochmuller H. Hum. Mol. Genet. 2001; 10: 3025-3035Crossref PubMed Scopus (109) Google Scholar, 21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar, 22Salviati L. Hernandez-Rosa E. Walker W.F. Sacconi S. DiMauro S. Schon E.A. Davidson M.M. Biochem. J. 2002; 15: 321-327Crossref Google Scholar). It is unclear why mammalian cells require two distinct Sco molecules for CcO maturation. Patients with mutations in SCO2 have a clinical presentation that is distinct from that of SCO1 patients (15Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.-P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar, 16Jaksch M. Ogilvie I. Yao J. Kortenhaus G. Bresser H.-G. Gerbitz K.-D. Shoubridge E.A. Hum. Mol. Genet. 2000; 9: 795-801Crossref PubMed Scopus (195) Google Scholar, 23Jaksch M. Horvath R. Horn N. Auer D.P. Macmillan C. Peters J. Gerbitz K.-D. Kraegeloh-Mann I. Muntau A. Karcagi V. Kalmanchey R. Lochmuller H. Shoubridge E.A. Freisinger P. Neurology. 2001; 57: 1440-1446Crossref PubMed Scopus (78) Google Scholar). SCO2 mutations are associated with neonatal encephalocardiomyopathy, whereas SCO1 patients present with neonatal hepatic failure and ketoacidotic coma. The distinctive clinical presentation is not a result of tissue-specific expression of the two genes, since SCO1 and SCO2 are ubiquitously expressed and exhibit a similar expression pattern in different human tissues (14Papadopolou L.C. Sue C.M. Davidson M.M. Tanji K. Nishion I. Sadlock J.E. Selby J. Glerum D.M. Van Coster R. Lyon G. Scalais E. Lebel R. Kaplan P. Shanske S. De Vivo D.C. Bonilla E. Hirano M. DiMauro S. Schon E.A. Nat. Genet. 1999; 23: 333-337Crossref PubMed Scopus (490) Google Scholar). Recent studies with immortalized fibroblasts from SCO1 and SCO2 patients suggest that Sco1 and Sco2 have non-overlapping but cooperative functions in CcO assembly (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). The evidence for this conclusion is that the CcO assembly pathway is blocked at a similar step in both SCO1 and SCO2 patient cell lines and that overexpression of either gene in the reciprocal patient cell line resulted in a dominant negative effect on CcO activity (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). Although Sco proteins are reported to be competent to bind copper ions, this function was questioned in a recent report on the crystal structure of human Sco1 (24Williams J.C. Sue C. Banting G.S. Yang H. Glerum D.M. Hendrickson W.A. Schon E.A. J. Biol. Chem. 2005; 280: 15202-15211Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Sco1 crystallized without bound copper, and the three residues implicated in Cu(I) binding to yeast Sco1 were not in optimal configuration for trigonal Cu(I) binding (24Williams J.C. Sue C. Banting G.S. Yang H. Glerum D.M. Hendrickson W.A. Schon E.A. J. Biol. Chem. 2005; 280: 15202-15211Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The authors suggested that Sco1 is not a copper metallochaperone essential for metal transfer to CoxII but stated that the protein may have a critical role in redox sensing. In this study, we analyze the copper binding properties of human Sco1 and Sco2 and report that copper binding appears to be important for the function of each protein. We report that both proteins have the ability to bind either Cu(I) or Cu(II) and that Cu(II) binding may be an important facet of function. Yeast Strains and Human Cell Lines—All yeast strains used were in W303 background (MAT a, ade2-1, his3-1,15, leu2,3,112, trp1-1, ura3-1). The strain cox17Δ-SCO2/COX17 for the yeast cytosolic assay was generated by transforming cox17 with pRS303-MET25-SCO2/COX17 (25Maxfield A.B. Heaton D.N. Winge D.R. J. Biol. Chem. 2004; 279: 5072-5080Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The cox17-SCO2/COX17 strain was cultured on plates or in liquid in complete medium lacking uracil for pYEF2 selection or lacking leucine for pRS315 selection. sco1Δ cells were cultured on plates or in liquid in complete medium lacking tryptophan for TNDW4(SCO1) selection (9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Cells were cultured with glucose, raffinose, or galactose as carbon sources as described before. DNA transformations were performed using a lithium acetate protocol. Primary cell lines from control SCO1 and SCO2 patient skin fibroblasts were immortalized and cultured as previously described (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). Plasmids— hSCO1 (lacking the first 333 bp) or hSCO2 (lacking the first 234 bp) amplified by PCR was cloned into pHis-parallel 2 to generate pHis-hSCO1 or pHis-hSCO2 by adding BamHI and SalI restriction sites to the 5′- and 3′-ends, respectively. The same hSCO fragment fused to a 5′ sequence encoding a poly-His purification tag was amplified from pHis-hSCO1 or pHis-hSCO2 by PCR with NotI and EcoRI restriction sites added to the 5′- and 3′-ends, respectively. The truncated SCO1 genes were subcloned into pYEF2 containing the GAL1 promoter and CYC1 terminator for transformation of cox17Δ-SCO2/COX17 cells. The plasmid is designated YEp-GAL1-hSCO1 or YEp-GAL1-hSCO2. Mutagenesis of SCO genes was carried out using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) on both pHis-hSCO1 and pTNDW4 (yeast SCO1). The resulting SCO1 mutants were sequenced, and those in pTNDW4 were subcloned to ensure there were no mutations in the plasmid backbones. The construction of YCp-MET25-hCOX17 and pRS303-MET25-SCO2/COX17 was similar to the construction of YCp-MET25-COX17 and YCp-MET25-SCO2/COX17 described previously. The Sco2-Cox17 fusion contains the 5,104 residues of Sco2, including the N-terminal mitochondrial import sequence and transmembrane domain of Sco2 fused to Cox17. The correct sequences of all plasmids generated in this study were confirmed by sequencing. Wild type hSCO1 and hSCO2 cloned into the gateway-modified retroviral expression vector pLXSH or pBABE were used as templates to generate specific point mutants using the QuikChange™ site-directed mutagenesis kit (Stratagene). The fidelity of all resultant constructs was confirmed by sequencing prior to their use in expression studies. Protein Purification—Recombinant human and yeast Sco and mutant Sco were purified from BL21 (DE3) transformants harboring pHis-hSCO or pTNDW2 (His-tagged SCO1 or mutant SCO1) as described previously by Nittis et al. (9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Nickel-NTA Superflow (Qiagen) was used for the purification of the His-tagged Sco proteins. Yeast transformants with YEp-GAL1-hSCO were cultured in raffinose medium to an A600 of 0.6. Galactose was then added to induce expression of the His-tagged Sco. Cells were harvested after 5 h for preparation of lysates by use of a French press and subsequent Ni2+-NTA purification from clarified samples. UV-visible Electronic Absorption and Electronic Paramagnetic Resonance Spectroscopies—Absorption spectra of a sample in a cuvette with a path length of 1 cm were recorded with a Beckman DU640 spectrophotometer. X-band EPR spectra were obtained on a 9-GHz Bruker EMX spectrometer. All samples were run at 77 K in a liquid nitrogen finger Dewar. Spin quantitation was determined relative to a 0.5 mm CuEDTA standard. Assays—The copper and nickel concentration of the protein samples was measured using a PerkinElmer Life Sciences (AAnalyst 100) atomic absorption spectrophotometer or a PerkinElmer Optima (3100XL) ICP spectrometer. A bathocuproine sulfonate assay was used to determine the Cu(I) content of the protein samples. The appearance of a Cu(bathocuproine sulfonate)2 complex was measured by monitoring the absorbance at 483 nm using a molar extinction coefficient of 12,250 cm-1 m-1. Protein was quantified by amino acid analysis after hydrolysis in 5.7 n HCl at 110 °C in vacuo on a Beckman 6300 analyzer. Oxygen consumption assays were performed on a YSI 5300A biological oxygen monitor. The rate of oxygen consumption of each strain was calculated based on the linear portion. Each strain was assayed repeatedly two or three times to get the average rate. Immunoblot Analysis—Protein (10-50 μg) from the mitochondrial fraction was electrophoresed on a 15% SDS-polyacrylamide gel system and transferred to nitrocellulose (Bio-Rad). Membranes were blocked in 1× phosphate-buffered saline (50 mm Na2PO4, 100 mm NaCl, pH 7.0), 0.01% Tween 20, and 10% milk solution prior to detection with appropriate antibodies and visualization with Pierce chemiluminescence reagents using a horseradish peroxidase-conjugated secondary antibody. Antiserum to porin (Por1) was from Molecular Probes, Inc. (Eugene, OR). Rabbit anti-Sco1 antiserum was generated as described previously (9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Immortalized human fibroblasts were differentially permeabilized using digitonin in order to generate mitochondrially enriched fractions (26Klement P. Nijtmans L.G. Van den Bogert C. Houstek J. Anal. Biochem. 1995; 231: 218-224Crossref PubMed Scopus (94) Google Scholar) and subsequently solubilized in phosphate-buffered saline containing 1.5% lauryl maltoside supplemented with complete protease inhibitor mixture (Roche Applied Science) as previously described (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). Equal amounts of protein were fractionated on 15% SDS-PAGE gels and transferred to nitrocellulose. Membranes were blotted with polyclonal antisera raised against human Sco1 (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar) and Sco2 (18Jaksch M. Paret C. Stucka R. Horn N. Muller-Hocker J. Horvath R. Trepesch N. Stecker G. Freisinger P. Thirion C. Muller J. Lunkwitz R. Rodel G. Shoubridge E.A. Lochmuller H. Hum. Mol. Genet. 2001; 10: 3025-3035Crossref PubMed Scopus (109) Google Scholar) and a monoclonal anti-porin antibody (Calbiochem). Following incubation with the relevant secondary antibody, immunoreactive proteins were detected by luminol-enhanced chemiluminescence (Pierce). Miscellaneous—Phoenix amphotropic cells (Dr. G. Nolan, Stanford University) were used to transiently produce and package all individual human cDNA constructs of interest. Subsequent infection and selection of fibroblast cell lines were essentially as previously described (21Leary S.C. Kaufman B.A. Pellechia G. Gguercin G.-H. Mattman A. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2004; 13: 1839-1848Crossref PubMed Scopus (196) Google Scholar). Protein concentration (27Bradford N.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), CcO (28Capaldi R.A. Marusich M.F. Taanman J.W. Methods Enzymol. 1995; 260: 117-132Crossref PubMed Scopus (125) Google Scholar), and citrate synthase (29Srere P.A. Methods Enzymol. 1969; 13: 3-26Crossref Scopus (2036) Google Scholar) activities were measured as described elsewhere. Recombinant Human Sco1 and Sco2 Expression—To study the biochemical properties of the globular domains of human Sco1 and Sco2, truncates were engineered lacking only the N-terminal mitochondrial targeting sequence and single transmembrane helix (Fig. 1). Soluble domains of each protein were used for our studies to avoid the difficulties of purifying and working with membrane proteins. Soluble domains of human SCO1 and SCO2 fused to a 5′ poly-His tag were expressed in Escherichia coli BL21 (DE3) cells for the purpose of purification by Ni2+-NTA chromatography and subsequent characterization of the two proteins. Protein recovered after imidazole elution was found to contain bound copper by atomic absorption spectroscopy. Each protein fraction was subsequently chromatographed on gel filtration to recover homogeneous Sco1 and Sco2. Both proteins eluted from gel filtration in fractions corresponding to monomeric molecules. The copper content of hSco1 and hSco2 was 1.0 and 0.8 mol eq, respectively. Dialysis of the copper-containing proteins overnight in 1 mm EDTA resulted in only a slight depletion of bound copper, with human Sco1 and Sco2 each retaining 0.8 mol eq of bound copper (TABLE ONE). No metal ions other than low levels of Ni(II), derived from the Ni2+-NTA resin used in the purification, were detected using ICP spectroscopy. Samples dialyzed against EDTA were depleted of the minimal levels of Ni(II). Cleavage of the poly-His tag followed by subsequent purification of the non-tagged protein retained 80% of the bound copper. Thus, the copper ions bound to the fusion protein were associated with the Sco1 protein and not the His purification tag. The copper ions were stably associated with both Sco proteins.TABLE ONECopper binding stoichiometry of human Sco1 and Sco2 purified from bacteria The Sco proteins were purified by a combination of Ni2+-NTA chromatography and gel filtration. Copper and protein were quantified by atomic absorption spectrometry and amino acid analysis, respectively.Copper/proteinCu(II)/proteinDialysis (1 mm EDTA)Dialysis (1 mm DTT), Cu(II)/proteinCopper/proteinCu(II)/proteinWT human Sco10.95 ± 0.030.35 ± 0.080.78 ± 0.050.17 ± 0.020.24 ± 0.04D259A human Sco11.13 ± 0.260.17 ± 0.020.12 ± 0.050.10 ± 0.03NDaND, not detectableH260A human Sco10.66 ± 0.090.34 ± 0.070.28 ± 0.06NDNDWT human Sco20.79 ± 0.060.35 ± 0.010.75 ± 0.070.34 ± 0.080.32 ± 0.03a ND, not detectable Open table in a new tab Absorption spectroscopy of purified Sco1 revealed a chromophore in the visible region of the spectrum with maxima at 360 and 480 nm (Fig. 2A). The visible absorption bands resembled the S-Cu(II) charge transfer bands of nitrosocyanin (30Lieberman R.L. Arciero D.M. Hooper A.B. Rosenzweig A.C. Biochemistry. 2001; 40: 5674-5681Crossref PubMed Scopus (67) Google Scholar, 31Basumallick L. Sarangi R. George S.D. Elmore B. Hooper A.B. Hedman B. Hodgson K.O. Solomon E.I. J. Am. Chem. Soc. 2005; 127: 3531-3544Crossref PubMed Scopus (87) Google Scholar), and the presence of bound Cu(II) in human Sco1 as purified was confirmed by EPR (Fig. 3A). Integration of the EPR signal revealed a Cu(II) content of 0.3 mol eq. Titration of human Sco1 with the Cu(I) chromogenic chelator bathocuproine sulfonate revealed a Cu(I) content of 0.7 mol eq. The stoichiometry in combination with EPR spectroscopy suggests that each monomeric Sco1 molecule contains either a bound Cu(I) or a bound Cu(II). The fraction of bound Cu(I) versus Cu(II) was similar in multiple isolates of hSco1. To determine the binding stability of the two valence states of copper, hSco1 dialyzed overnight with 1 mm EDTA revealed a Cu(II) content of 0.17 mol eq, whereas the total copper content dropped to 0.78 mol eq. Although the Cu(II) content was reduced 2-fold by the EDTA dialysis treatment, the chromophore content was unchanged. The dialysis result is consistent with the EPR spectrum that forms two gz values with different hyperfine splittings, indicating the presence of two Cu(II) species in human Sco1 (Fig. 3A). Although human Sco1 contains two Cu(II) species, only one generates the chromophore. Two distinct Cu(II) species were also observed in the Sco1 protein from Bacillus subtilis (32Balatri E. Banci L. Bertini I. Cantini F. Cioffi-Baffoni S. Structure. 2003; 11: 1431-1443Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar).FIGURE 3EPR spectrum of human and yeast Sco1 samples. A, EPR spectrum of human and yeast Sco1 proteins as isolated. The concentrations of the human and yeast proteins were 2.3 and 2.4 mm, respectively. Shown are the hyperfine splittings of the gz components. The presence of two gz components suggests the presence of two distinct Cu(II) species. B, the spectrum of the human Sco1 sample (0.67 mm) dialyzed against 1 mm EDTA and 1 mm DTT is shown. EPR spectra were recorded at 9.429 GHz with a power of 19.8 milliwatts with a modulation amplitude of 4 G.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Incubation of hSco1 with 1 mm DTT overnight reduced the Cu(II) content from 0.35 to 0.24 mol eq, but the chromophore content was unchanged (TABLE ONE). Thus, the Cu(II) species that is responsible for the visible chromophore is stable to dialysis in 1 mm EDTA and 1 mm DTT. The EPR spectrum of the CuSco1 sample after dialysis in EDTA and DTT is shown in Fig. 3B. This spectrum is characterized by only four hyperfine splittings of the gz component of the Cu(II) signal. This pattern of hyperfine splitting is consistent with a single Cu(II) species that gives rise to the visible absorption bands. Prolonged incubation of Sco1 at 4 °C did not alter the abundance of the Cu(II) chromophore. As mentioned, the chromophore was not bleached by 1 mm DTT but was abolished by dithionite. After bleaching by 2 mm dithionite followed by dialysis in buffer, the copper content was reduced by an increment corresponding to the quantity of copper originally present as Cu(II). Thus, upon reduction of Cu(II), the Cu(I) is unable to stably associate with the Sco1. Analogous experiments with human Sco2 found that the Cu(I) and Cu(II) adducts were indistinguishable from those of human Sco1. Collectively, these data argue that the unique function of each human Sco in the metallation of CcO is not attributable to differences in their copper-binding properties. The EPR spectrum of Cu(II)Sco1 (Fig. 3B) shows features that are typical of 14N ligand hyperfine interaction (a three-line pattern from the I = 1 nucleus superimposed on a four-line pattern from the I = 3/2 63,65Cu hyperfine) that is expected from a histidine nitrogen ligation of the metal. This is illustrated by the inset in Fig. 3B, which shows the second derivative of the gx-gy region of the Cu(II) EPR spectrum, with clear structure attributable to 14N ligand hyperfine to at least one such ligand. A more complete analysis of the EPR spectroscopy of Cu(II)Sco1, including multifrequency measurements will be presented in a subsequent publication. Previously, we reported the presence of Cu(I) in yeast Sco1, but only a percentage of the copper was reactive with the Cu(I) chelator bathocuproine sulfonate (9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To determine whether yeast Sco1 also contained bound Cu(II), the soluble domain of yeast Sco1 was recombinantly expressed and purified as described previously (9Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Sc
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