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• W2009486598 abstract "The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as CuA and CuB) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1·MelC2 binary complex function, six single substitution mutations were introduced into the CuA and CuB sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1·MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion. The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as CuA and CuB) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1·MelC2 binary complex function, six single substitution mutations were introduced into the CuA and CuB sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1·MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion. Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase that catalyzes both the O-hydroxylation of monophenols and the oxidation of O-diphenols to O-quinones (1Mason H.S. Annu. Rev. Biochem. 1965; 34: 595-634Crossref PubMed Scopus (170) Google Scholar,2Lerch K. Siegel H. Metal Ions in Biological Systems. 13. Marcel Dekker Inc., New York1981: 143-186Google Scholar). This enzyme is ubiquitous and is responsible for the biosynthesis of melanin pigment from tyrosine (2Lerch K. Siegel H. Metal Ions in Biological Systems. 13. Marcel Dekker Inc., New York1981: 143-186Google Scholar). The primary structures of tyrosinase from Streptomyces (3Bernan V. Filpula D. Herber W. Bibb M. Katz E. Gene ( Amst. ). 1985; 37: 101-110Crossref PubMed Scopus (120) Google Scholar, 4Huber M. Hintermann G. Lerch K. Biochemistry. 1985; 24: 6038-6044Crossref PubMed Scopus (90) Google Scholar, 5Huber M. Lerch K. Biochemistry. 1988; 27: 5610-5615Crossref PubMed Scopus (42) Google Scholar), Neurospora crassa (6Kupper U. Niedermann D.M. Travaglini G. Lerch K. J. Biol. Chem. 1989; 264: 17250-17258Abstract Full Text PDF PubMed Google Scholar), Rana nigromaculata (7Takase M. Miura I. Nakata A. Takeuchi T. Nishioka M. Gene ( Amst .). 1992; 121: 359-363Crossref PubMed Scopus (31) Google Scholar), Mus musculus (8Kwon B.S. Wakulchik M. Haq A.K. Halaban R. Kestler D. Biochem. Biophys. Res. Commun. 1988; 153: 1301-1309Crossref PubMed Scopus (128) Google Scholar, 9Müller G. Ruppert S. Schmid E. Schütz G. EMBO J. 1988; 7: 2723-2730Crossref PubMed Scopus (229) Google Scholar), and Homo sapiens (10Kwon B.S. Haq A.K. Pomerantz S.H. Halaban R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7473-7477Crossref PubMed Scopus (396) Google Scholar, 11Giebel L.B. Strunk K.M. Spritz R.A. Genomics. 1991; 9: 435-445Crossref PubMed Scopus (128) Google Scholar) have been determined and exhibit considerable heterogeneity. However, the catalytic domain of this enzyme from different sources all has a single binuclear copper center. In the last 20 years, substantial progress has been made to elucidate the role of the active site copper center involved in catalysis (for reviews see Refs. 2Lerch K. Siegel H. Metal Ions in Biological Systems. 13. Marcel Dekker Inc., New York1981: 143-186Google Scholar and 12Huber M. Lerch K. Linzen B. Invertebrate Oxygen Carriers. Springer-Verlag Inc., Berlin1986: 265-276Crossref Google Scholar). Apart from theStreptomyces tyrosinase, the mechanisms by which copper ions incorporated into the various sources of apotyrosinase are largely unknown (13Lee Y.-H.W. Hausinger R.P. Eichhorn G.L. Marzilli L.G. Mechanisms of Metallocenter Assembly. VCH Inc., New York1996: 223-234Google Scholar). The structural gene (melC2) for the tyrosinase ofStreptomyces antibioticus (14Katz E. Thompson C.J. Hopwood D.A. J. Gen. Microbiol. 1983; 129: 2703-2714PubMed Google Scholar) or Streptomyces glaucescens (15Hintermann G. Zatchej M. Hütter R. Mol. Gen. Genet. 1985; 200: 422-432Crossref PubMed Scopus (42) Google Scholar) is part of a polycistronic operon (melC), preceded by the melC1 gene, which encodes a conserved protein essential for the expression of melanin inStreptomyces (3Bernan V. Filpula D. Herber W. Bibb M. Katz E. Gene ( Amst. ). 1985; 37: 101-110Crossref PubMed Scopus (120) Google Scholar, 14Katz E. Thompson C.J. Hopwood D.A. J. Gen. Microbiol. 1983; 129: 2703-2714PubMed Google Scholar, 16Lee Y.-H.W. Chen B.-F. Wu S.-Y. Leu W.-M. Lin J.-J. Chen C.W. Lo S.J. Gene ( Amst. ). 1988; 65: 71-81Crossref PubMed Scopus (38) Google Scholar). In a series of investigations, our results showed that the MelC1 protein plays the dual roles of regulating copper incorporation and promoting the secretion of apotyrosinase via a transient MelC1·MelC2 complex (16Lee Y.-H.W. Chen B.-F. Wu S.-Y. Leu W.-M. Lin J.-J. Chen C.W. Lo S.J. Gene ( Amst. ). 1988; 65: 71-81Crossref PubMed Scopus (38) Google Scholar, 17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar, 18Leu W,-M. Chen L.-Y. Liaw L.-L. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20108-20113Abstract Full Text PDF PubMed Google Scholar, 19Chen L.-Y. Chen M.-Y. Leu W.-M., T.-Y. Tsai T.-Y. Lee Y.-H.W. J. Biol. Chem. 1993; 268: 18710-18716Abstract Full Text PDF PubMed Google Scholar). Evidence was also provided that indicated a conformational transition of MelC2 during the copper activation (19Chen L.-Y. Chen M.-Y. Leu W.-M., T.-Y. Tsai T.-Y. Lee Y.-H.W. J. Biol. Chem. 1993; 268: 18710-18716Abstract Full Text PDF PubMed Google Scholar). This function of MelC1 is reminiscent of that of the molecular chaperone involved in protein folding, assembly, secretion, and heat shock responses (20Ellis R.J. van der Vies S.M. Annu. Rev. Biochem. 1991; 60: 321-347Crossref PubMed Google Scholar, 21Rothman J.E. Cell. 1989; 59: 591-601Abstract Full Text PDF PubMed Scopus (629) Google Scholar, 22Hartl F.U. Martin J. Curr. Opin. Struct. Biol. 1995; 5: 92-102Crossref PubMed Scopus (154) Google Scholar). To gain insights into the molecular mechanism of the copper activation process in Streptomyces apotyrosinase, we recently set out to study the structure-function relationship of MelC1. Our results suggested that the signal peptide region as well as the histidine residues 102 and 117 of MelC1 played important roles in the activity of MelC1 (18Leu W,-M. Chen L.-Y. Liaw L.-L. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20108-20113Abstract Full Text PDF PubMed Google Scholar, 19Chen L.-Y. Chen M.-Y. Leu W.-M., T.-Y. Tsai T.-Y. Lee Y.-H.W. J. Biol. Chem. 1993; 268: 18710-18716Abstract Full Text PDF PubMed Google Scholar, 23Liaw L.-L. Lee Y.-H.W. Biochem. Biophys. Res. Commun. 1995; 214: 447-453Crossref PubMed Scopus (7) Google Scholar). Their mutations affected either the copper incorporation process or the release of tyrosinase into the medium. To further delineate the copper metallocenter assembly process of theStreptomyces tyrosinase, we examined in this study the role of copper-binding ligands of tyrosinase in the activation of MelC1·MelC2 binary complex. Our results indicate that all copper ligands are crucial for the activation of tyrosinase. Their lesions result in the formation of defective binary complexes that severely affect the discharge of MelC2 from the complexes and also lead to a moderate effect on the secretion of MelC1 or MelC2 in some mutants. Streptomyces lividans TK64 (SLP2−, SLP3−, pro-2, str-6) (24Hopwood D.A. Kieser T. Wright H.M. Bibb M.J. J. Gen. Microbiol. 1983; 129: 2257-2269PubMed Google Scholar) was used as the host for recombinant plasmids. Streptomycesplasmid pIJ702 (14Katz E. Thompson C.J. Hopwood D.A. J. Gen. Microbiol. 1983; 129: 2703-2714PubMed Google Scholar) containing the thiostrepton resistance determinant (tsr) and the melanin operon (melC) was kindly provided by Prof. E. Katz (Georgetown University). Plasmid pIJ702-117 is a derivative of pIJ702 carrying a mutation in the upstream regulatory region of the melC operon that results in the overexpression of the melC operon (25Leu W.-M. Wu S.-Y. Lin J.-J. Lo S.J. Lee Y.-H.W. Gene ( Amst. ). 1989; 84: 267-277Crossref PubMed Scopus (13) Google Scholar). Standard media, culture conditions, and transformation procedures forStreptomyces were previously described (26Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich1985Google Scholar). The Altered Site system (Promega) was used for in vitro mutagenesis of the melC2 gene as specified by the manufacturer. Plasmid pSELC2 was constructed by subcloning the 1.4-kilobaseSstI-EcoRV fragment of melC from pIJ702, into the SstI/SmaI digested pSELECT-1. Single-strand DNA from pSELC-2 was used as a template for site-directed mutagenesis. Oligonucleotides designed for site-directed mutagenesis are shown below. Each primer was designated by the position of the mutated amino acid in the MelC2 protein and by the one-letter symbols for the amino acids before and after the mutation. The mutated bases are shown in boldface and underlined type. All mutations were confirmed by DNA sequencing using the chain-termination method (27Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52668) Google Scholar): H37Q, 5′-TCACCACGCAGAACGCGTTC-3′; H53Q, 5′-CACCGGCCAGCGTTCGCCGTC-3′; H62N, 5′-CTGCCCTGGAACCGCAGATTTC-3′; H189Q, 5′-GTCAATCTGCAGAACCGGGTG-3′; H193Q, 5′-CCGGGTGCAGGTCTGGGTCGG-3′; and H215Q, 5′-GGCTGCACCAGGCCTACATCG-3′. The 1.18-kilobase SstI-PvuII fragment containing each of the melC2 mutations on pSELC2 derivatives was isolated and used to replace the corresponding segment on pIJ702-117. The derivatives produced were designated pIJ702S-H37Q, pIJ702S-H53Q, pIJ702S-H62N, pIJ702S-H189Q, pIJ702S-H193Q, and pIJ702S-H215Q, respectively. S. lividans TK64 harboring plasmid pIJ702-117 or its mutant derivatives were cultured in TSB medium (Difco) (50-ml culture) in the absence or presence of copper ion (100 μm) for 24 h at 30 °C. Preparation of mycelial extracts and culture supernatants, the assay of tyrosinase activity, and the detection of MelC1 and MelC2 proteins by immunoblot were described previously (18Leu W,-M. Chen L.-Y. Liaw L.-L. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20108-20113Abstract Full Text PDF PubMed Google Scholar). The immunoblot was detected by the enhanced chemiluminescence method (ECL system, Amersham Pharmacia Biotech) using the horseradish peroxidase-conjugated antibody as the secondary antibody (23Liaw L.-L. Lee Y.-H.W. Biochem. Biophys. Res. Commun. 1995; 214: 447-453Crossref PubMed Scopus (7) Google Scholar). The protein contents of the samples were determined by the Bradford method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar) using bovine serum albumin as the standard. The immunochromatography of the MelC1·MelC2 complex has been described elsewhere (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar, 18Leu W,-M. Chen L.-Y. Liaw L.-L. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20108-20113Abstract Full Text PDF PubMed Google Scholar). Briefly, the culture supernatants (1 ml) were incubated with anti-MelC1 antibody resins (volume 150 μl), which had been pre-equilibrated with buffer A (0.1 n sodium phosphate buffer (pH 7.2), 0.25 n NaCl). After extensive washing with buffer A, protein was eluted with 0.1 mglycine-HCl buffer (pH 2.8). The collected fractions (volume, 200 μl) were immunoblotted with anti-MelC1 or anti-MelC2 antiserum. The MelC1·MelC2 complex and tyrosinase were purified from the culture supernatants as described previously (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar), and their copper contents were analyzed by using a polarized Zeeman effect atomic absorption spectrometer (Hitachi model Z-8200) (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar). The detection sensitivity for the copper ion was in the range of 0.5–40 parts/billion. The emission spectra of the purified MelC1·MelC2 complex (10 μg/ml) and its copper-activated form were measured at room temperature in a fluorometer (Hitachi model F-4010) with an excitation wavelength of 280 nm (excitation bandpass, 3 nm; emission bandpass, 10 nm). All far-UV spectra were collected on a AVIV 60DS spectropolarimeter (AVIV Associates, Inc., Lakewood, NJ) in a 1-cm light path cell at 20 °C. Data were collected at a protein concentration of 1–2 μm. All protein samples were dialyzed against 5 mm sodium phosphate buffer (pH 7.2). Mean residue ellipticity, [θ]MRW(degree cm2 dmol−1), was determined from the formula [θ]MRW = ε/(10Cnl), where ε is the measured ellipticity in millidegrees, C is the protein concentration in mol/liter, l is the path length of the cuvette in cm, and n is the number of amino acid residues in the protein (29Schmid F.X. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford1990: 251-285Google Scholar, 30Woody R.W. Methods Enzymol. 1995; 246: 34-71Crossref PubMed Scopus (707) Google Scholar). According to previous reports (4Huber M. Hintermann G. Lerch K. Biochemistry. 1985; 24: 6038-6044Crossref PubMed Scopus (90) Google Scholar, 5Huber M. Lerch K. Biochemistry. 1988; 27: 5610-5615Crossref PubMed Scopus (42) Google Scholar, 31Jackman M.P. Hajnal A. Lerch K. Biochem. J. 1991; 274: 707-713Crossref PubMed Scopus (51) Google Scholar), the S. glaucescens tyrosinase contains 2 atoms of copper, CuAand CuB. His37, His53, and His62 are assigned to be the copper ligands for CuA, whereas His189, His193, and His215 are for the CuB site (4Huber M. Hintermann G. Lerch K. Biochemistry. 1985; 24: 6038-6044Crossref PubMed Scopus (90) Google Scholar, 5Huber M. Lerch K. Biochemistry. 1988; 27: 5610-5615Crossref PubMed Scopus (42) Google Scholar, 31Jackman M.P. Hajnal A. Lerch K. Biochem. J. 1991; 274: 707-713Crossref PubMed Scopus (51) Google Scholar). Because these copper ligands are also conserved in the S. antibioticus tyrosinase (Ref. 3Bernan V. Filpula D. Herber W. Bibb M. Katz E. Gene ( Amst. ). 1985; 37: 101-110Crossref PubMed Scopus (120) Google Scholar and Fig. 1), they may serve identical functions. In this study, we substituted each of these six histidine residues in MelC2 of the S. antibioticus melC operon with a noncoordinating glutamine or asparagine residue using site-directed mutagenesis. Six such mutations were obtained: 1) His37 to Gln37 (mutant H37Q), 2) His53 to Gln53 (mutant H53Q), 3) His62 to Asn62 (mutant H62N), 4) His189 to Gln189 (mutant H189Q), 5) His193 to Gln193 (mutant H193Q), and 6) His215 to Gln215 (mutant H215Q). When examined for melanin production on R2YE agar plates (containing 0.05% tyrosine), all mutants displayed Mel− phenotype and showed no detectable tyrosinase activities (data not shown). The loss of tyrosinase activity in these mutant strains was not because of the reduction of intracellular MelC2 protein under two different culturing conditions (with or without 100 μm copper ion) (Fig. 2, A and B). On the contrary, an increase of intracellular MelC2 protein (106–160% of wild type) was observed in all mutants except H53Q, where a slight reduction of MelC2 (78% of wild type) was observed when culturing in the absence of copper supplement (Fig. 2 A). Intriguingly, unlike MelC2, a completely different MelC1 expression pattern for the wild type and mutant strains was noted, depending on the culture condition. When cultured in supplemental copper ion (100 μm), both the intra- and extracellular levels of MelC1 in the wild type strain were markedly decreased as compared with those without copper ion supplement (compare lane WT in Fig. 2 B with the same lane in Fig. 2 A). However, this is not the case for the mutant strains; their MelC1 levels for both cellular fractions under the copper ion supplement conditions accounted for a 5–17-fold increase over that of wild type, although in some cases such as those of the mutants H53Q, H62N, H189Q, and H193Q, their MelC1 levels in the absence of copper ion adversely decreased to 35–63% of the wild type (Fig. 2 A). The reduction of MelC1 in the wild type strain presumably resulted from the aggregation of MelC1 after released from the binary complex by the copper ion (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar). The differential copper effect on the MelC1 levels in mutants as compared with the wild type may be indicative of a defect in their copper-activated complex. Moreover, the presence of multiple MelC1 species (14–15-kDa) in the intracellular fractions of the wild type and mutant strains (Fig. 2, A and B) might reflect the degradation or the different conformations of this intracellular protein as noted before (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar, 18Leu W,-M. Chen L.-Y. Liaw L.-L. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20108-20113Abstract Full Text PDF PubMed Google Scholar, 19Chen L.-Y. Chen M.-Y. Leu W.-M., T.-Y. Tsai T.-Y. Lee Y.-H.W. J. Biol. Chem. 1993; 268: 18710-18716Abstract Full Text PDF PubMed Google Scholar). Additionally, quantitation of MelC1 and MelC2 exported by immunoblot suggested that in the wild type strain, approximately 56–73% of total cellular MelC1 and MelC2 were secreted to the medium, whereas in some mutants, especially mutants H37Q, H53Q, H62N, and H189Q, the secretion of MelC1 or MelC2 protein decreased to 60–77% of the wild type (Fig. 2,A and B). Thus, along with a block of tyrosinase activity, mutation of the copper ligands also elicited a moderate effect on the export of MelC1 or MelC2 protein. Our previous work (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar) indicated that MelC1 forms a transient complex with apotyrosinase during the copper activation process and is discharged from the complex after the activation of tyrosinase. Because the copper ligand-defective mutants had lost their tyrosinase activity, it was likely that these mutations might have affected the binary complex formation. Analysis of the MelC1·MelC2 complex formation in an anti-MelC1 antibody column showed that the MelC1 and MelC2 proteins from all the mutants cultured without copper ion supplement formed a complex like the wild type (Fig. 3, C and D, lanes marked with −). Nevertheless, when cultured in the presence of supplemented copper ion (100 μm), the MelC2 protein of all the mutant strains, unlike the wild type, was still retained by an anti-MelC1 antibody column (Fig. 3, C and D, lanes marked with +), suggesting that the mutant form of the binary complex was hardly dissociated by the added copper ion. The lack of retention of wild type MelC2 by the anti-MelC1 antibody column was not because of the lower quantity or absence of MelC2 after copper ion activation, because essentially the same amount of MelC2 was present in the loading or flow-through fraction from the wild type and mutant strains (Fig. 3, A and B). It was more likely because of the fact that MelC2 from the wild type was dissociated from the binary complex after copper activation (17Chen L.-Y. Leu W.-M. Wang K.-T. Lee Y.-H.W. J. Biol. Chem. 1992; 267: 20100-20107Abstract Full Text PDF PubMed Google Scholar). This conclusion was further supported by fast protein liquid chromatography (FPLC) 1The abbreviations used are: FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis. analysis of the in vitro copper-activated binary complex (Fig. 4). Although the purified wild type binary complex (retention time, 34.9 min) displayed a discharge of MelC2 (retention time, 36.6 min) from the complex after copper ion addition, no such phenomenon was observed in the complexes derived from the mutants. Apart from mutant H193Q, the retention time of all mutant binary complexes was identical to that of the wild type (34. 9 min) and remained unchanged after copper addition. The retention time (36.3 min) for the H193Q binary complex was also independent of copper ion; however, its value was closer to that of MelC2 (Fig. 4, panel H193Q). This abnormal behavior of the H193Q binary complex was not because of the discharge of MelC2 from the complex (immunoblot analysis not shown) but rather implied a substantial change of this particular complex conformation. Additionally, SDS-PAGE and immunoblot analysis showed that afterin vitro copper activation, the wild type MelC1 markedly decreased as compared with that without copper activation, whereas the MelC1 of all the mutant forms remained the same (Fig. 5). Notably, this differential copper effect on the MelC1 of the wild type and mutants was in accord with thein vivo data (Fig. 2). Taken together, both in vivo and in vitro experiments strongly suggested that the copper activation process was defective in these copper ligand-defective mutants in such a way that the mutant MelC1 or MelC2 apparently could not be released from the complex after copper activation. The failure to resolve the MelC2 from the copper-activated complexes of the mutants may be because of the mutational influence of the copper incorporation into the complexes. To assess this possibility, the copper contents of the purified binary complexes and their in vitrocopper-activated species were examined. The purified complexes of the wild type and mutant strains contained essentially no copper ion (less than 0.09 atom/molecule) (Table I).In vitro activation led to the incorporation of approximately 2 atoms of copper/molecule of the complex in wild type, H189Q, and H215Q but only 1 atom of copper in the other four mutants. Therefore, whereas mutation at each of copper ligands in CuA site expectedly blocked copper incorporation into the CuA site, mutation at His189 and His215 in the CuB site did not affect the copper incorporation.Table ICopper content of the purified MelC1·MelC2 complex and its in vitro copper-activated products from wild type and copper ligand-defective mutant strainsSampleProtein concentrationCopper concentrationCopper contentnmatom/molecule proteinTyrosinase2551.3 ± 6.32.05 ± 0.25Wild type352.0 ± 1.40.06 ± 0.04H37Q12011.3 ± 0.60.09 ± 0.01H53Q602.5 ± 0.30.04 ± 0.01H62N1209.3 ± 2.80.08 ± 0.02H189Q600.63 ± 0.00.01 ± 0.00H193Q1208.19 ± 0.50.07 ± 0.00H215Q12016.1 ± 0.50.13 ± 0.00Wild type + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.3581.4 ± 8.72.33 ± 0.25H37Q + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.120110.2 ± 3.80.92 ± 0.03H53Q + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.6065.5 ± 5.21.09 ± 0.09H62N + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.120163.1 ± 5.41.36 ± 0.05H189Q + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.60136.2 ± 15.42.27 ± 0.26H193Q + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.120109.8 ± 1.10.92 ± 0.01H215Q + CuaThe binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water.120283.1 ± 7.72.36 ± 0.06The preparation and determination of the copper contents in the proteins are described under “Experimental Procedures.” All designations for mutant strains are identical to those described in the legend of Fig. 2. The data shown in this table represent the means ± S.D. of two to three determinations.a The binary complex was incubated in 100 μm copper sulfate at 4 °C for 24 h, and the resulting mixture was dialyzed extensively against the distilled water. Open table in a new tab The preparation and determination of the copper contents in the proteins are described under “Experimental Procedures.” All designations for mutant strains are identical to those described in the legend of Fig. 2. The data shown in this table represent the means ± S.D. of two to three determinations. Because the binary complexes of all the mutants had copper ion(s) incorporation, their defects in the discharge of MelC2 after copper activation might instead result from the incompetent conformational change during copper activation. To examine this possibility, intrinsic fluorescence spectroscopy in combination with CD spectroscopy was used to probe conformational changes of the binary complexes from the wild type and mutant strains. The MelC1·MelC2 binary complex contains 37 aromatic amino acid residues, of which 30 are in the MelC2 protein (tryptophan 12, tyrosine 6, and phenylalanine 12). Approximately one-third of the aromatic amino acid residues (tryptophan 5, tyrosine 2, and phenylalanine 6) are located around the binuclear copper sites in MelC2 (Fig. 1). We envisioned that the conformational change elicited either by copper ion incorporation or by the mutational effect might be revealed by the changes in the intrinsic fluorescence emission spectra. Fig. 6 B showed that the intrinsic fluorescence emission intensity (excitation at 280 nm, maximum emission at 337–339 nm) of the wild type binary complex was quenched by 30% as a result of copper insertion. A similar finding was reported for the tyrosinase of Neurospora, for which an approximately 60% quenching was found upon copper insertion (32Beltramin M. Lerch K. Biochem. J. 1982; 205: 173-180Crossref PubMed Scopus (19) Google Scholar). Notably, the complexes from all the mutants except H53Q were similar quenching ranging from 35 to 48% after copper insertion (Fig. 6 B). In contrast, a 1.5-fold enhancement of the fluorescence emission intensity was observed in the H53Q complex after copper activation. This indicated that the coordinated environment of the copper center in all the mutants, except H53Q, is similar to that in the wild type during copper insertion. However, none of intrinsic fluorescence emission intensity of the apoform of the mutant binary complex is identical to that of the wild type (Fig. 6 A). All their fluorescence intensities were enhanced (1.1–1.6-fold of the wild type) and had a maximum enhancement in mutants H62N and H193Q, suggesting that the native conformation of the mutant binary complex is perturbed by mutation. Thus, the fluorescence spectroscopy study suggested that although copper insertion into the mutant binary complex yielded conformational changes, the conformations of the apoforms from these mutant complexes were distinct from that of the wild type, which might result in a defect in the copper activation process. To ascertain whether the defect in copper activation of these mutants was because of gross structural perturbations, CD experiments were carried out. Fig. 7 showed the CD spectra for the mutants in the presence and absence of copper ion, which are superimposed with the spectra of the wild type binary complex in Fig. 8. The far-UV spectra (200–250 nm) of the wild type apoform was characterized by two minima with negative ellipticity near 208 and 230 nm, suggesting the presence of helical and other nonhelical structures including β-turn and random coil (29Schmid F.X. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford1990: 251" @default.
• W2009486598 created "2016-06-24" @default.
• W2009486598 creator A5047359756 @default.
• W2009486598 creator A5067104757 @default.
• W2009486598 date "1998-07-01" @default.
• W2009486598 modified "2023-10-03" @default.
• W2009486598 title "Roles of Copper Ligands in the Activation and Secretion ofStreptomyces Tyrosinase" @default.
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