From the Institute of Biochemistry, National Yang-Ming University,
Taipei 112, Taiwan, Republic of China
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INTRODUCTION |
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 (1,
2). This enzyme is ubiquitous and is responsible for the biosynthesis
of melanin pigment from tyrosine (2). The primary structures of tyrosinase from Streptomyces (3-5), Neurospora
crassa (6), Rana nigromaculata (7), Mus
musculus (8, 9), and Homo sapiens (10, 11) 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. 2 and 12). Apart from the
Streptomyces tyrosinase, the mechanisms by which copper ions
incorporated into the various sources of apotyrosinase are largely
unknown (13).
The structural gene (melC2) for the tyrosinase of
Streptomyces antibioticus (14) or Streptomyces
glaucescens (15) is part of a polycistronic operon
(melC), preceded by the melC1 gene, which encodes
a conserved protein essential for the expression of melanin in
Streptomyces (3, 14, 16). 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 (16-19). Evidence
was also provided that indicated a conformational transition of MelC2
during the copper activation (19). This function of MelC1 is
reminiscent of that of the molecular chaperone involved in protein
folding, assembly, secretion, and heat shock responses (20-22).
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 (18, 19, 23). 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 the
Streptomyces 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.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, and Culturing
Conditions--
Streptomyces lividans TK64
(SLP2
, SLP3
, pro-2, str-6) (24)
was used as the host for recombinant plasmids. Streptomyces
plasmid pIJ702 (14) 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 (25). Standard media,
culture conditions, and transformation procedures for
Streptomyces were previously described (26).
Plasmid Construction and in Vitro Mutagenesis--
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-kilobase
SstI-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 (27): 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'.
Construction of pIJ702-117 Mutant Derivatives--
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.
Assay of Tyrosinase and Immunoblotting Analysis--
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 (18). The immunoblot
was detected by the enhanced chemiluminescence method (ECL system,
Amersham Pharmacia Biotech) using the horseradish peroxidase-conjugated
antibody as the secondary antibody (23). The protein contents of the
samples were determined by the Bradford method (28) using bovine serum
albumin as the standard.
Immunoaffinity Chromatography of the MelC1·MelC2
Complex--
The immunochromatography of the MelC1·MelC2 complex has
been described elsewhere (17, 18). 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 M
glycine-HCl buffer (pH 2.8). The collected fractions (volume, 200 µl)
were immunoblotted with anti-MelC1 or anti-MelC2 antiserum.
Analysis of Copper Content--
The MelC1·MelC2 complex and
tyrosinase were purified from the culture supernatants as described
previously (17), and their copper contents were analyzed by using a
polarized Zeeman effect atomic absorption spectrometer (Hitachi model
Z-8200) (17). The detection sensitivity for the copper ion was in the
range of 0.5-40 parts/billion.
Fluorescence Spectroscopy--
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).
Circular Dichroism Spectroscopy--
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, [
]MRW
(degree cm2 dmol
1), was determined from the
formula [
]MRW =
/(10Cnl), where
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 (29, 30).
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RESULTS |
Alteration of the MelC2 Copper Ligands Blocked the Phenotypic
Melanin Formation but Moderately Affected the Export of MelC1 or
MelC2--
According to previous reports (4, 5, 31), the S. glaucescens tyrosinase contains 2 atoms of copper, CuA
and CuB. His37, His53, and
His62 are assigned to be the copper ligands for
CuA, whereas His189, His193, and
His215 are for the CuB site (4, 5, 31). Because
these copper ligands are also conserved in the S. antibioticus tyrosinase (Ref. 3 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).

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Fig. 1.
Amino acid sequence comparison of tyrosinases
copper centers. The amino acid sequences surrounding the copper
centers CuA (A) and CuB
(B) of the tyrosinases of S. antibioticus
(S. a.) (3), S. glaucescens (S. g.)
(4), frog (7), mouse (8), human (11), and N. crassa
(N. c.) (6) and hemocyanin of P. interruptus
(P. i.) (33) are aligned. Isofunctional residues are
shaded (T and S; S and A; D and E; K, R, and H; Y, F, and W;
and I, V, L, and M). The asterisks denote histidyl residues
that have been proposed to be ligands of the copper ions. The numbers
indicate the amino acid position. A special numbering scheme is used
for hemocyanin (33).
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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. 2A). 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.
2B with the same lane in Fig. 2A). 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. 2A). 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 (17).
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 (17-19). 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.

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Fig. 2.
Expression of MelC1 and MelC2 proteins in
Streptomyces tyrosinase copper ligand-defective
mutants. Streptomyces cultures were grown in TSB medium with
(B) or without (A) supplemented copper ion (100 µM) for 24 h at 30 °C. The intracellular
fractions were prepared by sonication. Extracellular proteins in the
culture medium were precipitated by 5 volumes of cold acetone, and the
recovered pellets were dissolved in the sampling buffer (39). Both
fractions were analyzed in 13.5% PAGE containing 0.1% SDS and
immunoblotted with both anti-MelC1 and/or anti-MelC2 antisera. Protein
from 25 µl of cultures was applied to each lane. The positions for
the MelC1 (C1) and MelC2 (C2) proteins are
indicated with arrows. The band intensity (arbitrary units)
of each MelC1 and MelC2 bands were determined densitometrically
(Molecular Dynamic) and normalized to the wild type level. The
percentages of secreted MelC1 and MelC2 are also indicated.
WT, wild type, TK64 (pIJ702-117); H37Q, TK64 (pIJ702S-H37Q);
H53Q, TK64 (pIJ702S-H53Q); H62N, TK64 (pIJ702S-H62N); H189Q, TK64
(pIJ702S-H189Q); H193Q, TK64 (pIJ702S-H193Q); and H215Q, TK64
(pIJ702S-H215Q).
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Effects of Copper Ligand Mutations on the Activation of
MelC1·MelC2 Complex--
Our previous work (17) 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 (17).

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Fig. 3.
Analysis of the MelC1·MelC2 complex
formation by immunoaffinity chromatography. The culture
supernatant (1 ml) of each culture grown either in TSB medium with (+)
or without ( ) supplemented copper ion (100 µM) were
incubated with anti-MelC1 antibody resins (150 µl) as described under
"Experimental Procedures." The loaded samples (A), the
flow-through (B), and the bound fractions (C and
D) were analyzed by SDS-PAGE and immunoblotted with
anti-MelC1 and/or anti-MelC2 antisera. For comparison, the same amounts
of protein were applied to each lane of the same panel. All
designations for mutant strains are identical to those described in the
legend of Fig. 2. The positions for the MelC1 (C1) and MelC2
(C2) proteins are indicated. The presence of dimeric form of
MelC1 in the wild type sample (marked with (C1)2
in lane WT of C) is consistent with previous
results (17).
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This conclusion was further supported by fast protein liquid
chromatography (FPLC)1
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.

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Fig. 4.
Analysis of the in vitro
copper-activated MelC1·MelC2 complex by FPLC gel filtration
column. Purified MelC1·MelC2 complex (10 µg) from each culture
supernatant was incubated in 20 mM Tris-HCl buffer (pH 7.2)
(buffer B) without ( ) or with (+) 100 µM copper sulfate
for 24 h at 25 °C. Portions (5 µg) of the samples were
chromatographed in a FPLC gel filtration column (Superose HR-12; size,
25 ml), which had been pre-equilibrated with buffer B containing 0.5 N NaCl, and the absorbance profile at 280 nm in the eluates
was monitored. The retention time for MelC1·MelC2 complex or MelC2,
if applicable, is indicated. All designations for mutant strains are
identical to those described under the legend of Fig. 2. The relative
retention times for several standard proteins in the same column were
as follows: bovine serum albumin, 31.3 min; ovalbumin, 33.1 min;
chymotrypsin, 37.3 min; RNase A, 38.3 min. WT, wild
type.
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Additionally, SDS-PAGE and immunoblot analysis showed that after
in 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 the
in 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.

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Fig. 5.
Analysis of the in vitro
copper-activated MelC1·MelC2 complex by SDS-PAGE and
immunoblot. Purified MelC1·MelC2 complex (10 µg) from each
culture supernatant was incubated in buffer B without ( ) or with (+)
100 µM copper sulfate for 24 h at 25 °C. The
proteins were separated for SDS-PAGE, analyzed by Coomassie Brilliant
Blue staining (A), and immunoblotted with anti-MelC2
(B) and/or anti-MelC1 antisera (C). The loaded
protein is 5 µg/sample in A and 0.5 µg/sample in
B and C. All designations for mutant strains are
identical to those described in the legend of Fig. 2. The positions for
the MelC1 (C1) and MelC2 (C2) proteins are
indicated. The presence of dimeric form of MelC1(marked with
(C1)2) in the wild type (WT), H37Q,
and H62N samples was noted here.
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Analysis of the Copper Contents in the Binary Complexes of the
Copper Ligand-defective Mutants--
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 vitro
copper-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.
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Table I
Copper content of the purified MelC1·MelC2 complex and its in vitro
copper-activated products from wild type and copper ligand-defective
mutant strains
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.
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Analysis of the Copper-induced Conformational Change in the Binary
Complexes of Mutant Strains--
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.
6B 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 (32). Notably, the complexes
from all the mutants except H53Q were similar quenching ranging from 35 to 48% after copper insertion (Fig. 6B). 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. 6A). 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.

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Fig. 6.
Intrinsic fluorescence spectra of
MelC1·MelC2 binary complexes from different strains. Samples of
purified MelC1·MelC2 binary complex (10 µg/ml each) from each
culture supernatant of different sources were incubated with or without
100 µM copper sulfate for 3 h at 25 °C and then
dialyzed against buffer B overnight. The resulting samples were
analyzed for their emission spectra on a fluorometer (Hitach model
F-4010) with excitation wavelength at 280 nm (see "Experimental
Procedures"). All spectra were normalized to the same protein
concentration. A, comparison of the intrinsic fluorescence
spectra of the apoform binary complexes from various sources.
B, comparison of the intrinsic fluorescence spectra of the
apoform and copper-activated form of binary complexes from various
sources. All designations for mutant strains are identical to those
described under the legend of Fig. 2. WT, wild type.
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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
-turn and random coil (29, 30)
(Fig. 7, panel WT). Apart from the intensity differences,
these features were also present in the spectra of all the mutant
apoforms. However, the CD spectra of H37Q and H193Q apoforms showed
significant deviation from that of the wild type (Fig. 8, A
and C), indicating minor secondary structural changes in
these two mutants. Addition of copper ion to the apoform of the wild
type binary complex led to a small, discernible decrease in the
negative ellipticity near 208-225 nm (Fig. 7, panel WT),
reflecting a decrease of helical content in the binary complex with a
concomitant increase of other structures after copper activation (29,
30). When compared with the wild type, the mutants were less likely to
show copper-induced spectral changes. This is especially the case for
mutants H37Q, H62N, H189Q, and H215Q (Fig. 7). Only the mutant complex
of H53Q or H193Q exhibited a significant copper-induced CD spectral
change near the 220-235 nm (H53Q) or 200-220 nm (H193Q) region (Fig.
7). Furthermore, when compared with the apoform, the CD spectra of all
the copper-activated mutant binary complexes differed considerably from
that of the wild type (Fig. 8, compare panels B and
D with panels A and C), suggesting
that none of the copper-activated forms of mutant strains displayed the
same conformational state as the wild type. Therefore, our results
showed that although copper insertion into the mutant form of the
binary complex might induce a structural change in certain mutants,
these changes appeared dissimilar to those in the wild type. This
incompetent binary complex might lead to the failure of the resolution
of MelC2 from the copper-activated complex and of the production of
active tyrosinase.

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Fig. 7.
CD spectral change of MelC1·MelC2 complex
during copper activation. Purified MelC1·MelC2 binary complex
(1-2 µM) from each culture supernatant was incubated
with or without 100 µM copper sulfate for 24 h at
4 °C and then dialyzed against 5 mM phosphate buffer (pH
7.2) overnight. The resulting samples were analyzed for their far-UV CD
spectra (see "Experimental Procedures"). Mean residue ellipticities
([ ]MRW) were plotted as a function of the wavelength
(nm). All designations for mutant strains are identical to those
described in the legend of Fig. 2. WT, wild type.
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Fig. 8.
Comparison of the CD spectra of the apoform
and copper-activated form of MelC1·MelC2 complexes from different
strains. The CD spectra of the apoform (A and
C) or copper-activated form (B and D)
of CuA- or CuB-defective mutants were compared
with those of wild type (WT). All data are from Fig. 7. All
symbols are identical to those described in the legend of Fig. 7.
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DISCUSSION |
Tyrosinases are evolutionally diverse enzymes, yet all of them
possess a binuclear copper center consisting of six essential copper
ligands. Of particular interest is the degree of equivalence of these
two copper sites (CuA and CuB) and the
mechanism of copper incorporation into this enzyme. Much of the
available information on copper incorporation into tyrosinase has come
from the studies of the Streptomyces system, the smallest
representative of this class of enzyme known so far (13). In the
present working model for the activation of Streptomyces
tyrosinase, MelC1 functions as a chaperone that maintains the state of
apotyrosinase that facilitates copper incorporation and secretion
(17-19). Earlier works have shown that the alterations of either the
signal peptide or several particular histidine residues
(e.g. 102 and 117) of MelC1 affected the stability of the
MelC1·MelC2 binary complex, resulting in a loss of tyrosinase
activity or its secretion (18, 19, 23). Although these studies have
delineated the role of the MelC1 in activation and secretion of
tyrosinase, the relative importance of MelC2 in these two aspects
remains uncharacterized.
In this study, we examined the role of copper ligands of MelC2 in the
formation of the functional binary complex by introducing a single
substitution mutation into each of six putative copper ligands in the
CuA or CuB site of S. antibioticus
tyrosinase (Fig. 1). All the mutants displayed Mel
phenotypes and had lost their tyrosinase activity. This result strongly
suggests that these conserved histidine residues serve the same
copper-binding function in S. antibioticus tyrosinase as in
S. glaucescens tyrosinase. In fact, analysis of the copper contents of all copper ligand-defective mutants from these two tyrosinases supports this view. The tyrosinases from
His189- and His215-defective mutants of
S. glaucescens and S. antibioticus all contain 2 copper atoms, whereas their CuA site and His193
mutations result in the loss of 1 copper atom (Ref. 31 and this work).
This also implies that the lability of copper-histidine prosthetic
group in the two copper sites of tyrosinase are different from one
another. Conceivably, the two copper sites are structurally and
functionally nonequivalent. Along this line, there are several other
clues to support this view (also see Ref. 13). A comparison of amino
acid sequences of tyrosinases from various sources, including Streptomyces (3, 4), Neurospora (6), R. nigromaculata (7), and mammals (8, 11) with a related, copper
protein hemocyanin (33), shows strong sequence homology in regions
containing the ligands of the CuB site but weaker homology
in regions containing the CuA site (Fig. 1). In addition,
the differential reactivity of two copper sites in mushroom tyrosinase
with citrate, oxalate, or copper ion is in agreement with this notion
(34-36). Finally, our observation of the differential impairment on
the function or property (e.g. copper content, stability,
secretion, or conformational status) of certain mutant binary complexes
(e.g. H37Q, H53Q, and H193Q) can be ascribed to differential
importance of copper ligands in maintaining the structural or
functional integrity of the two copper sites.
If the two copper sites of Streptomyces tyrosinase are
distinct from each other as inferred from our present study, such a feature of distinction may be also applicable to its copper activation process. If this is the case, it is pertinent to know whether the
insertion of a copper ion into only one site (presumably the higher
affinity one) is sufficient in eliciting a discharge of the MelC2 from
the complex. This working model for activation of apotyrosinase
proposes the presence of an MelC2 intermediate harboring only 1 atom of
copper. However, our present study argues against this view. Although
the introduced mutations in the CuA or CuB site
resulted in differential levels of copper content (Table I), all
mutants could not discharge MelC2 from their copper-activated complexes
(Figs. 3-5), suggesting that neither one of the copper ion insertions
alone can elicit a resolution of the MelC1·MelC2 binary complex and
that insertion of two copper ions is necessary but not sufficient for
the activation of tyrosinase. The defects of discharge of the mutant
MelC2 from its binary complex appear to result from the alterations of
the conformation of the binary complex, as evidenced by the changes in
their fluorescence emission or CD spectra (Figs. 6-8). Therefore, an
integrity of both copper centers is a determinant for formation and
discharge of active tyrosinase. Although the two copper sites may have
inherent structural and functional distinction, their roles in
activation of tyrosinase are not distinguishable in this study.
Interestingly, although these mutant proteins have deleterious effects
on the copper activation process, most of their conformations are still
competent for secretion (Fig. 2). This implicates that the binary
complex conformation may exist in either activation or export
subconformation, and presumably, the distinct features of these two
subconformations are modulated by both MelC1 and MelC2 proteins, very
likely through their different domains or different amino acid
residues. Notably, our previous findings of the differential effect of
activation or export of tyrosinase in MelC1 mutants are consistent with
this notion (17, 18, 23).
Apart from the Streptomyces tyrosinase, there have been a
few studies on the copper center assembly in the tyrosinase of
Neurospora (37, 38) and mushroom (36). Involvement of copper
metallothionein in the activation of the Neurospora
tyrosinase has been suggested (37, 38). In mushroom, the differential
kinetics for the incorporation of 2 copper atoms to the apotyrosinase
has been found (36). The requirement of a chaperone protein MelC1 for
the activation of Streptomyces tyrosinase raises the
question as to whether this is also applicable to other tyrosinases.
However, in view of the facts that 1) no MelC1 homologs have been found
for other tyrosinase systems; 2) the apotyrosinase of
Neurospora or mushroom can be readily reconstituted with
copper ion in vitro without the presence of a chaperone
protein (13, 38); and 3) the Streptomyces tyrosinase is the
smallest representative of this class known so far, it is therefore
likely that a MelC1-like chaperone function resides within the
eukaryotic tyrosinase. In other words, a portion of the apoprotein
sequence of these tyrosinases may perform similar functions as MelC1
for their copper center assembly. The fact that MelC1 can function as a
cis-chaperone for activation of the apotyrosinase of
Streptomyces2
supports this view. Therefore, the picture of the metallocenter assembly of tyrosinases from higher organisms may emerge through the
identification of the protein moiety important for the copper incorporation into the active site of their respective
apotyrosinases.
We are indebted to Dr. L. Kim for the
measurement and data analysis of CD spectra and to Dr. T.-C. Hung and
Y.-S. Tseng for the copper content analysis. We also thank Drs. C. W. Chen, D. J. Cork, and C. Weaver for critical reading of the
manuscript.