A Molecular Mechanism for Copper Transportation to Tyrosinase That Is Assisted by a Metallochaperone, Caddie Protein*

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.

Among the many biological systems that employ transition metal ions for their function, it is a requirement that specific metal co-factors be transported into the correct metalloenzyme. Proteins known as "metallochaperones" play key roles in this process. Although a few structures of the metallochaperone have now been determined (1)(2)(3)(4)(5)(6), the molecular mechanisms underlying specific metal transfers remain unclear with the exception of the metallochaperone involved in the copper transport to superoxide dismutase (1).
Tyrosinase (EC 1.14.18.1) belongs to a type-3 copper protein family harboring a catalytic center formed by dinuclear copper and catalyzes the ortho-hydroxylation of phenol and the subsequent oxidation of catechol to the corresponding quinone (7). The quinone product is a reactive precursor for the synthesis of melanin pigments. In mammals, this enzyme is responsible for skin pigmentation abnormalities, such as flecks and albinism (8). The development and screening of potent inhibitors of tyrosinase is therefore of particular interest to the cosmetics industry. We have also recently reported from our laboratory that trilinolein contained in sake lees is a strong tyrosinase inhibitor (9).
Tyrosinase is classified into the same protein family as catechol oxidase and hemocyanin, although catechol oxidase lacks monooxygenase activity. Hemocyanin acts as an oxygen carrier in arthropods and mollusks. During catalysis, the type-3 copper center of tyrosinase adopts three redox forms (7). The deoxy form (Cu(I)-Cu(I)) is a reduced species, which binds dioxygen to yield the oxy form. In the oxy form, molecular oxygen binds in the form of peroxide in a -2 : 2 side-on bridging (Cu(II)-O 2 2Ϫ -Cu(II)) mode, which destabilizes the O-O bond and activates it. The met form (Cu(II)-Cu(II)) is recognized as the resting enzymatic form, where the Cu(II) ions are normally bridged with small ligands, such as water molecules or hydroxide ions. In these three redox forms of tyrosinase, the oxy form can catalyze both monooxygenase and oxidase reactions, whereas the met form lacks monooxygenase activity.
Many strains classified into the genus Streptomyces produce a melanin-like pigment (10). The melanin-synthesizing operon of Streptomyces antibioticus is composed of two genes that encode MelC1 and MelC2 proteins (11). It has been demonstrated that apotyrosinase (MelC2) forms a stable complex with MelC1 (12). Although apotyrosinase is not activated by copper added from the outside, the addition of copper ions to the purified complex gives rise to the incorporation of two copper ions. Furthermore, during the in vitro activation of the MelC1-MelC2 complex, Cu(II)-bound MelC2 is discharged from the complex, but no trace of the released MelC1 protein is detectable. This suggests that the released MelC1 protein might form an aggregate to enable its separation from the protein complex. It is of interest to understand the molecular mechanism underlying the transactivation processes between MelC1 and MelC2.
We previously cloned a melanin-synthesizing gene from the chromosomal DNA of Streptomyces castaneoglobisporus HUT 6202 that produces a melanin pigment in high amounts. This gene forms an operon consisting of two cistrons (13), one being an open reading frame (ORF) consisting of 378 nucleotides designated orf378 and the other a tyrosinase-encoding gene, designated tyrC, which is located just downstream of orf378. We refer to ORF378 as "caddie" because this protein may carry copper ions for tyrosinase.
We have succeeded in determining the three-dimensional structure of S. castaneoglobisporus tyrosinase in complex with caddie at a very high resolution (14) (Fig. 1A). This represents the first crystal structure determination of any tyrosinase from prokaryotes or eukaryotes. We obtained the met form of Cu(II)bound tyrosinase in complex with caddie by soaking the native crystals in a CuSO 4 -containing solution. At the catalytic site of tyrosinase, each of two closely spaced copper ions is surrounded by three His residues through their N⑀ nitrogen atoms, as seen in the crystal structures of other type-3 copper protein members (15)(16)(17)(18)(19). Although the active centers of type-3 family proteins are similar in both their overall structure and their ability to bind to molecular oxygen, their enzymatic functions differ. This is explained by the variation in the substrate-binding pocket and the accessibility of the substrate to the active site (7, 20 -22).
During the maturation of tyrosinase, the transfer of the two closely spaced copper ions into the catalytic center is crucially important because the electrostatic repulsion between these two ions seems to inhibit the maturation process. We speculated that the caddie protein may act as a metallochaperone to assist the introduction of two copper ions into the tyrosinase catalytic center to complete the maturation. Hence, an understanding of the mechanism of copper transportation assisted by caddie is not only of interest from the viewpoint of structural biology but would also be useful in the design of novel tyrosinase inhibitors that block the maturation of these enzymes.
In our present study, structural changes in the active center of tyrosinase were crystallographically tracked by altering the Cu(II) soaking time. In addition, by preparing crystals of tyrosinase in complex with each of four caddie mutants whose capacity for the uptake of Cu(II) into the catalytic center is hindered, we were able to determine the copper-binding properties of these mutants. Based on these crystal structures determined at high resolutions, we propose a Cu(II)-transportation mechanism that is assisted by the tyrosinase-specific caddie protein.

EXPERIMENTAL PROCEDURES
Bacterial Strains-Escherichia coli DH5␣ and BL21(DE3)-pLysS strains were used as hosts for cloning and protein expression, respectively.
Mutagenesis-The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate caddie mutants. The PCR primers used containing the desired single mutations (underlined) were as follows (sense only): 5Ј-CGGCGTGCAGCTG-CAGGTGATGCGCAAC-3Ј (H82Q), 5Ј-GCAGCTGCACGTG-CTGCGCAACGCCG-3Ј (M84L), 5Ј-GCGTCGTCAGCCAG-TACGACCCGGTGC-3Ј (H97Q), and 5Ј-GCGTCGTCAGCCA-CTTTGACCCGGTGCCC-3Ј (Y98F). The pET-orf378 plasmid (23) used for the expression of His 6 -tagged caddie, was amplified using sense and antisense primers. The original plasmid was removed by DpnI digestion, and the mutated plasmid was then amplified in E. coli. By using the mutated plasmid as a template, the region containing the T7 promoter and mutated orf378 gene was amplified with the forward primer, 5Ј-GCAC-GCATGCGAAATTAATACGACTC AC-3Ј, and the reverse primer, 5Ј-CTATGCATGCCAAAAAACCCCTCAAGAC-3Ј (the underlines in each case indicate the SphI site). The amplified fragment was digested with SphI and inserted into the same site in pET-tyrC (23), a plasmid used for the expression of His 6tagged tyrosinase, to generate a construct that coexpresses tyrosinase and a caddie mutant. A plasmid in which the direction of the tyrosinase and caddie genes is in the opposite orientation was chosen. The introduction of the mutation was confirmed by DNA sequencing analysis.
Preparation of the Complex-The overproduction and purification of tyrosinase complexed with wild type caddie or a caddie mutant was performed as described previously (23).
HPLC Analysis-Purified complexes (10 M) were incubated in a 20 mM Tris-HCl buffer (pH 7.8) supplemented with 0.2 M NaCl and 50 M CuSO 4 . After the aggregates generated in the samples were removed by centrifugation, the resulting supernatant fluid was applied to HPLC using Superdex 200 10/300 GL (GE Healthcare) equilibrated with a 20 mM Tris-HCl buffer (pH 7.8) containing 0.2 M NaCl. The flow rate was set to 0.75 ml/min, and the elution profile was monitored at 280 nm.
Kinetic Analysis-The purified complexes (10 nM) were preincubated at 30°C in a 10 mM sodium phosphate buffer (pH 4, 6, or 8) containing the given concentrations of CuSO 4 . At specific times after a 1-ml portion of the solution was mixed with the same volume of a 100 mM sodium phosphate buffer (pH 6.28, A, overall structure. The tyrosinase and caddie proteins are shown in orange and cyan, respectively. Copper ions identified previously are indicated by green spheres. Residues of the ligands for these copper atoms are shown in a stick model. In the Cu(II)-free complex, the tyrosinase His 54 is disordered. Apart from Cu A and Cu B in the active site, an additional copper ion was found at the Cu C -or Cu D -binding site. Between the two binding states, the conformations of the side chains of the caddie His 82 , Met 84 , and His 97 are different. A detailed structure focused on the four copper-binding sites is shown in B. The coordination bonds and hydrogen bonds are indicated by dashed lines. Carbon atoms from the residues of tyrosinase and caddie are represented by orange and cyan, respectively. Copper atoms and water molecules (or hydroxide ions) are represented by green and red spheres, respectively. 6.25, or 5.37) containing 10 mM L-3,4-dihydroxyphenylalanine (L-DOPA) in a 1.0-cm cuvette, the increasing rate of absorbance at 475 nm (⌬A 475 /min) was immediately monitored to estimate the oxidase activity (24). The pH of the mixed solution, in which the oxidase reaction progressed, was 6.2. All kinetic experiments were performed in duplicate, and reproducibility within a deviation of 0.05 ⌬A 475 /min was confirmed.
Crystallography-Crystallization was conducted using a previously described method (14). For soaking experiments, crystals of a similar size (0.3-0.4 mm in one dimension) were selected. When the crystal of tyrosinase complexed with caddie was dissolved into a solution after the long Cu(II)-soaking experiment, it was confirmed that the solution exhibited catalytic activity toward L-DOPA. The diffraction intensities of the crystal were measured using synchrotron radiation from station BL26B2, BL38B1, or BL41XU at SPring-8 (Harima, Japan). The crystal was frozen using a nitrogen gas stream (100 K) or by liquid nitrogen and mounted onto a goniometer. The diffraction of each crystal was measured with a CCD camera equipped at the station, and the intensities were integrated and scaled using the HKL2000 program (25). The existence of the copper ion was confirmed using anomalous difference Fourier maps. The model was refined by conventional restrained methods using the CNS program (26). A subset of 5% of the reflections was used to monitor the free R factor (R free ) (27). Each refinement cycle included the positional parameters, individual isotropic B-factors, a correction using the flat bulk solvent model, and the addition of solvent molecules. After the CNS refinement was converged, the model was further refined using SHELXL-97 (28). At this stage, when the resolution of the data was higher than 1.25 Å, anisotropic temperature factors were introduced for all atoms. Otherwise, anisotropic temperature factors were introduced only for all sulfur and copper atoms. The distance between the copper ion and its ligand atom was not restrained. The occupancy of the copper ions was refined while keeping the temperature factor of the ion similar to that of the surrounding atoms. The model was revised using an electron density map created using XtalView software (29). Details of the data collection and refinement statistics are provided in Table 1.

Effects of the Soaking Time on the Tyrosinase Activity Levels-
The kinetics of tyrosinase complexed with the caddie protein were found to be multifaceted because the catalytic activity levels of this enzyme depended not only on the concentration of Cu(II) but also on the incubation time with the metal ions prior to the reaction. For the enzymatic kinetic study, the complex of tyrosinase with the caddie protein was preincubated for given times with CuSO 4 . HPLC analysis showed that, by incubation with Cu(II) at pH 7.8 for 2 h, a portion of tyrosinase was dissociated from the caddie protein as an active Cu(II)-bound form ( Fig. 2A). On the other hand, it was difficult to observe the released caddie protein, probably due to the aggregation, as found in a previous study using the MelC1 protein and tyrosinase from S. antibioticus (12).
The oxidase activity of tyrosinase was measured using L-DOPA as a substrate (final concentration of 5 mM). We spec-ulated that the activity of the enzyme immediately after the addition of the substrate might reflect the levels of mature tyrosinase, which comprises the sum of the Cu(II)-bound complex and Cu(II)-bound but released tyrosinase. We first investigated the effects of the pH of an incubation buffer containing 10 M CuSO 4 on tyrosinase activity. Tyrosinase complexed with caddie showed very low activity at pH 4, and the activity reached its maximum at 10 min (Fig. 2B). On the other hand, the maximal tyrosinase activity was obtained by preincubation for 30 min at pH 6 or 8, but the maximum level at pH 6 was lower than that at pH 8. Relative to the activity of equimolar caddie-unbound tyrosinase, the maximal activities obtained at pH 6 and 8 were 40 and 90%, respectively. This indicates that the copper ions were not completely introduced into the tyrosinase active center at the low pH conditions, even after a lengthened incubation with Cu(II).
HPLC analysis revealed that most tyrosinase molecules form a complex with the caddie protein after incubation with Cu(II) for 30 min, suggesting that the aggregation of caddie proceeds very slowly after the incorporation of the copper atoms. Surprisingly, the catalytic activity was gradually reduced by incubation with Cu(II) for longer than 30 min. This reduction in activity may be caused by the aggregation of a portion of Cu(II)bound released tyrosinase together with the aggregation of caddie. In fact, although the proportion of released tyrosinase to the complexed one was time-dependently increased by a longer incubation with Cu(II), the peak corresponding to the amount of dissociated tyrosinase was lower ( Fig. 2A).
We further measured the oxidase activity of tyrosinase after the preincubation of Cu(II)-free complexes in the presence of the various concentrations of CuSO 4 every 10 min. It was found that when the concentration of Cu(II) was lower, a longer incubation time was necessary to attain the maximum activity. Fig.  2C shows the maximal activities plotted against the Cu(II) concentrations. The EC 50 values, which are defined as an effective concentration of Cu(II) producing 50% of the maximal oxidase activity, were determined to be 0.3 M at pH 6 and 0.03 M at pH 8. This also indicates that an alkaline pH is suitable for the transfer of Cu(II).
Effects of Soaking Time on the Manner of Cu(II) Binding-We have previously shown that one copper ion (designated Cu A ) in the catalytic center of tyrosinase is surrounded by His 38 , His 54 , and His 63 residues (14). Another copper (Cu B ) is surrounded by His 190 , His 194 , and His 216 residues. Fig. 3A shows the structure of the tyrosinase catalytic site obtained by soaking the crystal of the complex in a Cu(II)-containing solution for 6 months (Protein Data Bank code 2AHK). In the absence of the copper ion (Protein Data Bank code 1WXC), His 54 , which is a ligand of Cu A , is clearly observed to adopt two conformations. The side chain of His 54 in one conformation is oriented toward the Cu Abinding site, whereas the side chain in the other conformation points toward that of His 97 in caddie. In both cases, a hydrogen bond between the side chain N␦ atom of the tyrosinase His 54 and the side chain carboxyl of the tyrosinase Asp 45 is formed. When His 54 assumes the former conformation, seven water molecules may be present at the catalytic site (Fig. 3B). Four of these (Wat 4 -Wat 7 ) have been shown to be lined between the side chain of tyrosinase Asn 191 and the main-chain carbonyl of tyrosinase Asp 45 . The side chain of the Tyr 98 residue in caddie is accommodated in the pocket of an active site of tyrosinase, like L-Tyr as a substrate. Both Wat 2 and Wat 3 form hydrogen bonds with the hydroxyl of caddie Tyr 98 . In addition, Wat 3 is hydrogen-bonded to the side chains of His 190 and His 216 residues in tyrosinase, which are ligands of Cu B , and Wat 2 forms three hydrogen bonds with the main-chain carbonyl of tyrosinase Thr 203 , the side-chain hydroxyl of tyrosinase Ser 206 , and Wat 1 . Wat 1 is present between the side chain of the tyrosinase His 38 , which is a ligand of Cu A , and the main-chain carbonyl of Gly 204 in tyrosinase. On the other hand, when His 54 takes the latter conformation, six water molecules might be present at the catalytic site (Fig. 3C). In detail, two water molecules (Wat 5 and Wat 6 ) disappear to avoid close contact with the side-chain of His 54 ; instead, Wat 8 is introduced between Wat 3 and Wat 4 . In addition, the electron density of Wat 7 is weaker when the side chain of the tyrosinase His 54 points toward the caddie His 97 residue.
In a previous study (14), we reported that the caddie protein has two copper-binding sites (Fig. 1, A and B). A copper ion (designated Cu C ), which is most frequently identified in the Cu(II)-soaked crystals of tyrosinase in a complex with caddie, binds to the N⑀ atom from His 82 of caddie. In addition, Cu C binds to the side-chain N␦ atom and the main-chain carbonyl from the His 68 and to the side-chain carboxyl from the Glu 67 residues of caddie, although these two residues are not always identified in an electron density map due to their high mobility. The Cu C -binding site is located near to the C terminus of the ␣2-helix of tyrosinase, which contains the Cu A -ligating His 38 residue. The negative charge on the caddie Glu 67 residue and the charge produced by a helix dipole effect may help the binding of the positively charged Cu(II) at this site. At this time, the N␦ atom of the His 82 side chain forms a hydrogen bond with the N␦ atom of His 97 in caddie, mediated by the nitrate ion derived from a precipitant solution. However, when the crystal was soaked in a solution containing Cu(II) for 6 months (Protein Data Bank code 2AHK), neither Cu C nor nitrate ions were found, whereas another copper ion (designated Cu D ), which adopts a trigonal planar coordination with the N⑀ atoms from the caddie His 82 and His 97 residues and a sulfur atom from the caddie Met 84 , alternatively emerged. To form the Cu D -binding site, the imidazole rings of His 82 and His 97 should be rotated around the bond between their C␤ and C␥ atoms and then get closer to one another. Hence, it is impossible for both Cu C and Cu D to coexist. The imidazole ring of caddie His 97 is located near the side chain of tyrosinase His 54 . Notably, in Cu(II)-free tyrosinase complexed with caddie, the side chain of the tyrosinase His 54 residue partially (ϳ50%) points toward the side chain of caddie His 97 . These clustered His residues, which extend from the solvent-exposed His 82 of caddie to the active center of tyrosinase, may bind to Cu(II) ions during the transfer of two metal ions to the catalytic center.
To evaluate the mechanism of copper transportation to tyrosinase assisted by caddie, we first analyzed the nature of Cu(II) binding in crystals soaked in a CuSO 4 -containing solution for various lengths of time (Table 1). Information on the identified copper ions is summarized in Table 2. Two crystal structures (ST1 and ST2) were obtained by Cu(II) soaking for about 20 h. In ST1, electron densities derived from copper are found only at the Cu B and Cu C sites ( Fig. 4A and supplemental Fig. S1A). The occupancy of Cu C is about 0.4, whereas that of Cu B is about 0.2. This result suggests that after Cu(II) is bound to the surface Cu C site, the metal ion is transported to the tyrosinase active center. The side chain of the tyrosinase His 54 is disordered, as found in the Cu(II)-free structure. As a result, Wat 5 and Wat 6 are absent, and the electron density of Wat 7 is weak. On the other hand, in ST2, electron densities from copper are mainly found at the Cu A , Cu B , and Cu C sites ( Fig. 4B and supplemental Fig. S1B).
The occupancies of Cu A , Cu B , and Cu C are about 0.3, 0.6, and 0.8, respectively. In addition, a weak electron density is evident at the Cu D -binding site, and the occupancy was calculated to be about 0.2. To form the Cu D site, the conformations of the side chains of the caddie His 82 , Met 84 , and His 97 residues must be changed. Although the side chain of the caddie Met 84 residue clearly adopts two conformations, the disordered structures of the caddie His 82 and His 97 residues were not confirmed, probably due to the low occupancy of the alternative conformations.
The side chain of the tyrosinase His 54 also adopts two conformations. Due to the disorder of His 54 , the electron densities at Wat 5 and Wat 6 are weak. The distance between Cu A and Cu B is 3.4 Å, and two molecules are present between these two copper atoms. The molecules that form the bridge are located near the sites of Wat 3 and Wat 8 , although a hydrogen bond between the latter molecule and Wat 4 is lost. The electron density of the molecule near the Wat 8 site is much weaker than that near the Wat 3 site. Although Wat 8 cannot coexist with the Cu A -oriented side chain of His 54 due to the close contact, the bridging molecule near the Wat 8 site seems to coexist with the side chain via an interaction with the Cu A atom. The electron densities from copper in ST3, which was obtained by Cu(II) soaking for about 40 h, are mainly observed at the Cu A , Cu B , and Cu C sites ( Fig. 4C and supplemental Fig.  S1C). The electron density of Cu B in ST3 is stronger than that in ST2. The occupancy was calculated to be about 0.8. Interestingly, the electron density of Cu A is significantly elongated, suggesting that the Cu(II) ion is split between two binding sites (Cu A-1 and Cu A-2 ). Cu A-1 is about 4.2 Å apart from Cu B , whereas Cu A-2 is about 3.4 Å apart from Cu B . The occupancies of Cu A-1 and Cu A-2 were calculated to be 0.3 and 0.4, respectively. The electron density near to the Wat 8 site in ST3 is stronger than that in ST2. However, within ST3, the electron density near the Wat 8 site is weaker than that near the Wat 3 site. Furthermore, the side chain of the tyrosinase His 54 is also disordered in ST3. Cu A-1 is maximally coordinated to His 38 , His 54 , and His 63 , whereas Cu A-2 is maximally coordinated to His 38 , His 54 , and two bridging molecules. Moreover, the electron density at the Wat 1 site is weakened, and the tyrosinase Gly 204 residue has two conformations. One is the same as that found in the Cu(II)-free structure, where its carbonyl oxygen is hydrogen-bonded to the N␦ atom of the His 38 residue via Wat 1 . As shown in Fig. 3A, a carbonyl oxygen of Gly 204 in the other conformation is directly bound to His 38 .
In ST4 and ST5, which were obtained by Cu(II) soaking for about 80 h, strong electron densities from copper are found at the Cu A-2 , Cu B , and Cu C sites (Fig. 4, D and E, and supplemental  Fig. S1, D and E). The occupancy of Cu A-2 is in the range of 0.6 -0.8, whereas that of Cu B is in the range of 0.8 to 0.9. Although an additional copper was found at the Cu D site after soaking for 6 months, the copper was mainly found at the Cu C site in ST4 and ST5. Furthermore, the electron densities of the two bridging molecules are equally strong. On the other hand, the Wat 2 density is lost. In accordance with the disappearance of Wat 2 , the hydroxyl oxygen of caddie Tyr 98 is directly bound to the hydroxyl of tyrosinase Ser 206 , as found in the crystal structure obtained by Cu(II)-soaking for 6 months (Fig. 3A). Interestingly, the structures of the tyrosinase His 54 and the caddie His 97 residues are different in ST4 and ST5. In ST4, as found in ST1-ST3, the side chain of the tyrosinase His 54 is disordered, and the side chain of the caddie His 97 is hydrogen-bonded to that of the caddie His 82 via a nitrate ion (Fig. 4D and supplemental Fig. S1D). As a result, the electron densities of Wat 5 and Wat 6 are not observed. On the other hand, in ST5, almost all of the side chain of the tyrosinase His 54 points toward Cu A-2 ( Fig.  4E and supplemental Fig. S1E). Furthermore, the conformation of the side chain of the caddie His 97 changes to point toward the Cu D -binding site, although the electron density of Cu D is not observed at the site. As a result, a large vacant space is generated between the tyrosinase His 54 and the caddie His 97 residues, and Wat 5 and Wat 6 are fully accommodated in the space.
Effects of Caddie Mutations on Tyrosinase Activity-The residues selected to mutate are His 82 , Met 84 , and His 97 , which are ligands of additional copper atoms (Cu C and Cu D ) bound to caddie, and Tyr 98 , whose hydroxyl group participates in the hydrogen bond network around the active center. His 82 , Met 84 , His 97 , and Tyr 98 were replaced by Gln, Leu, Gln, and Phe, respectively. The caddie mutants were thus named H82Q, M84L, H97Q, and Y98F, respectively. At first, the effects of these mutations on the Cu(II)-induced liberation of caddie from the complex were investigated by HPLC analysis (supplemental Fig. S2). We found that the H82Q, H97Q, and Y98F mutants were barely released from the complex, whereas the M84L mutant was more quickly released than the wild-type protein.
A kinetic experiment was also performed by using complexes of tyrosinase and the caddie mutants. When the oxidase activity was measured after incubation for a given time in the presence of 10 M CuSO 4 at pH 8, the activity of tyrosinase complexed with the H82Q or M84L mutants reached its maximum at 20 min, but that complexed with Y98F showed maximal activity at   AUGUST 26, 2011 • VOLUME 286 • NUMBER 34 occurring after the copper transfer. Tyrosinase might not be dissociated from H97Q because the copper transfer was hindered in this mutated complex. However, the reasons why the dissociation rate of tyrosinase was altered by other mutations remain unclear. Furthermore, it was obvious that when the concentration of Cu(II) was low (1 or 0.1 M), tyrosinase complexed with wild type or Y98F caddie has a slower maturation rate at the early phase (Figs. 5, B and C). On the other hand, the slow maturation phase was barely observed for tyrosinase when complexed with H82Q or M84L.

Copper Transportation to Tyrosinase by Caddie
The maximal activities of tyrosinase complexed with the wild-type, H82Q, M84L, or Y98F caddie proteins were plotted against the concentration of CuSO 4 (Fig. 5D). It was revealed that tyrosinase complexed with the caddie mutants has very low activity in the presence of 0.03 M CuSO 4 , whereas tyrosinase complexed with wild-type caddie has about 60% of the maximum activity. In the mutated complexes, the concentration of copper ions required for the activation of tyrosinase was found to be 1 order higher than that in the wild-type complex. The ED 50 values of the H82Q and Y98F complexes were estimated to be 0.1 M, whereas that of the M84L complex was 0.2 M.
Effects of Caddie Mutations on the Crystal Structure of the Complex-We analyzed the crystal structure of tyrosinase in complex with each caddie mutant (Table 1). In the crystal structure of tyrosinase complexed with H82Q, which was soaked in a CuSO 4 -containing solution for 80 h (ST6), strong electron densities were observed at the Cu A and Cu B sites. This indicates that two copper ions had been introduced into the active site in most of the molecules in the crystal (Fig. 4F). As in ST3, the electron density of Cu A indicated that the copper is split between two binding sites. Furthermore, Wat 1 and Wat 2 were found to be released from the active site, and a bridging molecule near the Wat 8 site was partially apparent. On the other hand, the side chain of the tyrosinase His 54 adopts a single conformation that points toward Cu A , and Wat 5 and Wat 6 are present between the tyrosinase His 54 and caddie His 97 residues. Additional copper is found near the Cu D -binding site (supplemental Fig. S1F), but the occupancy is very low (about 0.3). In the crystal structure of tyrosinase complexed with M84L, which was soaked in a CuSO 4 -containing solution for 80 h (ST7), the active-site geometry is also similar to that in ST6 (Fig. 4G). Additional copper with an almost full occupancy was found at the Cu C -binding site (supplemental Fig. S1G). From these structures, it is not clear why the concentration of Cu(II) required for the activation of tyrosinase was increased in the complexes with each of these caddie mutants, although the increased Cu(II) requirement is likely to be related to the destruction of additional copper-binding sites (the Cu C and Cu D sites in H82Q and the Cu D site in M84L).
Interestingly, in the crystal structure of tyrosinase complexed with H97Q, which was soaked in a CuSO 4 -containing solution for 80 h (ST8), the electron densities from copper are found only at the Cu B and Cu C sites ( Fig. 4H and supplemental  Fig. S1H). The occupancy of Cu C is about 0.8, whereas that of Cu B is about 0.4. This is in agreement with the result that tyrosinase complexed with the H97Q caddie mutant showed very low activity. In ST8, the side chain of the tyrosinase His 54 assumes one conformation that points toward the Cu A -binding site. As a result, Wat 8 is absent, whereas Wat 5 and Wat 6 are present at the dimer interface, although they are disordered. In addition, Wat 1 but not Wat 2 is present.
In the crystal structure of a complex between tyrosinase and the Y98F caddie mutant soaked in a CuSO 4 -containing solution for 80 h (ST9), we found a new copper-binding site, designated the Cu E -binding site, between His 54 of tyrosinase and His 97 of caddie ( Fig. 4I and supplemental Fig. S1I), and the existence of Cu A-1 , Cu B , Cu C , Cu D , and Cu E was clearly confirmed using an anomalous difference Fourier map (Fig. 6). Furthermore, the side chains of the caddie His 82 , Met 84 , and His 97 and tyrosinase His 54 residues were found to be disordered. The occupancies of Cu A-1 , Cu B , Cu C , Cu D , and Cu E are 0.6, 0.8, 0.6, 0.4, and 0.3, respectively. Among these copper atoms, Cu C and Cu D appear to be mutually exclusive because the caddie His 82 residue is a  ligand for the copper at either site, adopting a different conformer depending on which copper site is occupied. Similarly, Cu D and Cu E are mutually exclusive because the caddie His 97 residue must adopt a different conformer. Furthermore, Cu E and Cu A are mutually exclusive because the tyrosinase His 54 residue must adopt a different conformer. The partial occupancies of these coppers are also consistent with this concept. These results indicate that two copper ions are introduced from the Cu C -binding site, which is located on the molecular surface, into the active center of tyrosinase via the Cu D -and Cu E -binding sites. In ST9, Wat 1 and Wat 2 remain at the active center. Furthermore, only one bridging molecule, which is positioned near the Wat 3 site, was found between Cu A-1 and Cu B . Although weak electron density was also found near the Wat 8 site, the molecule assigned at the position is bound to neither Cu A-1 nor Cu B .

DISCUSSION
Structural Changes at the Active Center of Tyrosinase-During the maturation of tyrosinase, the introduction of the two closely spaced copper ions into the catalytic center is of crucial importance because the electrostatic repulsion between these two ions seems to inhibit the maturation of this enzyme. We considered the possibility that the caddie protein may act as a metallochaperone to accommodate two copper ions in the tyrosinase catalytic center. In our present study, we carried out kinetic and crystallographic analyses to elucidate the mechanism underlying copper transport to tyrosinase assisted by caddie. When compared with the kinetic results obtained in solution, copper transfer was very slow in the crystals. However, the known structure of the tyrosinase binuclear copper center emerged later in the crystal structures, and the crystals, after a long soaking time, showed tyrosinase activity, suggesting that the crystallographic observations are functionally significant. In all probability, the mobility of the residues involved in the copper transfer, which is lowered in the crystals, may be necessary for fast copper transfer. Through crystallographic analysis, we evaluated the structural changes at the tyrosinase active center, as illustrated in Fig. 7.
In the ST1 structure obtained from a crystal soaked in a harvesting solution containing 1 mM CuSO 4 for about 20 h, a copper ion was only introduced at the Cu B -binding site, in which the copper took a tetrahedral coordination with the side chains of His 190 , His 194 , and His 216 and a molecule near the Wat 3 site (Fig. 7C). This indicates that the Cu B -binding site is more stable than the Cu A -binding site. In fact, the Cu B -ligating residues have lower B-factors than the Cu A -ligating residues. However, in ST2, which was also obtained from a crystal soaked for about 20 h, the electron densities of both Cu A-2 and Cu B could be clearly observed. Although the occupancy of Cu B was calculated to be slightly higher than that of Cu A-2 , the sum of the occupancies of the two coppers was below 1.0. Assuming that the accommodation of the second copper in the active site is a rate-limiting step, it is reasonable to speculate that most complexes in the crystal contain one copper in the active center. Namely, at the early stages, Cu A-2 and Cu B appear to be mutually exclusive (Fig. 7, B, C, CЈ, and CЉ) because the electrostatic repulsion between the two coppers prevents the formation of a dicopper center. The partial copper occupancies of these two sites are also consistent with this. In ST2, as observed in the crystal structure of the Cu(II)-free complex, the electron density near the Wat 8 site is lower than that near the Wat 3 site, and the side chain of His 54 is disordered. These results indicate that a copper ion is introduced at the Cu A-2 site at first. The copper adopts a tetragonal coordination with the side chains of His 38 and His 54 and Wat 3 and Wat 8 (Fig. 7B). When the copper moves to the Cu B site, the structures of the Cu A-2 -binding site are disrupted. The Cu A-2 -oriented side chain of His 54 is too close to Wat 8 if simultaneously occupied. Therefore, His 54 takes the other conformation (Fig. 7CЈ), or Wat 8 is released from active site (Fig. 7CЉ).
In ST3, which was obtained from a crystal soaked for about 40 h, the electron density from copper is observed at the Cu A-1 , Cu A-2 , and Cu B sites. The sum of the occupancies of Cu A-1 , Cu A-2 , and Cu B is higher than 1.0, indicating that tyrosinase molecules in the crystal may contain one or two coppers in their active site. Moreover, as in ST2, the electron density near the Wat 8 site is lower than that near the Wat 3 site, and the side chain of His 54 is disordered. When one copper is present at the active site, the metal ion is expected to be positioned at either the Cu A-2 or the Cu B site. On the other hand, when two coppers are present at the active site, the coppers are positioned at the Cu A-1 and Cu B sites (Fig. 7, E and EЈ). At this time, Cu B adopts a tetrahedral coordination, whereas Cu A-1 assumes a trigonal coordination with the N⑀ atoms of His 38 , His 54 , and His 63 . It is possible that one (EЈ) or two bridging molecules (E) are present between Cu A-1 and Cu B , although the former geometry seems to be more stable than the latter.
In the crystals soaked in a Cu(II)-containing solution for about 80 h (ST4 and ST5) or 6 months (Protein Data Bank code 2AHK), copper atoms with a higher occupancy are accommodated in the Cu A-2 and Cu B sites. Furthermore, two bridging molecules display equally strong electron densities. These results indicate that the second copper is accommodated in the Cu A-1 site (Fig. 7E) and then moves toward the Cu A-2 site (Fig.  7F). Hereafter, we designate the geometry containing two Cu(II) ions at the Cu A-1 and Cu B sites as the met1 form and the geometry containing two Cu(II) ions at the Cu A-2 and Cu B sites as the met2 form. The distance between Cu A-1 and Cu B is in the range of 4.0 -4.5 Å, whereas the distance between Cu A-2 and Cu B is in the range of 3.2-3.5 Å. Two bridging molecules in the met2 form should be negatively charged hydroxide ions because the hydroxides seem to be important for weakening the electrostatic repulsion between the two coppers. On the other hand, one hydroxide may bridge the coppers in the met1 form. The second copper may be accommodated in the Cu A-1 site after the conversion of one of two bridging water molecules to the hydroxide ion. Subsequently, together with the conversion of the other water molecule to the hydroxide, the copper may approach Cu B , resulting in the generation of the met2 form.
An earlier EXAFS study on the met form of fungal tyrosinase suggests that the Cu-Cu distance is 3.4 Å (30), similar to the met2 form in this study. Furthermore, a previous x-ray absorption study on the met form of S. antibioticus tyrosinase (31), which is 82% identical to the S. castaneoglobisporus tyrosinase in terms of the amino acid sequence, suggests that two coppers lie about 3.4 Å apart with two bridging oxygens. The dihydroxo-bridged dicopper(II) center found in our present study may be a characteristic geometry of the met form of tyrosinase. On the other hand, in the met form of catechol oxidase, two cupric ions lie about 2.9 Å apart with one bridging molecule, which is considered to be a hydroxide ion (19). Each of the two cupric ions adopts a trigonal pyramidal coordination with three His residues and one bridging oxygen. The preferred geometries of the met forms, which are different between the catechol oxidase and tyrosinase, must be determined by the scaffolding of the dicopper center comprising six His residues.
Interestingly, the electron densities of Wat 1 and Wat 2 were found to be weakened in a time-dependent manner, suggesting that the two water molecules were removed in accordance with the generation of the met2 form at the active site. Removal of these molecules from the active site seems to be entropically advantageous and accelerate the uptake of the second copper into the active site. After the generation of the met2 form, the side chain of the tyrosinase His 54 is still disordered (Fig. 7, F, FЈ, and G) but was stabilized after the introduction of Wat 5 and Wat 6 . Namely, if these water molecules are absent, the side chain is disordered to cover the vacant space. For the full introduction of Wat 5 and Wat 6 , the side chain of the caddie His 97 must point toward the Cu D -binding site. The introduction of the second copper to the active site is likely to be intrinsically coupled with the formation of the Cu D -binding site and the introductions of Wat 5 and Wat 6 . In most cases, when the side chain of His 54 points toward the Cu A -binding site, Wat 8 is absent. Two Cu(II) ions may be transported into the active site together with the structural change of the His 54 side chain (AЈ to B and DЈ to E). Wat 3 and Wat 8 are likely to be deprotonated together with the protonation of His 54 (D and F steps). Furthermore, according to the formation of the met2 form (G), Wat 1 and Wat 2 seem to be released from the catalytic center. ST1 and ST2 may represent the structures prior to the first deprotonation, and they contain A, AЈ, CЈ, and CЉ geometries and B, CЈ, and CЉ geometries, respectively. In the ST3 crystal, some of the molecules seem to experience the first deprotonation, and the ratio of EЈ geometry increases. In the ST4 crystal, some of the molecules seem to experience two deprotonation steps, and the ratio of FЈ and G geometries increases. In the ST5 crystal, most of the molecules adopt a G geometry. Cu A is surrounded by His 38 , His 54 , and His 63 , whereas Cu B is surrounded by His 190 , His 194 , and His 216 . Cu A-2 is closer to Cu B than Cu A-1 . It is noteworthy that when Cu A-2 is bound to the active center, the coordination bond between His 63 and copper is lost.
To form the dihydroxo-bridged dicopper(II) center in tyrosinase, Wat 3 and Wat 8 must be converted to hydroxide ions by deprotonation. At the active center of tyrosinase, Wat 3 forms a hydrogen bond with Wat 8 , and Wat 8 is positioned near the Cu A -pointed side chain of the tyrosinase His 54 . Hence, Wat 3 and Wat 8 may be converted to hydroxide ions as His 54 is protonated (Fig. 7, D and F). It is important to note also that the side chain of the tyrosinase His 54 in each of the two conformations interacts with the negatively charged side chain of the tyrosinase Asp 45 . The positive charge generated on the His 54 residue may be relayed to the His 97 residue of caddie when the His 54 residue takes the other conformation. The charge may then move to the solvent molecule through a currently unknown route. This concept is also consistent with the experimental observation that an alkaline pH facilitates the activation of tyrosinase assisted by caddie. It is important to note also that the concentration of Cu(II) to activate tyrosinase complexed with the caddie Y98F mutant is higher than that in the case of tyrosinase complexed with wild-type caddie (Fig. 5D). Furthermore, the maximum activity of the Y98F complex, which is obtained under conditions of a high concentration of Cu(II), is lower than that of the wild-type complex (Fig. 5, A and D). At the active center of tyrosinase complexed with wild-type caddie, the hydroxyl oxygen of the caddie Tyr 98 forms hydrogen bonds with Wat 2 and Wat 3 , and Wat 2 and Wat 3 further bond to Wat 1 and Wat 8 , respectively. The lack of hydroxyl of the caddie Tyr 98 residue results in an imperfect hydrogen bond network, which may be the reason for the low affinity toward Cu(II) of tyrosinase complexed with the Y98F caddie mutant. In fact, Wat 1 and Wat 2 remain at the active center, whereas a bridging molecule near the Wat 8 site is absent in ST9.
Implications for the Mechanism of Cu(II) Transfer-Kinetic experiments demonstrated that mutations at the additional copper-binding site of caddie reduced the ability to activate tyrosinase. The effects of a mutation at His 82 , a ligand of Cu C and Cu D , and Met 84 , a ligand of Cu D , were moderate, whereas a mutation at His 97 , a ligand of Cu D and Cu E , completely abrogated tyrosinase activity. These results indicate that the binding of copper to the Cu E -binding site at the dimer interface is essential for Cu(II) transfer. On the other hand, binding to the Cu C -and Cu D -binding sites may be necessary for effective Cu(II) transfer.
As illustrated in Fig. 8, we propose a Cu(II) transfer mechanism assisted by caddie; first, a copper ion binds to the Cu C site (Fig. 8B) and moves to either the Cu A-2 -or the Cu B -binding site (Fig. 8C). The energy barrier to introduce the first copper into the active center may be low, and this step should progress without any assistance. Indeed, the transportation of a copper into the active site was observed even in the crystal structure of the inactive complex between tyrosinase and H97Q. To form a dicopper center in tyrosinase, a large energy barrier, which is formed by the electrostatic repulsion between the two copper ions, must be overcome. Therefore, a rate-limiting step for the maturation of tyrosinase is likely to be the uptake of the second copper into the active site.
The next step is the binding of the second copper to the Cu C -binding site (Fig. 8D), as indicated by ST2. Although it was difficult to trap the intermediate steps in the present crystallographic study, the second copper ion must be moved to the Cu E site via the Cu D site (Fig. 8E). At this time, because the coordination bond between Cu A-2 and His 54 is lost, the first copper completely moves toward the Cu B site. The third copper ion then binds to the Cu C -binding site (Fig. 8F). This intermediate state may be contained in the crystal structure of tyrosinase in complex with Y98F. As reported above, the caddie mutant has a low ability to introduce the second copper into the active site of tyrosinase, probably due to the imperfect hydrogen bond network. It is noteworthy that the Cu E site, formed by two ligands, has more difficulty fixing the copper than the Cu D site, formed by three ligands. Hence, the second copper bound to the Cu E site may easily move back toward the Cu D site. The third copper ion, which is newly bound to the Cu C site, may play a role in preventing Cu E from moving back to the Cu D site because the binding of the third copper blocks the formation of the Cu D site.
Next, in accordance with the movement of the third copper to the Cu D site, the second copper is introduced into the Cu A-1 site (Fig. 8G). This intermediate state is also likely to be contained in the crystal structure of tyrosinase complexed with Y98F. The second copper then moves to the Cu A-2 site, resulting in the formation of the met2 form. The formation of the met2 form is stimulated by the conversion of two water molecules (Wat 3 and Wat 8 ) to the hydroxide ions and the release of two other water molecules (Wat 1 and Wat 2 ), as reported above. Finally, two water molecules (Wat 5 and Wat 6 ) are introduced into the space between the tyrosinase His 54 and caddie His 97 residues and complete the maturation of tyrosinase (Fig. 8H). Cu A -and Cu D -binding sites are stabilized by the introductions of Wat 5 and Wat 6 . However, in the presence of the nitrate ion, the ion is introduced into the Cu D -binding site, and the third copper moves back to the Cu C -binding site prior to the introduction of Wat 5 and Wat 6 , as found in the crystal structures. Therefore, in most crystal structures prepared by Cu(II) soaking, an additional copper was found at the Cu C site, and the His 54 residue of tyrosinase was disordered (Fig. 8I).
Relative to tyrosinase in a complex with wild-type caddie, tyrosinase complexed with the mutant caddie proteins H82Q or M84L requires a high concentration of copper ions for maturation (Fig. 5D), whereas the maturation rates within 10 min are high (Fig. 5, B and C). As reported above, the Cu C and Cu D sites seem to provide the route for the uptake of external copper to the Cu E site, and the third copper bound to the Cu C site may contribute to the transfer of the second copper to the Cu A site. In these two mutated complexes, due to the destruction of Cu C and/or Cu D sites, the external copper may be directly bound to the Cu E site. As a result, copper should be transferred into the tyrosinase more quickly at an early stage under a high copper concentration. On the other hand, in a complex between tyrosinase and Y98F, the second copper may be transferred into the Cu E site via the Cu C and Cu D sites, as in the wild-type complex. However, because the hydrogen bond network is imperfect, the affinity between the Cu(II) ions and tyrosinase is low (Fig. 5D).
Nitrate ions may block the binding of Cu D , but they will help the formation of the Cu C -and Cu E -binding sites. We investigated the effects of the nitrate ion on the kinetics of tyrosinase complexed with caddie. As a result, the nitrate ion reduced the Cu(II) concentration required for the activation of tyrosinase, indicating that this ion stimulates the Cu(II) transfer. As sug-gested by our kinetic and crystallographic studies of tyrosinase complexed with H82Q, the movement of the second copper from the Cu E site to the Cu A site may occur without the assistance of the third copper. However, for effective Cu(II) transfer, the formation of an intermediate state, in which coppers bind to the Cu B , Cu C , and Cu E sites (Fig. 8F), may be a crucial step, and the nitrate may stabilize this state. Perhaps also, in the presence of the nitrate ion, the movement of the second copper from the Cu E to the Cu A site is not always coupled with the movement of the third copper from the Cu C to the Cu D site because the binding of copper to the Cu D site is blocked. However, at least in the case of tyrosinase complexed with Y98F, a coupled movement between the second and third coppers is likely to be required. In the near future, the Cu(II) transfer mechanism assisted by caddie proposed in the present study will be elucidated by computer simulation analysis or through the use of model compounds.