Activation Mechanism of the Streptomyces Tyrosinase Assisted by the

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Activation mechanism of the Streptomyces tyrosinase assisted by the caddie protein Yasuyuki Matoba, Shogo Kihara, Yoshimi Muraki, Naohiko Bando, Hironari Yoshitsu, Teruo Kuroda, Miyuki Sakaguchi, Kure'e Kayama, Hulin Tai, Shun Hirota, Takashi Ogura, and Masanori Sugiyama Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00635 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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Biochemistry

1

Activation mechanism of the Streptomyces tyrosinase assisted by the caddie protein

2 3

Yasuyuki Matoba,1,* Shogo Kihara,1 Yoshimi Muraki,1 Naohiko Bando,1 Hironari

4

Yoshitsu,1 Teruo Kuroda,1 Miyuki Sakaguchi,2 Kure’e Kayama,2 Hulin Tai,3 Shun

5

Hirota,3 Takashi Ogura,2 and Masanori Sugiyama1,*

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7

Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan

8 9

2

Picobiology Institute, Graduate School of Life Science, University of Hyogo, RSC-UH

10

Leading Program Center, Koto 1-1-1, Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148,

11

Japan

12

3

Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan

13 14 15

*To whom correspondence addressed. E-mail: [email protected] or

16

[email protected]

17 18

Abstract

19

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center,

20

catalyzes a rate-limiting reaction of melanogenesis: that is, the conversion of a phenol to

21

the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated

22

as a complex with a “caddie” protein that assists the transport of two copper ions into the

23

active center. In this complex, the Tyr98 residue in the caddie protein was found to be

24

accommodated in the pocket of the active center of tyrosinase, probably in a manner

1

ACS Paragon Plus Environment

Biochemistry

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similar to that of L-tyrosine as a genuine substrate of tyrosinase. Under physiological

26

conditions, the addition of the copper ion to the complex releases tyrosinase from the

27

complex, in accordance with the aggregation of the caddie protein. The release of the

28

copper-bound tyrosinase was found to be accelerated by adding reducing agents under

29

aerobic conditions. Mass spectroscopic analysis indicated that the Tyr98 residue was

30

converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated

31

that the conversion occurred through the formations of µ-η2:η2-peroxo-dicopper(II) and

32

Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic

33

conditions and Fourier transform infrared spectroscopic analysis using CO as a structural

34

probe under anaerobic conditions indicated that the copper transportation process to the

35

active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the

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caddie protein, which is triggered by the conversion of the Tyr98 residue to dopaquinone,

37

may ensure the generation of fully activated tyrosinase.

38 39

Introduction

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Specific metal co-factors must be transported into the correct metalloenzyme

41

among many biological systems that employ transition metal ions for their function. The

42

“metallochaperone” protein plays a role in the accomplishment of this process.

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Although a few metallochaperone structures have been determined,1−8 the particular

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molecular mechanism for the specific metal transfer is still unclear except for a

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metallochaperone involved in the copper transport to superoxide dismutase.1

46

Tyrosinase (EC 1.14.18.1), harboring an active center formed by a dinuclear

47

copper, catalyzes the conversion of a phenol to the corresponding ortho-quinone

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through the hydroxylation and subsequent oxidation reactions in addition to the

2


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oxidation of a catechol to the quinone (Fig. 1).9–12 The quinone product is a reactive

50

precursor of the melanin synthesis. A series of reactions occurs under the concomitant

51

reduction of dioxygen to water. Tyrosinase is classified into the type 3 copper protein

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family, like catechol oxidase13 and respiratory pigment hemocyanin.14 Catechol oxidase

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oxidizes catechols to the corresponding quinones but lacks hydroxylase activity.

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Hemocyanin acts as an oxygen carrier in arthropods and mollusks. The dicopper center

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of type 3 copper proteins adopts three redox forms.9–12 The deoxy form [Cu(I)–Cu(I)] is

56

a reduced species, which binds dioxygen to yield the oxy form. In the oxy form,

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dioxygen binds as a peroxide in a µ-η2:η2 side-on bridging mode [Cu(II)–O22-–Cu(II)].

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The met form [Cu(II)–Cu(II)] is recognized as a resting enzymatic form, in which

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Cu(II) ions are normally bridged with one or two small ligands, such as water molecules

60

or hydroxide ions. With respect to tyrosinase, the oxy form can catalyze the generation

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of ortho-quinone from both the phenol and the catechol substrates, whereas the met

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form does not catalyze the former reaction.

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64 65

Fig. 1. Reactions catalyzed by tyrosinase.

66 67

Our group has cloned a melanin-synthesizing gene from Streptomyces (S.)

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castaneoglobisporus HUT6202 that produces a large amount of melanin.15 This gene

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forms an operon composed of two cistrons: one being an open reading frame consisting

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of 378 nucleotides, designated orf378, and the other a tyrosinase-encoding gene,

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designated tyrC, which is located just downstream of orf378. We refer to ORF378 as a

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“caddie” because this protein helps to carry copper ions for the catalytic activity of

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tyrosinase. As observed in the S. antibioticus tyrosinase and its cognate MelC1

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protein,16 the Cu(II)-free tyrosinase forms a stable complex with the caddie protein.17

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Although apo-tyrosinase is not activated by copper added from the outside, the addition

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of copper ions to the complex gives rise to the incorporation of two copper ions into

77

apo-tyrosinase. Furthermore, the resulting Cu(II)-bound tyrosinase is discharged from

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the complex, but no trace of the released caddie protein is detectable in a solubilized

79

fraction, suggesting that the released caddie molecules aggregate.

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Fig. 2. Structure of a complex between tyrosinase and the caddie protein. A, Overall

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structure. Tyrosinase and the caddie are shown in orange and cyan, respectively. Copper

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ions, which include CuA and CuB at the active center and CuC, CuD, and CuE on the

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copper-transport pathway, are indicated by green spheres. Residues of the ligand for the

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copper ions and the Tyr98 residue in the caddie are shown as a stick model. B, A

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structural model around the active center of the met2 form of Cu(II)-bound tyrosinase.

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Carbon atoms from the residues of tyrosinase and the caddie are represented in orange

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and cyan, respectively. Copper ions and water molecules (or hydroxide ions) are

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represented by green and red spheres, respectively.

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We

previously

determined

the

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three-dimensional

structure

of

the

S.

93

castaneoglobisporus tyrosinase in a complex with a caddie at a very high resolution

94

(Fig. 2).18 This represents the first crystal structure of tyrosinase. In recent years, more

95

crystal structures of tyrosinase have been solved, e.g., from the insects Manduca sexta19

96

and Anopheles gambiae,20 the bacterium Bacillus megaterium,21 and the fungi Agaricus

97

bisporus22 and Aspergillus oryzae.23 Very recently, the crustacean and plant enzymes

98

with tyrosinase-like activity were crystallographically characterized.24–26

99

We have obtained the met form of Cu(II)-bound S. castaneoglobisporus tyrosinase

100

in a complex with a caddie by soaking the native crystals in a solution containing

101

CuSO4.18 At the active center of tyrosinase, each of the two closely positioned copper

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ions (CuA and CuB) is surrounded by three His residues through the Nε nitrogen atoms.

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Interestingly, in the crystal structure of the complex, the Tyr98 residue in the caddie

104

protein is accommodated in the active site pocket of tyrosinase (Fig. 2). Our group

105

previously performed kinetic and crystallographic studies to clarify the transferring

106

mechanism of Cu(II) atoms to the active center of tyrosinase assisted by a caddie.27 The

107

binding sites for the additional copper (CuC, CuD, and CuE) in the caddie protein (Fig.

108

2A) and the hydrogen-bonding network around the active center of tyrosinase (Fig. 2B)

109

were found to be important for the effective transfer of Cu(II).

110

In the present study, we demonstrate that the copper uptake to tyrosinase and the

111

subsequent release of tyrosinase progress more quickly with the addition of a reducing

112

agent under aerobic conditions. Furthermore, mass spectroscopic (MS), UV-vis, and

113

resonance Raman spectroscopic analyses indicated that the Tyr98 residue in the caddie is

114

likely

115

µ-η2:η2-peroxo-dicopper(II) and Cu(II)-semiquinone intermediates, which would

converted

to

reactive

dopaquinone

through

6


the

formations

of

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enhance the aggregation of the caddie and the release of tyrosinase. The dopaquinone

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formation must be a result of the catalytic activity of oxy-tyrosinase with a

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µ-η2:η2-peroxo-dicopper(II) center or its derivative bis-µ-oxo-dicopper(III). Electron

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paramagnetic resonance (EPR) analysis under anaerobic conditions and Fourier

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transform infrared (FT-IR) spectroscopic analysis using CO as a structural probe

121

indicated that the copper transportation process is a reversible event under anaerobic

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conditions. Based on these results, we propose a detailed activation mechanism of

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tyrosinase assisted by a caddie, which includes the incorporation of copper, conversion

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of the caddie Tyr98 residue to dopaquinone, and aggregation of the caddie.

125 126

Results

127

Effect of the addition of NH2OH. Tyrosinase from S. castaneoglobisporus was

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generated as a complex with a caddie after coexpression in an Escherichia coli host. In a

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previous study, we investigated the effect of the addition of Cu(II) to the complex.27 The

130

release of tyrosinase from the complex was evaluated via gel-filtration chromatography,

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where the released tyrosinase was eluted after the tyrosinase/caddie complex. On the

132

other hand, it was difficult to observe the released caddie in a solubilized fraction,

133

probably due to aggregation, as found in a previous study using the MelC1 protein and

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tyrosinase from S. antibioticus.16 After incubation with 50 µM CuSO4, tyrosinase slowly

135

dissociated from the complex, and the ratio of the released tyrosinase to the complexed

136

tyrosinase was time-dependently increased by the incubation with Cu(II).27 Based on

137

the peak areas on the chromatogram, the ratios were about 2 and 10 after 2 h and 6 h,

138

respectively. In this case, the released tyrosinase was mainly in the met form.

139

A reductant, NH2OH, can reduce aqueous Cu(II) to Cu(I). In addition, NH2OH can

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reduce met-tyrosinase to the deoxy form, but it cannot reduce dioxygen. Therefore, if

141

NH2OH is added to met-tyrosinase under aerobic conditions, oxy-tyrosinase that can

142

catalyze the hydroxylase reaction is generated by the binding of dioxygen to the deoxy

143

form. A gel-filtration chromatographic profile demonstrated that almost all of the

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tyrosinase molecules were released from the tyrosinase/caddie complex 30 min after the

145

addition of 50 µM CuSO4 and 1 mM NH2OH (Fig. 3), suggesting that the addition of

146

the reductant accelerates the copper-induced release of tyrosinase from the complex. In

147

this case, the main species of the released tyrosinase was the oxy form, as judged from

148

the shape of the UV-vis spectrum exhibiting a characteristic absorption maximum at

149

about 345 nm. The molar absorption coefficient of tyrosinase at 280 nm (73 mM-1 cm-1)

150

is slightly lower than that of the tyrosinase/caddie complex (83 mM-1 cm-1). However,

151

the peak area of the released tyrosinase is significantly lower than the estimate (Fig. 3),

152

suggesting that a part of the released tyrosinase aggregates with the caddie protein. A

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similar observation was found in the absence of NH2OH.27

154

155 156

Fig. 3. Gel-filtration chromatogram at 280 nm against retention time. The release of

157

tyrosinase from the complex was analyzed by gel-filtration HPLC after incubation with

158

50 µM CuSO4 in the absence or presence of 1 mM NH2OH for 30 min. Although

159

tyrosinase was released in the absence of NH2OH, a chromatographic pattern similar to

8


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that in the case with NH2OH was obtained only after incubation for at least 6 h.27

161 162

In our previous study,27 we also estimated the time to construct the dicopper center

163

at the active center of tyrosinase after the addition of copper to the complex based on

164

the oxidase activity toward L-DOPA. It was found that when the concentration of Cu(II)

165

was lower, a longer incubation time was necessary to attain the maximum activity. For

166

example, oxidase activities were maximized at 30 and 40 min by pre-incubation with 10

167

and 0.1 µM CuSO4, respectively, at pH 8.0. In the present study, the addition of the

168

reductant reduced the time required to attain the maximum activity (Fig. 4A). For

169

example, the activity was maximized at 10 and 5 min at pH 6 and pH 8, respectively, by

170

the pre-incubation with 10 µM CuSO4 and 1 mM NH2OH (Fig. 4A). Similar to the case

171

without the reductant,27 the activation rate was increased by the increase in pH. The

172

catalytic activity gradually decreased by the incubation after reaching a maximum. In

173

particular, the activity decreased very quickly under acidic pH conditions. Figure 4B,

174

which shows the oxidase activity after the 10 min pre-incubation, indicates that

175

tyrosinase was more activated by the lower concentrations of CuSO4 at pH 8 than at pH

176

6. Detailed descriptions of the results are shown in the Supporting information.

177

178 179

Fig. 4. Oxidase activities of tyrosinase after the addition of CuSO4 to the

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180

tyrosinase/caddie complex in the presence of NH2OH. A, Time-dependent activation

181

of tyrosinase after pre-incubation with 10 µM CuSO4 and 1 mM NH2OH. The effect of

182

the incubation buffer pH was also investigated. Diamonds, squares, and triangles

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indicate the results at pH 4, 6, and 8, respectively. B, Copper requirement for the

184

activation of tyrosinase. The purified complex was incubated at pH 6 or 8 for 10 min

185

with the indicated concentrations of CuSO4 and 1 mM NH2OH.

186 187

Conversion of the caddie Tyr98 residue to dopaquinone. Other research groups have

188

reported that tyrosinase catalyzes the conversion of solvent-exposed Tyr residues in

189

some peptides and proteins to dopaquinone.28–30 In these cases, the high reactivity of the

190

dopaquinone residue generated in proteins caused aggregation of the proteins via

191

intermolecular covalent bond formation, which occurs by the self-condensation of

192

quinones or through their coupling with the amine or thiol groups in the proteins. Thus,

193

we hypothesized that some Tyr residues in the caddie are converted to dopaquinone by

194

the catalytic activity of tyrosinase, which induces aggregation of the caddie. It is

195

important to note that the met form cannot act on the Tyr residue, since the met form

196

lacks hydroxylase activity.9−12 The observation that the addition of NH2OH accelerated

197

the aggregation of the caddie supports this hypothesis.

198

In our previous crystal structures of tyrosinase with a caddie,18,27 the side chain of

199

the caddie Tyr98 residue is accommodated in the pocket of the active center of tyrosinase

200

(Fig. 2), probably in a manner similar to that of L-tyrosine as a genuine substrate of

201

tyrosinase,12,31 although the residue is solvent-exposed after the separation from

202

tyrosinase. A mutant caddie, Y98F, in which the Tyr98 residue is replaced by Phe, did

203

not separate from the complex upon the addition of CuSO4.27 Theses results suggest that

10


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Biochemistry

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the Tyr98 residue of the caddie is a candidate residue to be converted to dopaquinone to

205

induce aggregation of the caddie. In fact, the conversion of the caddie Tyr98 residue to

206

dopaquinone was suggested by the spectroscopic analysis performed in the presence of

207

a quinone-trapping reagent, 3-methyl-2-benzo-thiazolinone-hydrazone (MBTH) (Fig.

208

S1).32

209

We measured the mass spectra of the peptide fragments that were generated after

210

treatment of the tyrosinase/caddie complex with trypsin. When using the complex

211

previously incubated with CuSO4 and NH2OH, mass peaks of the peptides from the

212

caddie were hardly detected. Aggregation of the caddie, which was caused by the

213

addition of these reagents, is likely to make analysis difficult. As described later, the

214

aggregation was found to be suppressed under weak alkaline conditions and a low

215

temperature. In addition, it is known that NADH can reduce o-quinones to catechols.33

216

Thus, incubation of the complex with CuSO4 and NH2OH was performed at pH 9 and

217

15°C, and then NADH was added prior to the trypsin digestion. As a result, mass peaks

218

of the peptides from the caddie were detected. The addition of NADH was necessary to

219

obtain the mass peaks from the caddie protein. NH2OH is also reported to reduce the

220

ortho-quinones to catechols.34 However, NH2OH may react differently with the quinone

221

in our experiment, where the reagent formed a covalent complex with the quinone even

222

in the presence of NADH (see the Supporting information).

223

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224 225

Fig. 5. MS spectra of the peptide fragments including the Tyr98 residue of the

226

caddie. Matrix-assisted laser desorption/ionization MS spectra of the peptide prepared

227

from the complex (20 µM) after treatment with 60 µM CuSO4, 1 mM NH2OH, and 10

228

mM NADH under air (A) and 18O2 atmosphere conditions (B). The monoisotropic peak

229

of the unmodified peptide was detected at m/z = 2196.7 (data not shown). Electrospray

230

ionization MS/MS spectra of the monoisotropic peptide with an increment of m/z = 16

231

are shown in C. Peak intensity is represented as a percentage relative to the intensity of

232

the highest peak in the range. In C, “Y +16” represents a tyrosine residue modified with

233

an increment of m/z = 16. Based on the b and y peptide fragments as indicated above,

234

the amino acid sequence of the peptide was confirmed.

235 236

When comparing the mass spectra obtained from the complex incubated with and

237

without CuSO4, a mass shift was observed at only one peak (monoisotropic m/z =

12


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Biochemistry

238

2196.7). This peak corresponds with the mass of a peptide fragment of the caddie

239

(protonated form of Asn86−Arg105, calculated monoisotropic m/z = 2197.1), which

240

contains the Tyr98 residue. When the complex was incubated with copper, three peaks

241

with an increment of m/z = 16, 31, or 46 were observed (Fig. 5A). The mass increment

242

of the first peak suggests that the enzymatically modified Tyr98 residue was converted to

243

DOPA from dopaquinone. When tyrosinase complexed with the Y98F caddie mutant

244

was analyzed, no significant shift in the mass value was observed. The MS/MS analysis

245

of the monoisotropic peak with an increment of m/z = 16 indicated that the peptide is

246

modified at the Tyr98 residue (Fig. 5C). Furthermore, the m/z of the corresponding peak

247

increased by 2 in the experiment performed under the

248

indicating that the oxygen atom added to the Tyr98 residue was derived from dioxygen.

249

Detailed descriptions of the results are given in the Supporting information.

18

O2 atmosphere (Fig. 5B),

250 251

Spectroscopic analysis to detect the intermediate species. Generally, when the

252

tyrosinase/caddie complex was incubated with CuSO4, the UV-vis spectrum of the

253

solution showed a drastic increase in absorbance at all the measured wavelengths

254

(250–700 nm) due to the aggregation of the caddie. The aggregation of the caddie seems

255

to make spectroscopic analysis difficult. However, the aggregation was suppressed

256

under weak alkaline conditions (about pH 9) and a low temperature (below 20°C).

257

Therefore, we performed further spectroscopic analysis at pH 9 and below 20°C. Under

258

these conditions, the shape of the UV-vis spectrum was scarcely changed after the

259

addition of CuSO4.

260

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261 262

Fig. 6. UV-visible spectra of tyrosinase complexed with a Y98F-mutated (A) or

263

wild-type caddie (B). The tyrosinase/Y98F-mutated caddie and tyrosinase/wild-type

264

caddie complexes (15 µM) were incubated with 60 µM CuSO4 and 1 mM NH2OH for 1

265

h at 20°C and 4°C, respectively. Black and gray lines indicate the spectra obtained

266

immediately before and 1 h after the addition of NH2OH, respectively. The pale gray

267

line in B represents the spectrum obtained 1 h after the temperature was increased to

268

20°C after incubation at 4°C for 1 h.

269 270

To generate oxy-tyrosinase, tyrosinase (15 µM) complexed with the Y98F-mutated

271

caddie was incubated with 60 µM CuSO4 in the presence of 1 mM NH2OH under

272

aerobic conditions at 20°C. The treatment gave a characteristic UV-vis spectrum within

273

1 h (Figs. 6A & S2A). An intense absorption peak at 345 nm and an additional weak

274

peak at 580 nm seem to be due to the formation of a µ-η2:η2-peroxo-dicopper(II)

275

species.10 Considering that the molar absorption coefficient of oxy-tyrosinase or

276

oxy-hemocyanin is about 20 mM-1 cm-1 at 345 nm,10 approximately 80% of the

277

tyrosinase complexed with the Y98F-mutated caddie was converted to the oxy form.

278

When the gas phase of the sample was replaced by purging nitrogen gas, the absorbance

279

at 345 nm disappeared. Furthermore, when the purged solution was returned to air, the

14


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absorbance at 345 nm returned to the former value very quickly, indicating that the

281

equilibrium between the deoxy and oxy forms is dependent on the O2 concentration.

282

The formation of oxy-tyrosinase was also confirmed by resonance Raman

283

spectroscopic analysis with 355 nm excitation using a concentrated protein solution

284

(110 µM) in the presence of 340 µM CuSO4 and 1 mM NH2OH. Before measurement of

285

the resonance Raman spectrum, we confirmed that the UV-vis spectrum of the Raman

286

samples displayed the same shapes with those of the diluted solution, as shown in

287

Figure 6. This indicates that the coppers were transferred to the active site under the

288

concentrated conditions. The Raman difference spectrum indicated that there are three

289

isotope-sensitive Raman bands at 747, 532, and 1,080 cm−1 with 16O2, which shifted to

290

708, 502, and 1,020 cm−1, respectively, upon

291

bands are assignable to symmetric O−O vibration, Cu−peroxide vibration, and the first

292

overtone, respectively, of the µ-η2:η2-peroxo-dicopper(II) core, as previously reported,35

293

although the wave numbers of the latter two bands seem to have large errors due to the

294

ambiguity of the shapes.

18

O2 substitution (Fig. 7A). These three

295

15


Biochemistry

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Page 16 of 49

296 297

Fig. 7. Resonance Raman spectra of tyrosinase complexed with a Y98F-mutated

298

(A) or wild-type caddie (B and C). A and B represent the resonance Raman spectra of

299

tyrosinase complexed with a Y98F-mutated and wild-type caddie, respectively, obtained

300

by excitation at 355.0 nm. Measurement of the spectra was initiated after the addition of

301

340 µM CuSO4 and 1 mM NH2OH to the 110 µM tyrosinase/caddie complex and

302

continued for 30 min. Experiments were performed under 16O2 and 18O2 atmospheres at

303

15°C. C represents the resonance Raman spectra obtained by excitation at 413.1 nm.

304

Measurement of the spectra was initiated about 30 min after the addition of CuSO4 and

305

NH2OH to the tyrosinase/wild-type caddie complex. Experiments were performed using

306

isotope-unlabeled tyrosine/caddie complexes under

307

isotope-labeled tyrosine/caddie complexes under a 16O2 atmosphere.

16

O2 and

18

O2 atmospheres and

308 309

The same treatment was applied to the tyrosinase/wild-type caddie complex (15

310

µM) except for the incubation at 4°C. The absorbance at 345 nm was increased to the

16


Page 17 of 49

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Biochemistry

311

maximum within 1 h under aerobic conditions (Figs. 6B & S2B), indicating the

312

formation of a µ-η2:η2-peroxo-dicopper(II) species. The formation of oxy-tyrosinase at

313

this stage was also confirmed by resonance Raman spectroscopic analysis using a

314

concentrated protein solution. There were three isotope-sensitive Raman bands at 745,

315

532, and 1,063 cm−1 with 16O2, which shifted to 703, 505, and 1,002 cm−1, respectively,

316

upon 18O2 substitution (Fig. 7B). However, the 345 nm peak was much wider than that

317

in the Y98F-mutated complex, and an additional weak peak was detected at about 630

318

nm in the UV-vis spectrum.

319

The broad 345 nm peak suggests the coexistence of molecular species different

320

from µ-η2:η2-peroxo-dicopper(II). After the absorption spectrum remained unchanged at

321

4°C, the temperature was increased to 20°C. As a result, the absorbance peak at 345 nm

322

decreased, and a peak at 400 nm emerged (Figs. 6B & S2B). The EPR spectrum at 77 K

323

of the concentrated solution with the 400 nm absorption band did not show any peak.

324

On the other hand, the resonance Raman spectrum of the concentrated solution obtained

325

by 413 nm excitation showed two notable peaks at 590 and 1,383 cm-1 (Fig. 7C). The

326

peak at around 1,380 cm-1, which has also been reported in the case of a final product

327

formed between a functional low-molecular-weight model of tyrosinase and

328

2,4-di-tert-butylphenolate,36,37 is diagnostic of metal-bonded semiquinones and

329

attributed to the C–O/ring mixed mode.37,38 The 590 cm-1 peak in the lower energy

330

region is likely due to the Cu–O mode associated with a ring motion.37 Unfortunately,

331

we could not detect an isotope shift of the peaks upon

332

However, we detected an isotope shift for the 590 cm-1 peak when using the 18O-labeled

333

tyrosinase/caddie complex, in which all hydroxyl oxygen atoms of the Tyr residues were

334

replaced with

18

18

O2 substitution (Fig. 7C).

O (Fig. 7C). Therefore, we assumed that the absorption at 400 nm is

17


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

335

due in part to the formation of a Cu(II)-semiquinone complex, in which the

336

semiquinone generated at the caddie Tyr98 residue is bound to Cu(II). The 400 nm peak

337

was especially clearly detected in the absorption spectrum after the decrease in the

338

absorption of the µ-η2:η2-peroxo-dicopper(II) species at 345 nm.

339

The absorption spectrum observed in this study is different from the

340

Cu(II)-semiquinone complex obtained from the model compound.36,37 However, it was

341

reported that absorption spectra of Cu(II)-semiquinone complexes are dependent on the

342

coordination geometry around Cu(II).39,40 In addition, although the semiquinone in the

343

Cu(II)-semiquinone models is bidentately bound to Cu(II), current resonance Raman

344

analysis using

345

Cu(II)-semiquinone at the caddie Tyr98 residue, in which an original tyrosine-oriented

346

oxygen, but not a newly added oxygen, is bound to Cu(II). Furthermore, since the intact

347

semiquinone or quinone species is known to have an absorption at about 400 nm,41 the

348

emergence of the peak at 400 nm may be partly due to the increase in intact species. The

349

quantification of Cu(II)-bound semiquinone, intact semiquinone, and intact quinone

350

species requires assumption. At least based on the resonance Raman analysis, the

351

Cu(II)-dopasemiquinone species was present at both 4°C and 20°C. On the other hand,

352

no clear band attributable to the C−O stretching vibration of the intact quinone was

353

observed in the resonance Raman spectra in the 1,640−1,680 cm-1 range, where the

354

band would shift by oxygen isotope labeling (Fig. 7C).42,43

18

O2 and

18

O-tyrosine suggests the possibility of monodentately bound

355 356

Reversibility in the anaerobic copper-transportation process. The dicopper active

357

center of tyrosinase is generally silent in the EPR spectrum.10 However, the signals with

358

g⁄⁄ = 2.25 and A⁄⁄ = 19 mT were observed in the EPR spectrum when an equivalent

18


Page 18 of 49

Page 19 of 49

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Biochemistry

359

amount of Cu(II) was anaerobically added to the tyrosinase/caddie (100 µM), indicating

360

a square planar type 2 copper-binding site in the complex (Fig. 8A). Judging from the

361

copper coordination structures, Cu(II) atoms may preferentially bind at the CuC site

362

rather than at the CuA or CuB site in the active center, as suggested by our previous

363

study.27 When two-fold Cu(II) was treated to the tyrosinase/caddie complex, similar

364

signals were observed with larger intensities in the EPR spectrum, indicating that the

365

CuC site was not fully occupied with Cu(II) by the equivalent addition of Cu(II) to the

366

tyrosinase/caddie complex (Fig. 8A). The EPR signals observed after adding two-fold

367

Cu(II) indicate that, although the dicopper state may be formed in a part of the

368

tyrosinase/caddie complex, the ratio is less than 1.0. The transportation of Cu(II) to the

369

active center under anaerobic conditions is likely a reversible event in the

370

tyrosinase/caddie complex, since the addition of two-fold Cu(II) was not sufficient for

371

the formation of a dicopper center. The dicopper state is unlikely to be formed without

372

the help of additional Cu(II) ions. In fact, a previous study suggested that three Cu(II)

373

ions are necessary to construct a dicopper center at the active site of tyrosinase

374

complexed with a caddie protein.27

375

CO is frequently used as a structural probe to investigate the active-site

376

environments

in

metal-binding

proteins.

When

CO

377

tyrosinase/wild-type caddie complex (0.5 mM) mixed with an equimolar amount of

378

Cu(II) (0.5 mM) and an excess of NH2OH (10 mM) under anaerobic conditions, the

379

C−O frequency of the CO-bound tyrosinase/wild-type caddie complex was detected at

380

2,075 cm-1 in the FT-IR spectrum (Fig. 8B, spectrum c). However, when CO was added

381

to the tyrosinase/wild-type caddie complex in which two-fold Cu(II) was treated in the

382

same way, a shoulder peak emerged at 2,068 cm-1 in the spectrum (Fig. 8B, spectrum

19


was

added

to

the

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

383

d), indicating that the bands at 2,075 and 2,068 cm-1 are attributable to the C−O

384

stretching (νC−O) bands of the CO bound to a copper ion (presumably CuB) of the

385

monocopper and dicopper states, respectively. The decrease in the νC−O frequency of the

386

CO-bound dicopper site compared to that of the CO-bound monocopper site is

387

apparently caused by the positive charge of the cuprous ion (presumably CuA) sitting

388

close to the bound CO.44 In the case using the Y98F-mutated complex, the two bands

389

were detected at 2,068 and 2,060 cm-1, which may correspond to the νC−O bands of the

390

monocopper and dicopper states, respectively (Fig. 8B, spectra e and f). The lower

391

νC−O frequency in the tyrosinase/Y98F-mutated caddie complex as compared to that in

392

the tyrosinase/wild-type caddie complex may be due to the removal of the electrostatic

393

effect to CO caused by the oxygen atom of the Tyr98 residue in the wild-type caddie.44

394

395 396

Fig. 8. EPR (A) and FT-IR (B) spectra of tyrosine/caddie complexes. A, The EPR

397

spectra of the tyrosinase/wild-type caddie complex (100 µM) after anaerobic incubation

398

on ice with (a) 100 or (b) 200 µM CuSO4 for 2 h are shown. The spectra were measured

399

at 77 K. B, The FT-IR spectra of the tyrosinase/wild-type caddie complex (c, d) and

400

tyrosinase/Y98F-mutated complex (e−h) are shown. The tyrosinase/caddie complexes

20


Page 20 of 49

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Biochemistry

401

(0.5 mM) were anaerobically incubated on ice with CuSO4 ((c) 0.5, (d) 1.0, (e) 0.5, (f)

402

1.0, and (g) 1.5 mM) and 10 mM NH2OH for 30 min under CO atmospheres. (h) The

403

spectrum of the CO-bound tyrosinase/Y98F-mutated caddie complex obtained by the

404

aerobic addition of five-fold CuSO4 to the complex, dialysis, concentration, and

405

treatment with 10 mM NH2OH under CO atmosphere is also shown. The spectra were

406

measured at 288 K.

407 408

Surprisingly, in the presence of CO and NH2OH, the population of the dicopper

409

state in the tyrosinase/Y98F-mutated caddie complex appears to be higher than that in

410

the tyrosinase/wild-type caddie complex, although the mutation was suggested to be a

411

disadvantage for the transportation of Cu(II) in the absence of NH2OH.27 We assumed

412

that the binding of CO to the dicopper center pushed out one (CuA) of the two copper

413

atoms from the active center to an additional site (CuC, CuD, or CuE) due to a steric

414

hindrance. The steric effect of the introduced CO on the CuA ion in the

415

tyrosinase/wild-type

416

tyrosinase/Y98F-mutated caddie complex due to the immobilization effect of the Tyr98

417

hydroxyl group on CO by an electrostatic interaction.

caddie

complex

may

be

larger

than

that

in

the

418

The intensity of the νC−O band at 2,060 cm-1 in the FT-IR spectrum was stronger

419

when three-fold Cu(II) was added to the tyrosinase/Y98F-mutated caddie complex

420

together with NH2OH than when two-fold Cu(II) was added to the complex under the

421

same conditions (Fig. 8B, spectra f and g). On the other hand, when the

422

tyrosinase/Y98F-mutated caddie complex was dialyzed under aerobic conditions after

423

the addition of five-fold Cu(II) in the absence of NH2OH and subsequently treated with

424

CO in the presence of NH2OH, the intensity of the 2,060 cm-1 band decreased to an

21


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

425

intensity similar to that obtained by the addition of two-fold Cu(II) in the presence of

426

NH2OH under anaerobic conditions (Fig. 8B, spectrum h). Based on the FT-IR results,

427

the formation of the dicopper center appears to be a reversible event in the

428

tyrosinase/caddie complex, independently of the oxidation state of the coppers, and the

429

dicopper state would be easily lost by dialysis (for Cu(II)) or CO addition (for Cu(I)).

430 431

Discussion

432

We previously performed crystallographic and mutational studies to clarify the

433

transfer mechanism of Cu(II) into the active center of tyrosinase mediated by a caddie

434

protein.27 These studies suggested that the formation of a dicopper center in tyrosinase

435

is a reversible event in the case of Cu(II). In the present study, we demonstrated that the

436

copper transfer was stimulated by the addition of NH2OH as an external reductant under

437

aerobic conditions (Figs. 3 & 4). Cu(I), but not Cu(II), might be a suitable species for

438

transfer into the active center of tyrosinase. In addition, an alkaline pH is suitable for the

439

transfer of both Cu(I) and Cu(II) (Fig. 4), probably because His residues acting as

440

copper ligands become deprotonated. On the other hand, the present study suggests that,

441

under anaerobic conditions, the formation of a dicopper center in tyrosinase is a

442

reversible event, even in the case of Cu(I) (Fig. 8). This implies that the dicopper center

443

of the deoxy or met form of tyrosinase is not a converged structure when it is generated

444

in the complex.

445

UV-vis and resonance Raman spectroscopic analyses demonstrated that

446

oxy-tyrosinase with a µ-η2:η2-peroxo-dicopper(II) center was generated at an early stage

447

after the addition of copper and a reductant under aerobic conditions (Figs. 6 & 7). In

448

the case of the Aspergillus tyrosinase, the addition of Cu(II) to the copper-free form has

22


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Biochemistry

449

been shown to generate a His-Cys-linked met form via a µ-η2:η2-peroxo-dicopper(II)

450

intermediate.45 In this case, the Cys residues in the enzyme seem to act as internal

451

reductants to reduce Cu(II) to Cu(I). The construction of µ-η2:η2-peroxo-dicopper(II)

452

appears to be an important step for the maturation of both the Streptomyces and

453

Aspergillus tyrosinases. However, there seems to be a major difference in that Cu(I) and

454

Cu(II) are likely suitable species incorporated into the Streptomyces and Aspergillus

455

tyrosinases, respectively, which may reflect the difference of the environment where the

456

enzymes mature.

457

Our group has suggested that, after two water molecules around the first Cu(II) are

458

converted to hydroxide ions, the second Cu(II) is effectively incorporated into the active

459

center, leading to the generation of the met2 form with bis-µ-hydroxo-dicopper(II).27

460

The hydroxide ions between the copper ions may play a role in weakening the

461

electrostatic repulsion. In the case of dimerization of the low-molecular-weight

462

mononuclear Cu(I)-ligated compound, it has been estimated that two Cu(I) ions interact

463

with each other after dioxygen binds to one of the two Cu(I) ions for generation of the

464

µ-η2:η2-peroxo-dicopper(II) species.46 Similarly, in tyrosinase, the second Cu(I) seems

465

to be incorporated into the active center after the formation of the Cu(II)-superoxide

466

complex generated by the binding of dioxygen to the first Cu(I), leading to the creation

467

of the oxy form (Fig. 9). Dioxygen may play a role in weakening the electrostatic

468

repulsion between the copper ions. Although we tried to detect the Cu(II)-superoxide

469

complex using a tyrosinase/caddie complex in which one-fold Cu(II) was added under

470

the reducing condition, the expected formation was not captured, probably due to the

471

short lifetime. It is important to note that the µ-η2:η2-peroxo-dicopper(II) site in the oxy

472

form resembles the bis(µ-hydroxo)-dicopper(II) site in the met2 form in terms of

23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

473

geometry and total charge. However, the binding of dioxygen to the first Cu(I) atom in

474

the oxy-form-generation process may occur faster than the conversion of two water

475

molecules around the first Cu(II) to hydroxide ions in the met2-form-generation process.

476

This may explain why the copper transfer to the active center of tyrosinase was

477

stimulated in the presence of NH2OH and dioxygen (Fig. 4).

478

479 480

Fig. 9. Proposed activation mechanism of tyrosinase complexed with a caddie

481

protein. When Cu(II) is added to copper-free tyrosinase (Ty) complexed with a caddie

482

(Cad), Ty is converted to the met, deoxy, or oxy form (met-Ty, deoxy-Ty, or oxy-Ty,

483

respectively). In the presence of external reductants, the oxy-Ty/Cad and deoxy-Ty/Cad

484

complexes are primarily generated under aerobic and anaerobic conditions, respectively,

485

whereas the met-Ty/Cad complex is mainly generated in the absence of external

486

reductants. Only the oxy-Ty/Cad complex is converted to deoxy-Ty/q-Cad (deoxy form

487

of Ty and quinone-containing Cad). After q-Cad is dissociated from deoxy-Ty, q-Cad

488

aggregates, and deoxy-Ty is converted to oxy-Ty. The resulting oxy-Ty may replace

24


Page 24 of 49

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Biochemistry

489

unoccupied Ty (Ty), half-occupied Ty (Ty[Cu(II)]), or met-Ty complexed with Cad, as

490

indicated by gray lines, resulting in the release of Ty, Ty[Cu(II)], or met-Ty. In addition,

491

met-Ty may be generated by the non-physiological oxidation of deoxy-Ty, as indicated

492

by the gray dotted line, since the active site of Ty is more solvent-accessible than that in

493

the Ty/Cad complex.

494 495

Early studies suggest that, after the uptake of copper ions into tyrosinase, the

496

caddie protein dissociates from the complex, and the released caddie aggregates.16,17

497

One possible explanation for this phenomenon is that the formation of a dicopper site in

498

tyrosinase reduces the size of the active-site pocket, stimulating the release of the caddie.

499

However, the reduction of the pocket size is not confirmed, due to the absence of the

500

crystal structure of the caddie-unbound tyrosinase. The Tyr98 residue in the caddie

501

protein is accommodated at the active site of tyrosinase, in which the hydroxyl group of

502

the caddie Tyr98 residue forms hydrogen bonds with the hydroxyl group in the

503

tyrosinase Ser206 residue and with the bridging molecule between CuA and CuB (Fig.

504

2B). Therefore, the wild-type caddie must have a higher interaction energy with

505

tyrosinase than the Y98F-mutated caddie, where the Tyr98 residue is replaced by Phe.

506

However, the previous study demonstrated that the copper-induced dissociation of the

507

caddie does not occur when using the Y98F mutant.27 In the present study, we observed

508

that the addition of NH2OH under aerobic conditions accelerates the dissociation of the

509

caddie (Fig. 3), which may be triggered by the formation of reactive dopaquinone at the

510

caddie Tyr98 residue lying on the molecular surface (Fig. 2). These results indicate that

511

the formation of dopaquinone at the caddie Tyr98 residue may induce temporal

512

dissociation of the dopaquinone-containing caddie from the complex. Although the

25


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 49

513

detailed mechanism for the dissociation is not clear, it is easily assumed that the

514

dissociated quinone-containing caddie protein aggregates.

515

The dopaquinone seems to be formed through the µ-η2:η2-peroxo-dicopper(II)

516

intermediate (Figs. 6 & 7). Considering the reversibility of the binding of dioxygen to

517

the deoxy form of tyrosinase, the µ-η2:η2-peroxo-dicopper(II) center is also unlikely to

518

be a converged structure, like the dicopper centers of the met and deoxy forms.

519

Dissociation of the quinone-containing caddie protein may ensure generation of the

520

fully activated tyrosinase, whose active center includes two copper ions and can

521

accommodate the low-molecular-weight substrate without the interface from the caddie

522

protein,

523

µ-η2:η2-peroxo-dicopper(II) center.

since

the

dissociation

step

occurs

after

the

formation

of

the

524

The present study suggests that the species having an absorption peak at 400 nm,

525

such as dopasemiquinone or dopaquinone, is accumulated under alkaline pH and low

526

temperature conditions that inhibit the separation of the quinone-containing caddie. One

527

of the possible species accumulated is a Cu(II)-bound dopasemiquinone, whose signal

528

was amplified in resonance Raman spectroscopic analysis, although the quantification

529

of Cu(II)-semiquinone, intact semiquinone, and intact quinone species requires

530

assumption. The radical species, such as the tyrosine radical or dopasemiquinone, have

531

been considered non-physiological products in the tyrosinase reaction, inhibiting the

532

production of the physiological dopaquinone.12 However, other research groups have

533

suggested that Cu(II)-semiquinone is a possible final intermediate in the tyrosinase

534

reaction cycle,36,37 which is formed just before the separation into dopaquinone and

535

deoxy-tyrosinase. If so, at the dicopper center where Cu(II)-semiquinone is present, a

536

copper ion coordinated by the semiquinone is Cu(II), but the other one may be reduced

26


Page 27 of 49

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Biochemistry

537 538

to Cu(I). The present study also suggests that the upshift in temperature stimulates the from

the

µ-η2:η2-peroxo-dicopper(II)

539

conversion

intermediate

to

the

540

Cu(II)-dopasemiquinone-containing species. In the Cu(II)-semiquinone state, the caddie

541

protein may interact with tyrosinase by the mediation of Cu(II). The following

542

one-electron-transfer step to convert Cu(II)-dopasemiquinone into the deoxy form of

543

tyrosinase and the quinone-containing caddie, and thereby the separation of the two

544

proteins, is likely accelerated by the acidification and/or by the further increase in

545

temperature.

546

It is important to note that the aggregation of the caddie progressed slowly, even in

547

the absence of an external reductant.27 This seems inconsistent with the fact that only

548

the oxy-tyrosinase can act on the Tyr residue.9–12 We propose the reaction that occurs in

549

the absence of the reductants, as shown in Figure 9. In this scheme, the residues in

550

tyrosinase or the caddie might act as internal reducing agents to convert some of the

551

Cu(II) to Cu(I). As a result, a small amount of tyrosinase complexed with the caddie

552

would be converted to the oxy form without the help of an external reductant, whereas a

553

large part of tyrosinase complexed with the caddie is converted to the met form. In the

554

former complex, the Tyr98 residue of the caddie is converted to dopaquinone by the

555

catalytic activity of oxy-tyrosinase, and the resulting deoxy-tyrosinase and

556

quinone-containing caddie are separated. The released quinone-containing caddie

557

aggregates, while deoxy-tyrosinase is converted to the oxy form in a manner dependent

558

on the concentration of dissolved dioxygen.

559

If this were the only route to separate tyrosinase from the complex, all of the free

560

tyrosinase enzymes should be in a deoxy or oxy form. However, the main species of the

27


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

561

free tyrosinase formed in the absence of a reductant is in the met form. In addition, the

562

interaction energy between tyrosinase and the caddie seems weak, since the buried

563

accessible surface area on the dimer formation is small compared to the values observed

564

for the other protein dimers.18,47 Therefore, we propose the hypothesis that the free

565

oxy-tyrosinase could replace the copper-free- or met-tyrosinase, forming a complex

566

with the unmodified caddie protein, resulting in the release of the copper-free or met

567

form. A self-subunit-swapping mechanism was also demonstrated to be required to

568

activate low-molecular-mass nitrile hydratase using a cobalt-transporter chaperone.48,49

569

This concept, which involves the release of copper-free tyrosinase, agrees with the

570

previous observation27 that all of the tyrosinase molecules complexed with caddie were

571

not activated in the absence of an external reductant, especially under low pH conditions.

572

However, there may be another route to produce met-tyrosinase in the absence of a

573

reducing reagent. The met-tyrosinase would be generated by the non-physiological

574

oxidation from deoxy-tyrosinase after the release of the caddie protein, since the active

575

site is more solvent-accessible than that in the tyrosinase/caddie complex.

576

Although the Streptomyces tyrosinases are found in intra- and extra-cellular

577

fractions, they contain no signal sequences.50−52 On the other hand, most caddie-like

578

proteins possess a typical leader peptide, suggesting a mechanism in which

579

apo-tyrosinase forms a binary complex with the caddie-like protein, followed by the

580

incorporation of copper and transport across the cytoplasmic membrane.50 The

581

twin-arginine translocation (TAT) pathway is known to allow the transport of proteins in

582

their folded conformation.53,54 Proteins secreted in this pathway display a characteristic

583

twin-arginine motif in the leader peptide sequence. Caddie-like proteins also have the

584

motif and may be transported by the TAT route.55,56 Therefore, the copper transfer into

28


Page 28 of 49

Page 29 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

585

the active center of tyrosinase assisted by a caddie is likely to begin in the cell. Since the

586

reducing condition is kept inside the cell by the abundant reductants, the copper transfer

587

into tyrosinase would be stimulated. However, the concentrations of copper and

588

dissolved dioxygen in the cell are lower than those on the outside. Under these

589

conditions, only some of the tyrosinase molecules complexed with the caddie may be

590

converted to the oxy form, followed by separation from the complex. Due to the

591

absence of a signal sequence in tyrosinase, the free tyrosinase remains in the cell.

592

Tyrosinase in the cellular fraction might be important for the detoxification of catecholic

593

compounds.57 On the other hand, other forms of tyrosinase, including the copper-free

594

and the deoxy forms, are secreted with the caddie protein. Subsequent activation

595

processes would occur in the extracellular fraction.

596

In the present study, we demonstrated that the conversion of the caddie Tyr98

597

residue to dopaquinone progresses through the formation of µ-η2:η2-peroxo-dicopper(II)

598

and Cu(II)-dopasemiquinone intermediates. Despite extensive studies using the crystal

599

structures of tyrosinase, low-molecular-weight model systems, and computer

600

simulations, the catalytic mechanism of tyrosinase has not been understood at the

601

atomic level. We are now performing crystallographic studies to elucidate the structural

602

basis for the tyrosinase reaction.

603 604

Experimental procedures

605

Construction of tyrosine auxotroph E. coli for protein expression. E. coli JW2581,

606

one of the BW25113 derivatives in the Keio collection,58 in which the tyrA gene is

607

replaced by a kanamycin resistance gene, was kindly provided by the National Institute

608

of Genetics, Japan. A tyrosine-auxotroph E. coli BL21 derivative, used for the

29


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 49

18

609

expression of the tyrosinase/caddie complex containing

610

group of the Tyr residue, was constructed using a Quick & Easy E. coli Gene Deletion

611

Kit (Gene Bridges). A DNA fragment containing the upstream region of tyrA, the

612

kanamycin resistance gene, and the downstream region of tyrA was amplified from the

613

JW2581

614

5ʹ-CCCCATATGGATGGCTCTTTTGATGTAGAAGC-3ʹ,

615

5ʹ-GACTATGCCGTCGTACCGATTGAAAATACCAG-3ʹ. The amplified PCR product

616

was introduced into E. coli BL21 harboring the pRED/ET plasmid to cause homologous

617

recombination. One of the resulting kanamycin-resistant clones was confirmed to be

618

auxotrophic for tyrosine.

chromosome

by

PCR

with

O oxygen at the hydroxyl

a

forward

primer,

and

a

primer,

reverse

619 620

Preparation of the complex. The overproduction and purification of tyrosinase,

621

complexed with the wild-type caddie or the caddie Y98F mutant, were performed

622

according to a previously described method.17,18,27 For expression of the isotope-labeled

623

complex, pET-mel2, an expression plasmid for the tyrosinase/caddie complex, was

624

introduced into the tyrosine-auxotroph E. coli BL21 derivative, and E. coli was grown

625

in an Overnight Express Autoinduction System 2 medium (Novagen) supplemented

626

with 175 µg mL-1

627

isotope introduction was calculated to be about 94% based on the peak area on the

628

electrospray ionization MS spectrum.

18

O-labeled L-tyrosine (Taiyo Nippon Sanso). Efficiency of the

629 630

HPLC analysis. A purified complex (10 µM) was incubated in a 20 mM Tris-HCl

631

buffer (pH 7.8) containing 0.2 M NaCl and 50 µM CuSO4 in the presence or absence of

632

1 mM NH2OH at 30°C. After the aggregation generated in the sample was removed by

30


Page 31 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

633

centrifugation, the supernatant was injected onto a gel-filtration Superdex 200 10/300

634

GL HPLC column (GE Healthcare), which was equilibrated with a 20 mM Tris-HCl

635

buffer (pH 7.8) containing 0.2 M NaCl. The flow rate was set to 0.75 mL min-1, and the

636

absorbance profile at 280 nm was monitored.

637 638

Kinetic analysis. A purified complex (10 nM) was preincubated at 30°C in a 10 mM

639

sodium phosphate buffer (pH 4, 6, or 8) containing the indicated concentrations of

640

CuSO4 with 1 mM NH2OH. At the given time, a portion (1 mL) of the solution was

641

mixed with the same volume of 100 mM sodium phosphate buffer (pH 6.28, 6.25, or

642

5.37) containing 10 mM L-DOPA in a 1 cm cuvette, and the increasing rate of

643

absorbance at 475 nm (∆A475 min-1), which is due to dopachrome formation,32 was

644

immediately monitored to estimate the oxidase activity. The pH of the mixed solution,

645

in which the oxidase reaction progressed, was 6.2. All kinetic experiments were

646

performed in duplicate.

647 648

Mass spectrometry. Measurements of mass spectra were kindly performed at the

649

Natural Science Center for Basic Research and Development, Hiroshima University. A

650

purified complex (20 µM) was incubated for 1 h at 15°C in a 0.1 M sodium phosphate

651

buffer (pH 9) containing 60 µM CuSO4 and 1 mM NH2OH under an air or

652

atmosphere. The tyrosinase reaction was terminated by the addition of HCl, EDTA, and

653

NADH at final concentrations of 0.2 M, 10 mM, and 10 mM, respectively. The proteins

654

were denatured by the addition of a 10-fold volume of 6 M urea, followed by

655

replacement of the buffer with 50 mM Tris-HCl (pH 7.5), containing 1 mM CaCl2 and

656

10 mM NADH, using Amicon (0.5 mL, 3-kDa cutoff, Millipore), and digestion with 5

31


18

O2

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 49

657

µg mL-1 trypsin (sequencing grade, Promega). The resulting samples were desalted

658

using

659

desorption/ionization MS spectra were measured using AXIMA-CFR plus (Shimazu).

660

Electrospray ionization MS and MS/MS spectra were measured using an LTQ Orbitrap

661

XL (Thermo Fisher Scientific).

the

C18

resin-packed

ZipTip

(Millipore).

Matrix-assisted

laser

662 663

UV-vis spectrometry. UV-vis spectroscopic analysis was performed using a JASCO

664

V-550 spectrophotometer with a Peltier-controlled thermostated cell holder. A purified

665

complex (20 µM) was placed in a 1 cm quartz cuvette at 4 or 20°C and incubated in a

666

0.1 M sodium phosphate buffer (pH 9) containing 60 µM CuSO4 and 1 mM NH2OH.

667

For the detection of quinone formation, a purified complex (10 µM) was incubated at

668

15°C in a 20 mM Tris-HCl buffer (pH 7.9) containing 0.2 M NaCl, 50 µM CuSO4, 1

669

mM NH2OH, and 20 mM MBTH in the presence or absence of 0.1% (w/v) SDS.

670 671

Resonance Raman spectrometry. Resonance Raman spectra excited at 355.0 or 413.1

672

nm were measured. Before the measurement, a purified complex (110 µM) was

673

incubated in a 0.1 M sodium phosphate buffer (pH 9) containing 340 µM CuSO4 and 1

674

mM NH2OH at a temperature between 4 and 20°C for the indicated times. The gas

675

phase was replaced before the addition of NH2OH. To prevent photo damage, the

676

sample cells were rotated.

677 678

EPR spectrometry. The tyrosinase/wild-type caddie complex in 0.1 M sodium

679

phosphate buffer (pH 9) was degassed with a vacuum line and purged with 1 bar of N2.

680

The subsequent anaerobic addition of 1 or 2 equivalents of CuSO4 to the protein

32


Page 33 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

681

solution was performed using a glove box (YSD-800L, UNICO, Tsukuba). The final

682

concentration of the tyrosinase/wild-type caddie complex was 100 µM. The sample

683

solutions were anaerobically transferred into a 4 mm ϕ EPR tube in the glove box,

684

further degassed with the vacuum line, purged with 1 bar of N2, and incubated on ice for

685

2 h. EPR spectra were measured at 77 K with an EPR spectrometer (JESFA100N,

686

JEOL). The frequency, microwave power, and modulation width were 9.170 MHz, 5

687

mW, and 0.5 mT, respectively.

688 689

FT-IR spectrometry. The tyrosinase/wild-type caddie or tyrosinase/Y98F-mutated

690

caddie complex (0.5 mM) in 0.1 M sodium phosphate buffer (pH 9) containing 0.5, 1.0,

691

or 1.5 mM CuSO4 and 10 mM NH2OH was anaerobically incubated on ice for 30 min.

692

After replacing the gas phase with CO with a vacuum line, the sample solution was

693

further incubated on ice for 30 min. The tyrosinase/Y98F-mutated caddie complex

694

(0.022 mM) in 0.1 M sodium phosphate buffer (pH 9) containing 0.11 mM CuSO4 was

695

aerobically incubated at 4°C for 3 h. After dialysis of the sample solution against a

696

copper-free buffer, the complex was concentrated. The gas phase was replaced with CO,

697

NH2OH was anaerobically added to the sample solution, and the sample solutions were

698

incubated on ice for 30 min. The obtained sample solutions were transferred

699

anaerobically into an infrared cell with CaF2 windows. FT-IR spectra were measured at

700

15°C using an FT-IR spectrometer (FT-IR 6100V, JASCO, Tokyo) equipped with an

701

MCT detector. A cryostat system (CoolSpeK IR USP-203IR-A, Unisoku, Hirakawa)

702

was used to control the temperature of the cell. Before the FT-IR measurements, the

703

sample solutions were incubated at 15°C for 10 min in the cryostat system.

704

33


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

705

Supporting information

706

Detailed descriptions of the effect of NH2OH on the oxidase activity of tyrosinase,

707

detection of quinone, results of MS analysis, and UV-vis spectrum in a range of 250 to

708

500 nm are given in the supporting information.

709 710

Acknowledgments

711

We are grateful to Ms. T. Amimoto at the Natural Science Center for Basic

712

Research and Development, Hiroshima University, for the measurement of MS spectra;

713

Dr. S. Yanagisawa at the University of Hyogo for the discussion of the resonance

714

Raman spectra; and Professor M. Abe at Hiroshima University and Professor H.

715

Masuda and Dr. D. Nakane at the Nagoya Institute of Technology for the initial

716

measurements of EPR spectra. E. coli JW2581 was kindly provided by the National

717

Institute of Genetics, Japan. This study was partly supported by grants (Nos. 25109530

718

and 15H009470 to Y. M.; 25109540 and 15H00960 to T. O.; and 15H00945 to S. H.,

719

Stimuli-Responsive Chemical Species) for Scientific Research on Innovative Areas

720

from MEXT of Japan. Y. M., S. H., and T. O. are visiting scientists at RIKEN.

721 722

References

723

(1)

Rosenzweig, A. C. Nat. Struct. Biol. 1999, 6, 724−729.

724 725 726 727 728

Lamb, A. L.; Wernimont, A. K.; Pufahl, R. A.; Culotta, V. C.; O'Halloran, T. V.;

(2)

Portnoy, M. E.; Rosenzweig, A. C.; Rae, T.; Huffman, D. L.; O'Halloran, T. V.; Culotta, V. C. J. Biol. Chem. 1999, 274, 15041−15045.

(3) Wernimont, A. K.; Huffman, D. L.; Lamb, A. L.; O'Halloran, T. V.; Rosenzweig, A. C. Nat. Struct. Biol. 2000, 7, 766−771.

34


Page 34 of 49

Page 35 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

729

(4)

49365−49370.

730 731

(5)

(6)

(7)

(8)

740 741 742 743 744 745 746

Watanabe, S.; Kawashima, T.; Nishitani, Y.; Kanai, T.; Wada, T.; Inaba, K.; Atomi, H.; Imanaka, T.; Miki, K. Proc. Natl. Acad. Sci. USA 2015, 112, 7701–7706.

738 739

Watanabe, S.; Matsumi, R.; Atomi, H.; Imanaka, T.; Miki, K. Structure 2012, 20, 2124–2137.

736 737

Löw, C.; Neumann, P.; Tidow, H.; Weininger, U.; Haupt, C.; Friedrich-Epler, B.; Scholz, C.; Stubbs, M. T.; Balbach, J. J. Mol. Biol. 2010, 398, 375–390.

734 735

Srinivasan, V.; Netz, D. J.; Webert, H.; Mascarenhas, J.; Pierik, A. J.; Michel, H.; Lill, R. Structure 2007, 15, 1246−1257.

732 733

Remaut, H.; Safarov, N.; Ciurli, S.; van Beeumen, J. J. Biol. Chem. 2001, 276,

(9)

Sanchez-Ferrer, A.; Rodriguez-Lopez, J.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1–11.

(10) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563–2606. (11) Solomon, E. I.; Chen, P.; Metz, M.; Lee, S. K.; Palmer, A. Angew. Chem. Int. Ed. 2001, 40, 4570–4590. (12) Rolff, M.; Schottenheim, J.; Decker, H.; Tuczek, F. Chem. Soc. Rev. 2011, 40, 4077–4098.

747

(13) Gerdemann, C.; Eicken, C.; Krebs, B. Acc. Chem. Res. 2002, 35, 183–191.

748

(14) van Holde, K. E.; Miller, K. I. Adv. Protein Chem. 1994, 47, 1–81.

749

(15) Ikeda, K.; Masujima, T.; Suzuki, K.; Sugiyama, M. Appl. Microbiol. Biotechnol.

750 751 752

1996, 45, 80–85. (16) Chen, L. Y.; Leu, W. M.; Wang, K. T.; Lee, Y. H. J. Biol. Chem. 1992, 267, 20100–20107.

35


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

753 754 755 756 757 758

(17) Kohashi, P. Y.; Kumagai, T.; Matoba, Y.; Yamamoto, A.; Maruyama, M.; Sugiyama, M. Protein Expression Purif. 2004, 34, 202–207. (18) Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. J. Biol. Chem. 2006, 281, 8981–8990. (19) Li, Y.; Wang, Y.; Jiang, H.; Deng, J. Proc. Natl. Acad. Sci. USA 2009, 106, 17002–17006.

759

(20) Hu, Y.; Wang, Y.; Deng, J.; Jiang, H. BMC Biol. 2016, 14, 2.

760

(21) Sendovski, M.; Kanteev, M.; Ben-Yosef, V. S.; Adir, N.; Fishman, A. J. Mol. Biol.

761 762 763 764 765

2011, 405, 227–237. (22) Ismaya, W. T.; Rozeboom, H. J.; Weijn, A.; Mes, J. J.; Fusetti, F.; Wichers, H. J.; Dijkstra, B. W. Biochemistry 2011, 50, 5477–5486. (23) Fujieda, N.; Yabuta, S.; Ikeda, T.; Oyama, T.; Muraki, N.; Kurisu, G.; Itoh, S. J. Biol. Chem. 2013, 288, 22128–22140.

766

(24) Masuda, T.; Momoji, K.; Hirata, T.; Mikami, B. FEBS J. 2014, 281, 2659–2673.

767

(25) Bijelic, A.; Pretzler, M.; Molitor, C.; Zekiri, F.; Rompel, A. Angew. Chem. Int. Ed.

768 769 770 771 772 773 774 775 776

Engl. 2015, 54, 14677–14680. (26) Molitor, C.; Mauracher, S. G.; Rompel, A. Proc. Natl. Acad. Sci. USA 2016, 113, E1806–1815. (27) Matoba, Y.; Bando, N.; Oda, K.; Noda, M.; Higashikawa, F.; Kumagai, T.; Sugiyama, M. J. Biol. Chem. 2011, 286, 30219–30231. (28) Freddi, G.; Anghileri, A.; Sampaio, S.; Buchert, J.; Monti, P.; Taddei, P. J. Biotechnol. 2006, 125, 281–294. (29) Anghileri, A.; Lantto, R.; Kruus, K.; Arosio, C.; Freddi, G. J. Biotechnol. 2007, 127, 508–519.

36


Page 36 of 49

Page 37 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

777 778 779 780 781 782

(30) Tessari, I.; Bisaglia, M.; Valle, F.; Samorì, B.; Bergantino, E.; Mammi, S.; Bubacco, L. J. Biol. Chem. 2008, 283, 16808–16817. (31) Decker, H.; Schweikardt, T.; Tuczek, F. Angew. Chem. Int. Ed. Engl. 2006, 45, 4546–4550. (32) García-Molina, F.; Muñoz, J. L.; Varón, R.; Rodríguez-López, J. N.; García-Cánovas, F.; Tudela, J. J. Agric. Food Chem. 2007, 55, 9739–9749.

783

(33) Brown, R. S.; Male, K. B.; Luong, J. H. Anal. Biochem. 1994, 222, 131–139.

784

(34) Hamann, J. N.; Rolff, M.; Tuczek, F. Dalton Trans. 2015, 44, 3251–3258.

785

(35) Ling, J. S.; Nestor, L. P.; Czernuszewicz, R. S.; Spiro, T. G.; Fraczkiewicz, R.;

786

Sharma, K. D.; Loehr, T. M.; Sanders-Loehr, J. J. Am. Chem. Soc. 1994, 116,

787

7682–7691.

788 789 790 791 792 793

(36) Mirica, L. M.; Vance, M.; Rudd, D. J.; Hedman, B.; Hodgson, K. O.; Solomon, E. I.; Stack, T. D. Science 2005, 308, 1890–1892. (37) Op't Holt, B. T.; Vance, M. A.; Mirica, L. M.; Heppner, D. E.; Stack, T. D.; Solomon, E. I. J. Am. Chem. Soc. 2009, 131, 6421–6438. (38) Barreto, W. J.; Barreto, S. R.; Ando, R. A.; Santos, P. S.; DiMauro, E.; Jorge, T. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2008, 71, 1419–1424.

794

(39) Thompson, J. S.; Calabrese, J. C. Inog. Chem. 1984, 24, 3171–3176.

795

(40) Verma, P.; Weir, J.; Mirica, L.; Stack, T. D. P. Inorg. Chem. 2011, 50, 9816–9825.

796

(41) Wendt, F.; Näther, C.; Tuczek, F. J. Biol. Inorg. Chem. 2016, 21, 777–792.

797

(42) Moënne-Loccoz, P.; Nakamura, N.; Steinebach, V.; Duine, J. A.; Mure, M.;

798 799 800

Klinman, J. P.; Sanders-Loehr, J. Biochemistry 1995, 34, 7020–7026. (43) Nakamura, N.; Moënne-Loccoz, P.; Tanizawa, K.; Mure, M.; Suzuki, S.; Klinman, J. P.; Sanders-Loehr, J. Biochemistry 1997, 36, 11479–11486.

37


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

801 802

(44) Li, T.; Quillin, M. L.; Phillips, G. N. Jr.; Olson, J. S. Biochemistry 1994, 33, 1433–1446.

803

(45) Fujieda, N.; Ikeda, T.; Murata, M.; Yanagisawa, S.; Aono, S.; Ohkubo, K.; Nagao,

804

S.; Ogura, T.; Hirota, S.; Fukuzumi, S.; Nakamura, Y.; Hata, Y.; Itoh, S. J. Am.

805

Chem. Soc. 2011, 133, 1180–1183.

806 807

(46) Itoh, S.; Nakao, H.; Berreau, L. M.; Kondo, T.; Komatsu, M.; Fukuzumi, S. J. Am. Chem. Soc. 1998, 120, 2890–2899.

808

(47) Jones, S.; Thornton, J. M. Prog. Biophys. Mol. Biol. 1995, 63, 31–65.

809

(48) Zhou, Z.; Hashimoto, Y.; Shiraki, K.; Kobayashi, M. Proc. Natl. Acad. Sci. USA

810

2008, 105, 14849–14854.

811

(49) Zhou, Z.; Hashimoto, Y.; Kobayashi, M. J. Biol. Chem. 2009, 284, 14930–14938.

812

(50) Leu, W. M.; Chen, L. Y.; Liaw, L. L.; Lee, Y. H. J. Biol. Chem. 1992, 267,

813

20108–20113.

814

(51) Ikeda, K.; Masujima, T.; Sugiyama, M. J. Biochem. 1996, 120, 1141–1145.

815

(52) Claus, H.; Decker, H. Syst. Appl. Microbiol. 2006, 29, 3–14.

816

(53) Cline, K.; Henry, R.; Li, C.; Yuan, J. EMBO J. 1993, 12, 4105–4114.

817

(54) Robinson, C.; Cai, D.; Hulford, A.; Brock, I. W.; Michl, D.; Hazell, L.; Schmidt,

818 819 820 821 822

I.; Herrmann, R. G.; Klösgen, R. B. EMBO J. 1994, 13, 279–285. (55) Schaerlaekens, K.; Scherková, K.; Lammertyn, E.; Geukens, N.; Anné, J.; van Mallaert, L. J. Bacteriol. 2001, 183, 6727–6732. (56) Schaerlaekens, K.; van Mellaert, L.; Lammertyn, E.; Geukens, N.; Anne, J. Microbiology 2004, 150, 21–31.

823

(57) Yang, H. Y.; Chen, C. W. PLoS One 2009, 4, e7462.

824

(58) Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K.

38


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Biochemistry

A.; Tomita, M.; Wanner, B. L.; Mori, H. Mol. Syst. Biol. 2006, 2, 2006.0008.

825 826 827

TOC

828

39


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 1 38x15mm (300 x 300 DPI)


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Biochemistry

150x138mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 3 106x113mm (300 x 300 DPI)


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Biochemistry

Fig. 4 76x28mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5 152x115mm (300 x 300 DPI)


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Biochemistry

Fig. 6 82x33mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 7 168x141mm (300 x 300 DPI)


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Biochemistry

Fig. 8 145x105mm (300 x 300 DPI)


Biochemistry

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199x110mm (300 x 300 DPI)


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Biochemistry

TOC 85x36mm (300 x 300 DPI)


Activation Mechanism of the Streptomyces Tyrosinase Assisted by the

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