Tyrosinase is the most well-studied dicopper oxygenase, and the primary sequences of the cDNA for tyrosinases from Streptomyces glaucescens,25 Neurospora crassa,26 Rona nigromaculata,27 Mus musculus,28and human melanocyte29have been reported. However, there is no crystal struc- ture presently available for any tyrosinase. On the other hand, the crystal structures of sweet potato catechol oxidase in the resting dicopper(II) state (met form), the reduced dicopper(I) state (deoxy form), and a complex with the inhibitor, phenylthiourea, have been solved.18 It is a monomeric 39 kDa enzyme with an overall elliptic shape which is composed primarily of-helices.18The four- helix bundle in the core of the enzyme holds the catalytic dicopper center and is surrounded by two other-helices and some short-strands.18Each of the two active-site copper ions, termed CuA and CuB, is coordinated by three histidine imidazoles provided by the four helices of the -bundle.18 Interestingly, the imidazole ring of His 109, one of the supporting ligands of CuA, is covalently cross-linked to the sulfur atom of Cys 92, forming a novel thioether linkage as shown inFigure 1.18 Such a His–Cys cross-link also exists in tyrosinase from Neurospora crassa30 and in some
hemocyanins.24,31The His–Cys bridge may regulate the conformation of the imidazole rings during the catalytic cycle, but details about its function are not clear at present.
In the resting state (met form) of catechol oxidase, the two cupric ions are separated by 2.9 A˚
and are bridged by a hydroxide ion (Figure 1).18 Both cupric ions exhibit a four-coordinate distorted trigonal pyramidal geometry with His 109 and His 240 in the apical positions for CuA and CuB, respectively.18These results are in agreement with the EXAFS data for the resting form of the enzyme32 indicating a similar structure of the enzyme in solution. The EPR silence of the resting enzyme32reflects an antiferromagnetic interaction between the two cupric ions through the bridging hydroxide (Figure 1). EPR nondetectable met-tyrosinase probably has a similar active site structure with a hydroxide-bridging ligand between two cupric ions, although the EXAFS data on met-tyrosinase indicated a CuCudistance of 3.4 A˚ that is longer than that of catechol oxidase (2.9 A˚).33The overall structure of the active site of catechol oxidase is remarkably similar to that of met-hemocyanin.15–19 Paramagnetic 1H-NMR studies have provided additional infor- mation about the active site structure of met-tyrosinase.34
In the reduced state of catechol oxidase, the distance between the two cuprous ions is increased significantly to 4.4 A˚, while the histidine residues move only slightly and no significant conform- ational change is observed for any other part of the enzyme (Figure 2).18The CuA site exhibits a distorted trigonal pyramidal geometry with one water molecule, while the CuB site has a three- coordinateT-shaped geometry.18
8.15.2.2 Peroxo Intermediates: Structure and Properties
Treatment of met-tyrosinase (EPR nondetectable dicopper(II) form) with hydrogen peroxide under aerobic conditions produces a characteristic absorption spectrum exhibiting an intense peak at 345 nm ("ẳ 18,000 M1cm1) together with a weak broad band at 600 nm ("ẳ 1,200 M1cm1).35–39 The same species can be generated by reduction of the met form with hydroxylamine followed by oxygenation with O2.40 The oxygenated product is also EPR silent,35,40 and provides a resonance-enhanced Raman band at 755 cm1with 16O2that shifts to 714 cm1upon18O2substitution.40The spectroscopic features of oxy-tyrosinase are essentially the same as those of oxy-hemocyanin,38 suggesting that the oxy-tyrosinase has a (-2:2-peroxo) dicopper(II) structure (A in Scheme 1) as in the case of oxy-hemocyanin.22,41,42Reaction of the met form of catechol oxidase with hydrogen peroxide also provides a similar absorption spectrum (343 nm ("ẳ6,500 M1cm1) and 580 nm ("ẳ450 M1cm1)) due to a similar peroxo dicopper(II) species.32 An EXAFS study has shown that the CuCudistance in the peroxo intermediate of catechol oxidase is 3.8 A˚,32 which is a little longer than that of oxy-hemocyanin (3.6 A˚).22,23
His109
Cys92
OH
His88
CuA
His118
His240
His274
His244 CuB
Figure 1 Active site structure of the resting dicopper(II) form of catechol oxidase from sweet potato.18
Details about the spectroscopic characterization of the (-2:2-peroxo) dicopper(II) complexes are presented later. It has been noted that the dioxygen binding of tyrosinase is reversible as in the case of oxy-hemocyanin (Scheme 1).32,35,36Thus, the oxy form of the enzyme is converted into the deoxy dicopper(I) state by releasing O2 when the sample is placed under anaerobic conditions (Scheme 1).35,36 Such a reversible dioxygen binding of tyrosinase, however, makes it difficult to study the single-turnover reaction between the peroxo species and substrates.
8.15.2.3 Enzymatic Reaction Pathways
Proposed reaction pathways of the phenolase and catecholase cycles are shown inScheme 2.1,2,14,16,17
As described above, oxygenation of the deoxy enzyme generates the oxy form of the enzyme having the (-2:2-peroxo)dicopper(II) structure (path (a)). Then, coordination of a phenolate (deprotonated form of the substrate) to one of the cupric ions of the peroxo inter- mediate occurs to give an oxy-P intermediate (P denotes phenolate substrate) (path (b)). This phenolate coordination step is what differentiates the enzymatic reactivity of tyrosinase and catechol oxidase from the mere dioxygen binding reaction of hemocyanin. A large difference in rate of displacement of the peroxide ligand in the oxy enzymes by exogenous ligands has been observed for tyrosinase relative to hemocyanin, demonstrating that the substrate accessibility is much higher in oxy-tyrosinase than in oxy-hemocyanin.38,43,44In this respect, findings of pheno- lase and catecholase activity in the activated hemocyanins are of great interest.15,45 Arthropod hemocyanin has been shown to gain the catecholase activity in the presence of NaClO4,46and the serine protease-treated hemocyanin from a chelicerate has also been demonstrated to exhibit both the phenolase and catecholase activities.47 In the former case, perchlorate ion may induce a conformational change of the enzyme, allowing the substrate incorporation into the dinuclear copper-active site.46In the latter case, on the other hand, the cleavage of anN-terminal peptide by
H O2
His109 Cys92
His88
His118 His274
His240 His244
CuA CuB
Figure 2 Active site structure of the deoxy dicopper(I) form of catechol oxidase from sweet potato.18
CuII O H
CuII H2O2
CuII O O
CuII
–O2
oxy (A) met
+O2
CuI
deoxy CuI
Scheme 1
the limited proteolysis opens the entrance to the active site of the enzyme (highly conserved phenylalanine; Phe-49 in this case).47The substrate binding process has been examined in tyrosi- nase by using a substrate analogue (inhibitor) such as mimosine to demonstrate that it can bind to at least one copper ion and induce a geometric change of the copper center from a tetragonal to a
CuII CuII O H R
O O
CuII CuII O
O R
O
H+ H+
H2O, R
O O
R
OH
CuI CuI CuII CuII
O O O2
H2O, H+
R
O O
R
OH OH R
OH OH
H+ CuII CuII
O H
2 H+
CuII CuII HO
O O
R
O H2O,
R
O O
deoxy oxy
oxy-P met-C
oxy-C met
Catecholase cycle
Phenolase cycle
(a)
(b)
(c) (d)
(e) (f)
(g)
Scheme 2
trigonal bipyramidal structure.48,49It has also been suggested that there are additional interactions between the aromatic ring of the inhibitor and the hydrophobic residues in the enzyme pocket, stabilizing the trigonal bipyramidal geometry.49Such a geometric change may be required for the following hydroxylation process (path (c)). Based on computer modeling using the X-ray struc- tures of hemocyanin, the phenol substrate has been suggested to bind to the CuA site, inducing close contact of the ortho-position of the phenyl ring of the phenol substrate to one of the oxygen atoms of the peroxo ligand.15,45Such a conformation might be essential for the efficient aromatic hydroxylation in tyrosinase.
Hydroxylation of the bound phenolate (path (c)) then occurs to give the catecholate product that may coordinate to the dicopper(II) unit as a bridging ligand as shown in Scheme 2.
An isotope-labeling experiment using18O2has clearly demonstrated that the oxygen atom incorp- orated into the product derives from dioxygen.50 Furthermore, kinetic experiments employing 3,5-ditritiated tyrosine have shown that there is little kinetic isotope effect, indicating that the CH bond cleavage of the ortho-position of the aromatic ring is not involved in the rate-determining step.51,52 Based on the model studies described later, this process is believed to involve rate- determining electrophilic attack of the peroxo group to the substrate arene ring.53 Inner-sphere electron transfer in the met-C intermediate (C denotes catecholate product) generates the quinone product and the dicopper(I) deoxy site (path (d)), which further reacts with dioxygen to construct the phenolase cycle (Scheme 2).
In the catecholase cycle, both the oxy and met sites can react with catechol derivatives, oxidizing them to the corresponding quinones (paths (f) and (g) inScheme 2).54Electron-donating substituents on the catechol ring have been shown to enhance the reactivity.55However, steric and polarity effects of the substituents may also be important for the observed difference in reactivity among the substrates.54
For the catecholase cycle of catechol oxidase, modeling of the substrate-binding process has been performed based on the X-ray structure of an inhibitor complex shown in Figure 3.17,18 It has been proposed that the substrate binds to the CuB site of the oxy form of the enzyme, where one of the hydroxyl groups of the catechol substrate is deprotonated. A nearby Glu 236 has been suggested to be the proton acceptor from the substrate.18 Hydrophobic interactions between the substrate and the amino acid residues Phe 261, Ile 241, and His 244 in the enzyme active site also have been postulated to contribute to the substrate binding.18In this model, the CuA site exhibits a square pyramidal geometry with His 88, His 118, and O22
as the equatorial ligands and His 109 as the axial ligand, whereas the CuB site has an octahedral structure in which the equatorial
PTU His109
Cys92
His88
His118 His274
His240 His244
CuA CuB
Figure 3 Active site structure of the inhibitor (phenylthiourea, PTU) bound catechol oxidase from sweet potato.18
plane is occupied by His 240, His 244, and O22 and the axial positions are filled by the monodentate substrate and His 274. From this complex, electron transfer from the bound catecholate to the peroxide ligand and simultaneous OO bond cleavage occur to produce the o-quinone product, water, and the met form of the enzyme (path (f)). The protonated Glu 236 and the second non-coordinating hydroxyl group of the substrate might donate protons to the peroxo ligand to promote OO bond cleavage (step (f )). Another molecule of catechol then reacts with the resulting dicopper(II) site (met form) to give the deoxy dicopper(I) and anothero-quinone product (path (g)), of which the former reacts with dioxygen to complete the catalytic cycles.18
Steady state kinetics as well as stopped-flow studies on the oxidation of phenol and catechol derivatives have so far been performed extensively.14,53–66However, detailed mechanistic informa- tion about the individual steps of phenolase and catecholase cycles has yet to be obtained due to the following side reactions: the self-inhibition by the phenol substrate (binding of phenols to the met form of the enzyme, not shown in Scheme 2), nonenzymatic intramolecular cyclization, disproportionation between the substrate and product, and polymerization of the product.