Tyrosinase copper sites

The enzyme requires two copper ions per subunit for full expression of activity (18), but, unlike tyrosinase and hemocyanin, there is an absence of magnetic coupling between the two Cu(II) sites and both appear to be separate, isolated mononuclear copper sites (17). The process of dioxygen binding and activation appears to involve interaction of the doxygen molecule with only one copper ion, and it is also found that a proton is requir for the hydroxylation of substrate (19). 

The hemocyanlns which cooperatively bind dioxygen are found in two invertebrate phyla arthropod and mollusc. The mollusc hemocyanlns additionally exhibit catalase activity. Tyrosinase, which also reversibly binds dioxygen and dlsmutates peroxide, is a monooxygenase, using the dloxygen to hydroxylate monophenols to ortho-diphenols and to further oxidize this product to the quinone. Finally, the multicopper oxidases (laccase, ceruloplasmin and ascorbate oxidase) also contain coupled binuclear copper sites in combination with other copper centers and these catalyze the four electron reduction of dloxygen to water. 

Characterization of the Type 2 Depleted Derivative of Laccase. The model for the coupled blnuclear copper site in hemocyanln and tyrosinase (Figure 7) may now be compared to the parallel site in laccase which contains a blue copper (denoted Type 1 or Tl), a normal copper (Type 2, T2), and a coupled binuclear copper (Type 3, T3) center. As shown in Figures 8a and b, native laccase has contributions from both the Tl and T2 copper centers in the EPR spectrum (the T3 cupric ions are coupled and hence EPR nondetectable as in hemocyanln), and an intense absorption band at associated with the Tl center (a thlolate —> Cu(II) CT transition).(14) The only feature in the native laccase spectra believed to be associated with the T3 center was the absorption band at 330 nm (e 3200 M cm ) which reduced with two electrons, independent of the EPR signals.(15) Initial studies have focussed on the simplified Type 2 depleted (T2D) derlvatlve(16) in which the T2 center has been reversibly removed. From Figure 8 the T2 contribution is clearly eliminated from the EPR spectrum of T2D and the Tl contribution to both the EPR and absorption spectrum remains. 

One of the questions surrounding the mechanism of tyrosinase concerns the initial site of attack. As a control, LFMD simulations of a model for the sTy active site, Meim6 (Fig. 28), give identical behavior for each Cu center consistent with its symmetry. In contrast, the LFMD simulations clearly distinguish the two copper sites in the sTy enzyme which must result from the protein environment (Fig. 29). 

Figure 92 (a) Structural mechanism for the hydroxylation of monophenolic substrates by oxytyrosinase (b) reaction coordinate diagram for associative ligand substitution at the copper site of tyrosinase... 

Several diverse metal centres are involved in the catalysis of monooxygenation or hydroxylation reactions. The most important of these is cytochrome P-450, a hemoprotein with a cysteine residue as an axial ligand. Tyrosinase involves a coupled binuclear copper site, while dopamine jS-hydroxylase is also a copper protein but probably involves four binuclear copper sites, which are different from the tyrosinase sites. Putidamonooxin involves an iron-sulfur protein and a non-heme iron. In all cases a peroxo complex appears to be the active species. 

This protein contains a coupled binuclear copper site that appears to be very similar to that found in hemocyanin (Section 62.1.12.3.8).1399 Tyrosinase catalyzes the hydroxylation of monophenols, and also behaves as an oxidase in the oxidation of orfho-diphenols. The deoxy protein [copper(I)] binds dioxygen to give oxytyrosinase, which is a Cu11 peroxide species with antiferromagnetic coupling between the two Cu11 centres. The oxybinuclear site is diamagnetic to the most sensitive detectors. 

A series of hemocyanin and tyrosinase active site derivatives (Fig. 23) can be prepared61"66), allowing systematic variation of the binuclear copper active site and chemical perturbation for spectral studies. In the simplest derivative, met-apo, one copper has been removed and the remaining copper oxidized to the spectroscopically accessible Cu(II). Next in complexity is a mixed-valent binuclear copper site. The Cu(II), in this half-met derivative, exhibits open-shell d9 spectroscopic features and the Cu(I), though spectroscopically inaccessible, can still be studied by comparison to the met-apo derivative. Two derivatives have formally binuclear cupric sites met, which is EPR-non-detect-able, and dimer, which exhibits an intense broad EPR signal. Spectroscopic study of these derivatives has led to the present picture of the coupled binuclear copper protein active site shown at the bottom of Fig. 23. 

Spectral studies of the native enzyme are difficult to interpret due to simultaneous contributions from all three types of copper. The EPR spectrum (Fig. 38) of native laccase98) shows features attributable to the type 2 (g = 2.237, gx = 2.053, A = 206 x 10-4 cm-1) and type 1 (gz = 2.300, gy = 2.055, g = 2.030, Az = 43 x 10-4 cm-1) coppers. The type 3 site is EPR-non-detectable and diamagnetic (-2 J >550 cm-1)56,57). This site should be compared to the met derivative of hemocyanin and tyrosinase only in the sense that it appears to contain two anti-ferromagnetically coupled copper(II) s lacking an EPR signal. A characteristic Blue band of the type 1 copper is seen in the optical spectrum at 614 nm (c = 5700 M-1 cm-1). The shoulder observed at 330 nm (e 2800 M-1 cm-1) was originally associated with the type 3 copper site, as it reduces with two electrons at... 

Active sites containing two copper ions that are antiferromagneti-cally coupled in the oxidized state are often referred to as type 3 copper sites (129). It has recently become evident that these centers cannot all be considered alike and that in the blue copper oxidases the type 3 sites are in fact part of a tricopper cluster these will be considered in Section VA. The proteins containing dinuclear type 3 copper sites comprise hemocyanin and a number of oxygenase enzymes, of which the best known are tyrosinase and dopamine j8-hydroxylase. 

The Copper Sites of Tyrosinase and Laccase. The information suggesting that copper pairs exist at the active site of tyrosinase was reviewed on page 285. Tyrosinase has been the classic Cu(I) enzyme, and there... 

Figure 5) and the copper-copper distance is 3.6 A. The geometry of the type-3 copper site in tyrosinase is very similar to that of hemocyanin due to the similar spectroscopic features and has now been derived from the X-ray structure of a bacterial tyrosinase. ... 

The crystal structures of tyrosinase from Streptomyces castaneoghbisporus HUT 6202 and catechol oxidase from the sweet potato Ipomoea batata have been determined. They confirm that the coordination of the type-3 copper site in tyrosinase and catechol oxidase is very similar to that found in hemocyanin. This had been deduced before from the similarity of spectroscopic properties and a comparison of many tyrosinase and hemocyanin primary structures. On the basis of the biological source of the proteins seven different domain organizations could be identified. Plant catechol oxidases of different organisms have a sequence identity of about 40-60%. The sequence identity between catechol oxidases and mulluscan hemocyanins is about 35% over almost the whole length of the sequences. In contrast, the sequence identity between plant catechol oxidases and other type-3 copper proteins from any nonplant source is limited to the two copperbinding regions. 

Remarkably, reduced T2D Lc does not react with atmospheric 02. The behavior of the dinuclear T3 center thus contrasts starkly with that displayed by other reduced dinuclear copper sites of enzymes such as hemocyanin and tyrosinase or synthetic model dinuclear complexes (see Chapter 8.15). " These promptly perform a two-electron reduction of O2 to the peroxide level (reversibly, in the case of hemocyanin). 

Spectroscopic and biochemical studies reveal that the active site of tyrosinase is very similar to the dinuclear copper site in hemocyanins (Hcs) (13), which are dioxygen carriers in the hemolymph of mollusks and arthropods. 

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