Tyrosinase dioxygen binding

Evidence tom a variety of sources indicates that the active site of tyrosinase is very similar to that of hemocyanin, a dioxygen-binding protein found in molluscs and arthropods (15,16). This type of active site contains two copper ions, which are cuprous in the deoxy state, and which reversibly bind dioxygen, forming the oxy form of the enzyme or protein in which a peroxy ligand bridges between two cupric ions. 

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 goal of our research on the multicopper oxidases has been to determine the spectral features of the type 3 (and type 2) centers, to use these spectral features to define geometric and electronic structural differences relative to hemocyanin and tyrosinase, and to understand how these structural differences contribute to their variation in biological function. The hemocyanins and tyrosinases reversibly bind and activate dioxygen whereas the multicopper oxidases catalyze its four-electron reduction to water. 

Figure 1 Schematic representation of the dioxygen binding dinuclear copper enzymes Hemocyanin (He), Tyrosinase (Tyr), and Catechol oxidase (Co)...

Details about the spectroscopic characterization of the (p-rj rj -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 i) 32,3s,36 fonn of the enzyme is converted into the... 

A apart. Dioxygen binding results in the two-electron reduced peroxide in a side-on bound dicopper(II) structure (Fig. 1). Crystallographic information is (also) established for the reduced, as well as the oxidized met forms (3-5). Based on their spectroscopic similarities, a similar active-site structure is proposed for the monooxygenase tyrosinase (Tyr) X-ray structures are available for catechol oxidase (CO) depicting a similar active-site structure to He and Tyr (6). However, unlike the reversible binding reactivity of hemocyanin, tyrosinase catalyzes the aromatic hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (l-DOPA), and can further perform catechol oxidase activity which oxidizes the catechol to the quinone (Fig. 2) (7). 

Our biomimetic investigations have focused on the metalloproteins hemocyanin (He) (11-17) and tyrosinase (11,12,14,16,18,29), which contain two copper ions in their active center. The function of hemocyanin is to bind and transport dioxygen in the hemolymph of molluscs and arthropods. Studies employing EXAFS spectroscopy have shown that in the deoxy form, two (19-21) or three (13,21) imidazole units fiom protein histidine residues coordinate to each cuprous ion. Upon addition of O2 to give oxy-Hc, considerable changes take place in the coordination sphere giving rise to tetragonally coordinated Cu(II) ions... 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

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. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

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. 

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