Coupled binuclear copper sites

Laccase contains four copper atoms that have been classified as type 1 or blue (Tl), type 2 or normal (T2), and type 3 (T3) or coupled binuclear copper sites, where the coppers are antiferromagnetically coupled through a bridging ligand (Fig. 4.4). 

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. 

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. 

Chemical and spectral studies have indicated that in addition to bridging exogenous ligands, there is an endogenous protein bridge at the coupled binuclear copper site. Evidence for this is seen with the half-met derivative and the binuclear cupric derivatives met and dimer. 

A charge transfer transition is found at 425 nm in both met and oxy (vide infra) and is characteristic of the coupled binuclear copper site. Elimination of sulfur ligation leaves the most reasonable assignment of this feature as a phenolate-to-copper(II) CT transition (see Table 4). This indicates that tyrosine is bound to the binuclear cupric site and the most likely candidate for the endogenous bridge. 

When the coupled binuclear copper site in the protein becomes structurally characterized by x-ray crystallography, single-crystal spectroscopy should provide a more detailed picture of its electronic structure and bonding, as has been achieved for the Blue Copper site described in the previous section. 

Fig. 42. Spectroscopically effective active site representations of the coupled binuclear copper site (left) and the type 3 site in Rhus laccase (right) where OR and R represent endogenous protein bridges in the respective sites...

Lerch, K., and German, U. A. (1988). Evolutionary relationships among copper proteins containing coupled binuclear copper sites. In Oxidases and Related Redox Systems (K. T. S., Mason, H. S., and Morrison, M., Eds.), pp. 331-348. A. R. Liss, New York. 

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