Laccase copper sites

Figure 18. Comparison of half-met hemocyanin with the half-met type 3 (in T2D) laccase copper sites. A EPR spectra and binding constants of exogenous azide binding. B Spectroscopically effective structural models for exogenous ligand binding to the half-met derivatives and their relation to differences in dioxygen reactivity.

Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J. 2002. Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nature Struct Biol 9 601-605. 

Co-immobilization of this redox polymer with a fungal laccase from Trametes versicolor, possessing a Tl copper site reduction potential of +0.57 V vs Ag/AgCl ( +0.77 vs NHE), was achieved using a diepoxide cross-linker, in an approach... 

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. 

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. 

It is noteworthy that the proximity of the copper sites in ceruloplasmin, and, indeed, the involvement of most of the correct ligand histidines, were predicted some time ago by Ryden (1982, 1984) strictly on the basis of sequence homologies to plastocyanin. A similar prediction was made for laccase based on sequence similarities around the cysteine regions (Briving et al, 1980). Proximity of the type II site to the type III site (e.g., a trinuclear site) was also predicted by Solomon and co-workers (Allen-dorf et al., 1985 Spira-Solomon et al, 1986) on the basis of spectroscopic analysis of azide binding to laccase. What could not have been foreseen... 

The blue oxidases contain these three types of copper together The stoichiometry is straightforward with laccase which contains one type-1 and one type-2 copper, and one type-3 dimeric copper site . One would expect two laccase-like sites in ascorbate oxidase and in ceruloplasmin, but the presence of respectively 3 and 1 and 1 and 3 type-1 and type-2 copper atoms has been deduced. Ceruloplasmin shows oxidase activities towards different substrates, like Fe (ferroxidase) and aromatic amines. It plays, moreover, an active role in the transport of copper With the proper precautions against the action of proteinases it can be isolated as a single polypeptide chain... 

Some proteins contain more than one copper site, and are therefore among the most complicated and least understood of all. The active site known as type 4 is usually composed of a type 2 and a type 3 active site, together forming a trinuclear cluster. In some cases, such proteins also contain at least one type 1 site and are in this case termed multicopper oxidases, or blue oxidases [3], Representatives of this class are laccase (polyphenol oxidase) [7-9], ascorbate oxidase (Figure 5.Id) [10], and ceruloplasmin [11], which catalyze a range of organic oxidation reactions. 

The multicopper oxidases (laccase, ascorbate oxidase, and ceruloplasmin) catalyze a four-electron reduction of dioxygen to water (285-287). Consistent with the four-electron stoichiometry, the enzymes contain four copper ions. One of the copper ions is type I, causing an intensely blue color of the proteins, thus the enzymes of this family are referred to as blue oxidases. They also contain a monomeric copper site that exhibits normal spectroscopic features, whereas the other two copper... 

In the presence of molecular oxygen, and only then, the type 1 copper is reoxidized in a first-order process. The type 1 copper is generally thought to be deeply embedded inside the protein, therefore a direct interaction between oxygen and this copper site is improbable. This, together with the fact that no laccase molecule contained more than one reduction equivalent suggests the following ... 

Figure 17. Comparison of the reactivity and magnetism ofdeoxy and met hemocyanin and the laccase type 3 copper site in the T2D derivative.

Upon oxidation of T2D, spectral changes104 are also observed for the type 1 copper site (Fig. 41), indicating intersite interaction. The type 1 parallel hyperfine increases to 42.9 x 10-4 cm-1, intensity of the Blue band decreases (Ac614 —300 M-1 cm-1) and the resonance Raman spectrum of the Blue site shows a significant increase in intensity of a 382 cm-1 vibration. These changes demonstrate that the geometry of the type 1 site is affected by oxidation of the type 3 copper in T2D laccase. 

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...

Fig. 3. Geometries of the type 1 copper sites of various blue copper proteins. The trigonal planar geometry is the type 1 site of laccase from Coprinus cinereus (PDB Code 1A65). The trigonal bipyramidal geometry shown is the copper site of azurin from Pseudomonas aeruginosa (PDB Code lAZU). The trigonal pyramidal/distorted tetrahedral sites are of the stellacyanin from Cucumis sativus (PDB Code IJER), NNSO site, and of the plastocyanin from Populus nigra (PDB Code IPLC) NNSS site.

B. Structural Model of the Copper Sites for Ceruloplasmin Fungal Laccases, Ascorbate Oxidases, and Related Proteins... 

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