Laccase binding

Laccase active site with proposed mode of substrate / Intermediate binding... 

Figure 17.3 Anatomy of a redox enzyme representation of the X-ray crystallographic structure of Trametes versicolor laccase III (PDB file IKYA) [Bertrand et al., 2002]. The protein is represented in green lines and the Cu atoms are shown as gold spheres. Sugar moieties attached to the surface of the protein are shown in red. A molecule of 2,5-xyhdine that co-crystallized with the protein (shown in stick form in elemental colors) is thought to occupy the broad-specificity hydrophobic binding pocket where organic substrates ate oxidized by the enzyme. Electrons from substrate oxidation are passed to the mononuclear blue Cu center and then to the trinuclear Cu active site where O2 is reduced to H2O. (See color insert.)...

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. 

Low Temperature MCD Studies of Exogenous Ligand Binding to Native Laccase. Native laccase reduces dloxygen to water but the reduced T3... 

Figure 16 Comparison of exogenous ligand binding modes In l/2-met hemocyanln (left) and 1/2-met laccase (right).

The binding of azide to the T3 and T2 sites in native laccase has been probed via absorption and low temperature MCD spectroscopic studies of the —> Cu(II) charge transfer transitions in the... 

Figure 20. Summary of N3 /F binding competition experiments for native laccase. Continued on next page.

Figure 21. Comparison of the spectroscopically effective models for azide binding at the blnuclear copper active site in hemocyanln and the trlnuclear copper cluster site in laccase.

When the complete amino acid sequence of ceruloplasmin was determined, an internal threefold repeat suggested gene triplication (Ta-kahashi et al., 1983). Moreover, sequence similarity to the small blue copper domains suggested that there were at least two domains with blue copper-binding sites. Analysis of fragments of laccase sequence indicated that there might be a relationship of this to small blue proteins and ceruloplasmin as well (Ryden, 1988). 

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 reduction of laccase by O J obtained by pulse radiolysis in the presence of Oj was followed by the decrease of the absorbance at 614 nm of type-1 Cu. Only a very partial reduction was observed (up to 7%). The binding of F anions to type-2 Cu lowered the reduction and the reoxidation rates... 

Laccase contains one type 1 Cu and one type 2 Cu in addition to the type 3 pair. The copper can be reversibly removed from the type 2 site (to give T2D-laccase). Reconstitution may be accomplished by adding CuS04 or Cu1 under anaerobic conditions.958 Loss of type 2 copper has little effect on the redox potentials of the type 1 and type 3 copper, or on the electron-transfer reactivity of the type 1 copper. It appears that type 2 Cu is a substrate-binding site in the reduction pathway for the blue copper.959... 

It is possible to prepare R. vernicifera laccase which is reversibly depleted in the type 2 copper. This is a great aid in understanding the role of type 2 copper in the mechanism. Type 2-depleted laccase may exist with the type 3 site in the oxidized or reduced form. The spectral features of the blue type 1 copper change with change in the oxidation state of the type 3 copper. This intersite structural interaction may relate to the electron-transfer pathway between type 1 and type 3 copper.1337 The type 2 site has been implicated in the binding of polyphenolic substrates.959... 

Kinetic studies with laccase have shown that the enzyme must be reduced by the organic substrate before reaction with dioxygen occurs. The first electron from the substrate is accepted by the type 1 Cu2+, and the second by the type 2 Cu2+. The electrons from these reduced sites are then transferred to the type 3 copper pair, which then binds dioxygen with reduction to peroxide. It is possible that the type 2 and type 3 centres are in the same cavity, which only becomes accessible to the solvent when the type 1 Cu+ is oxidized. 

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