Type 2 depleted laccase

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. 

On adding dioxygen to the fully reduced laccase of the lacquer tree Rhus vemicifera, the type-1 Cu and the type-3 Cu-pair were oxidized in the ms range and an optical intermediate was observed at 360 nm At liquid helium temperatures an EPR signal was observed, which was tentatively interpreted as due to O ", as a result of its very short relaxation time and of the increase of its linewidth when the reduced laccase of the fungus Polyporus versicolor was treated with 0 A similar paramagnetic oxygen intermediate was also observed with the laccase of another lacquer tree Rhus succedanea and with ceruloplasmin. The decay of the intermediate at 25 °C (tj = 1 s at pH 5.5 with R. succedanea laccase) was accompanied by the reoxidation of the type-2 Cu >. One would expect, however, such an intermediate to be extremely reactive (See Sect. 3.3), while it was stable in tree laccase depleted of type-2 Cu(II)... 

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... 

Fig. 39. Optical absorption spectra (room temperature) of native, type-2-depleted (T2D), T2D + 30-fold excess H202 (TT2D) and TT2D + 100-fold excess NJ laccase (pH = 6.0 protein concentration 1.5 mM) (from Ref. 104)...

AO = Ascorbate oxidase (h)Cp = (human) Ceruloplasmin CT = Charge transfer Hp = Hephaestin GPl = Glycosyl-phosphatidylinositol Lac = Laccase MCO = Multicopper oxidase T1(2,3)D = Type 1 depleted (and/or type 2 or type 3) Tf = Transferrin. 

Laccases (p-diphenol O2 oxidoreductase EC 1.10.3.2) catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. However, the actual substrate specificities of laccases are often quite broad and vary with the source of the enzyme [116,117]. Laccases are members of the blue copper oxidase enzyme family. Members of this family have four cupric (Cu +) ions where each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. In the blue copper oxidases the Cu + domain is highly conserved and, for some time, the crystallographic structure of ascorbate oxidase, another member of this class of enzymes, has provided a good model for the structure of the laccase active site [124,125]. The crystal structure of the Type-2 Cu depleted laccase from Coprinus cinereus at 2.2. A resolution has also been elucidated [126]. 

V Ducros, AM Brzozowski, KS Wilson, SH Brown, P 0stergaard, P Schneider, DS Yaver, AH Pedersen, GJ Davies. Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution. Nature Struct. Biol. 5(4) 310-316, 1998. 

Ducros V, Brzozowski AM, Wilson KS, Ostergaard P, Schneider P, Svendson A, Davies GJ. 2001. Structure of the laccase from coprinus cinereus at 1.68 A resolution evidence for different "type 2 Cu-depleted" isoforms. Acta Cryst Sect D 57 333-336. 

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