## 3 Optical Properties of Laccase  
### UV-Vis Absorption Spectroscopy  
Most lacases have important optical properties due to the presence of copper atoms in the catalytic center, as stated earlier. Most of the kinetic and spectroscopic studies on lacases are summarized in a book by [PERSON] [45] as well as in several reviews [33; 46]. As already mentioned, lacases are also categorized by their optical properties: blue, yellow, and white lacases. Most of the studies reported so far are on blue lacases. In blue lacases, the T1 copper, responsible for the blue color of cupoproteins, has an absorption maximum of about 600 nm [40; 47]. These lacases are reported in a variety of organisms, including _Pleurotus pulomarius_[48], _Pleurotus ostreatus_[49; 50], _Coriolus versicolor, Panus phlebia Radiata, Phlebia tremellosa, Agaricus bisporus_[51], _Thermus_ sp. [52], and _Trametes versicolor_[53]. Those lacases that do not exhibit the absorption feature around 600 nm are known as white or yellow lacases, and they have rarely been studied [54; 55]. They differ from blue lacases because they oxidize non-phenic substrates in the absence of mediator molecules [55], which are required in cases of blue lacases. White lacases have absorption at around 400 nm and contain one copper ion, one iron ion and two zinc ions. They have been observed in _Pleurotus ostreatus_[49; 50], _Trametes hirsuta_[56; 57], and _Myrothecium verrucaria_[58]. The yellow lacases contain the copper atoms in a modified oxidation state and have been reported in _Panus tigirus_[51], _Daedalea flavida_[59], _Trametes_ sp. [60], _Pleurotus ostreatus_[61], and _Sclerotinia scleroorum_[54]. As for the copper of type 2, it exhibits weak absorption in the visible spectrum. Finally, the two copper atoms of type 3 are characterized by absorption around 330 nm. A typical UV-Vis absorption spectrum for a blue lacase has been reported by [PERSON] et al. [62], in their study of the ligninolytic systems of _Coriolus hirsutus_ (a blue lacase) and their role in lignin degradation. The absorption spectrum of lacase showed a signal at approximately 600 nm that is related to T1 copper atoms (Figure 3). The contribution of the copper atoms of type 3 is not clear, but it is associated with a small shoulder that is present at around 330 nm [62]. In 2000, [PERSON] et al. [63] obtained spectroscopic insights into the effect of oxygen in aqueous solutions of two types of lacases: _Coriolus hirsutus_ and _Coriolus zonatus_. These authors report the optical absorption spectrum of _Coriolus hirsutus_ in a neutral aqueous solution of the _Coriolus hirsutus_  
Figure 3: Absorption spectrum of _Coriolus hirsutus_ lacase; investigated samples were prepared by dissolving 0.7 mg of the enzyme in 20 mM sodium acetate buffer pH 4.5 (reprinted with permission from Ref. [62]). lution saturated with air in Figure 1 of their paper. In that figure, changes in the 360-600 nm range are evident when the solution is found to be saturated with helium. This phenomenon turns out to be reversible; in fact, after the passage of air, the absorption band returns to the initial state [63]. The same was observed for _Coriolus zonatus_. [PERSON] et al. [53] investigated the absorption properties of three different types of laccases: blue (_Trantees versicolor_), white (_M. verrucaria_ ITCC 8447), and yellow (_Stropharia_ sp. ITCC 8422) laccase. Their spectra show significant differences. The absorption spectrum of _Trantees versicolor_ presents a peak at 605 nm (Type I Cu atom), whereas, in the case of white and yellow laccases, the peak is absent and there is a weak absorption band at 400 nm [43]. [PERSON] et al. [64] presented a work highlighting the optical properties of the laccases from several basidiomycetes (_Trantees ochraca_, _Trantees hirsuta_, _Coriolopsis fulvocinerea_ and _Cerena maxima_). Spectroscopic investigations were accomplished to understand the characteristics of the active sites of the different laccases mentioned above.