Laccase copper

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.

Figure 21. Laccase copper centers required for dioxygen reactivity. A XAS of fully reduced T2D laccase and fully reduced T2D laccase following exposure to dioxygen. B XAS of reduced TIHg laccase and reduced TIHg laccase following exposure to dioxygen. C Summary of the reactivity of deoxy T2D, fully reduced T2D, and reduced TIHg laccase with oxygen.

Another problem to be elucidated is the role of copper in cytochrome a. In the known copper enzymes such as tyrosinase and laccase, copper is an important component of the prosthetic group, but is released from the protein moiety by dialysis against potassium cyanide. In the case of cytochrome a, however, the mode of combination of copper must be different, since very little copper is released from the protein moiety by dialysis. The best known method of releasing the copper is by acid treatment. The role of copper in the electron trasnferring system is still obscure, though Cohen and Elvehjem (1934), Yoshikawa (1937), Schultze (1939, 1941), Gallagher et al. (1956), and Gubler et al. (1957) observed, from dietary experiments, that copper-deficient tissues and yeast have a low cytochrome oxidase activity and a decreased content of hemin a. 

Laccase, 6,699 copper, 6,654 cytochrome oxidases concerted electron transfer, 6,683 fungal... 

C13-0112. Fungal laccase is an enzyme found in fungi that live on rotting wood. The enzyme is blue and contains 0.40% by mass copper. The molar mass of the enzyme is approximately 64,000 g/mol. How many copper atoms are there in one molecule of fungal laccase ... 

Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C. 2002. Crystal structure of a four-copper laccase complexed with an arylamine Insights into substrate recognition and correlation with kinetics. Biochemistry 41 7325-7333. 

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. 

Piontek K, Antorini M, Choinowski T. 2002. Crystal structure of a laccase from the fungus Trametes versicolor at 1.90 A resolution containing a full complement of coppers. J Biol Chem277 37663-37669. 

Fungal laccases (benzenediokoxygen oxidoreductase, EC 1.10.3.2) belong to the multicopper blue phenoloxidases. They comprise glycosylated proteins expressed in multiple forms and variable molecular weight, ranging from 59 to 110 kDa. Laccase is expressed as multiple constitutive and induced isoenzymes [30, 64]. The enzyme contains four copper atoms (Cu), in different states of oxidation (I, II, III) [65], which play an important role in the catalytic mechanism. Laccase oxidizes different compounds while reducing O2 to H20, a total reduction of four electrons. 

The catalytic cycle of laccase includes several one-electron transfers between a suitable substrate and the copper atoms, with the concomitant reduction of an oxygen molecule to water during the sequential oxidation of four substrate molecules [66]. With this mechanism, laccases generate phenoxy radicals that undergo non-enzymatic reactions [65]. Multiple reactions lead finally to polymerization, alkyl-aryl cleavage, quinone formation, C> -oxidation or demethoxylation of the phenolic reductant [67]. 

Many multiple copper containing proteins (e.g., laccase, ascorbate oxidase, hemo-cyanin, tyrosinase) contain so-called type III copper centers, which is a historical name (cf. Section 5.8 for type I and type II copper) for strongly exchange-coupled Cu(II) dimers. In sharp contrast to the ease with which 5=1 spectra from copper acetate are obtained, half a century of EPR studies on biological type III copper has not produced a single triplet spectrum. Why all type III centers have thus far remained EPR silent is not understood. 

Malmstrom, B.G., Mosbach, R., and Vanngard, T. 1959. An electron spin resonance study of the state of copper in fungal laccase. Nature 183 321-322. 

We focus here on the use of oxygenases, particularly the blue copper oxygenases, such as laccase and bilirubin oxidase, which can biocatalytically reduce oxygen directly to water at relatively high reduction potentials under mild conditions. First, however, we will briefly consider reports on the use of hydrogen peroxide as an oxidant in biocatalytic fuel cells. 

Catalytic reduction of oxygen directly to water, while not as yet possible with traditional catalyst technology at neutral pH, is achieved with some biocatalysts, particularly by enzymes with multi-copper active sites such as the laccases, ceruloplasmins, ascorbate oxidase and bilirubin oxidases. The first report on the use of a biocatalyst... 

Laccase was first isolated by Yoshida in 1883 [43] from tree lacquer of Rhus ver-nicifera. Laccases can thus be classified according to their source plant, fungal or, more recently, bacterial or insect [44], The laccase enzyme active site contains four copper ions classified into three types based upon their geometry and coordinating ligands, denoted... 

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 is one of the main oxidizing enzymes responsible for polyphenol degradation. It is a copper-containing polyphenoloxidase (p-diphenoloxidase, EC 1.10.3.2) that catalyzes the oxidation of several compounds such as polyphenols, methoxy-substituted phenols, diamines, and other compounds, but that does not oxidize tyrosine (Thurston, 1994). In a classical laccase reaction, a phenol undergoes a one-electron oxidation to form a free radical. In this typical reaction the active oxygen species can be transformed in a second oxidation step into a quinone that, as the free radical product, can undergo polymerization. 

The laccase molecule is a dimeric or tetrameric glycoprotein, which contains four copper atoms per monomer, distributed in three redox sites. More than 100 types of laccase have been characterized. These enzymes are glycoproteins with molecular weights of 50-130 kDa. Approximately 45% of the molecular weight of this enzyme in plants are carbohydrate portions, whereas fungal laccases contain less of a carbohydrate portion (10-30%). Some studies have suggested that the carbohydrate portion of the molecule ensures the conformational stability of the globule and protects it from proteolysis and inactivation by radicals (Morozova and others 2007). 

Figure 4.4. Schematic representation of laccase trinuclear copper active center.

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). 

Tatiana V, Pegasova P, Zwart O, Koroleva V, Stepanova EV, Rebrikovc DV and Lamzinb V. 2003. Crystallization and preliminary X-ray analysis of a four-copper laccase from Coriolus hirsutus. Acta Crystallogr... 

Reinhammar B (1984) Laccase. In Lontie R (ed) Copper proteins and copper enzymes, vol 3. CRC, Boca Raton, pp 1-36... 

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

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