Laccase blue-copper oxidases

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

Blanford, C.F., Foster, C.E., Heath, R.S., and Armstrong, F.A. (2008) Efficient electrocatalytic oxygen reduction by the blue copper oxidase, laccase, directly attached to chemically modified carbons. Faraday Discussions, 140, 319-335. 

Figure 1. Proposed mechanism for the catalytic cycle and dioxygen reduction site structure in the blue copper oxidase, laccase (after ref. 19, with permission).

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... 

Ceruloplasmin is a member of the family of blue copper oxidases which also contains laccase and ascorbate oxidase. The relationship... 

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

The redox potential of blue copper oxidases varies from species to species. The high redox potential of around 700 mV in fungal laccase is primarily attributed to nonaxial methionine ligand, a geometry that stabilizes the reduced state. Other factors such as solvent accessibility, dipole orientation, and hydrogen bonding also play an important role. ... 

Laccase is perhaps the metallo-enzyme most widely used for this aim. Laccases are a family of multicopper ( blue copper ) oxidases widely distributed in nature Many laccases have fungal origin, while others are produced in plants. They contain four Cu(II) ions, and catalyse the one-electron oxidation of four molecules of a reducing substrate with the concomitant four-electron reduction of oxygen to water . In view of their low redox potential, which is in the range of 0.5-0.8 V vs. NHE depending on the fungal source laccases typically oxidize phenols (phenoloxidase activity) or anilines. 

Laccase, 36 318, 329, 40 122 see also Blue copper oxidases amino-acid sequences, 40 141 anaerobic reduction, 40 158-160 biological function, 40 124 electrochemistry, 36 360 fungal, 40 145-152 evolution, 40 153-154 inhibition, 40 162 kinetic properties, 40 157-162 molecular and spectroscopic properties, 40 125-126... 

So-called blue multinuclear copper oxidase enzymes, such as laccase and ascorbate oxidase, catalyze the stepwise oxidation of organic substrates (most likely in successive one-electron steps) in tandem with the four-electron reduction of O2 to water, i.e. no oxygen atom(s) from O2 are incorporated into the substrate (Eq. 4) [15]. Catechol oxidase, containing a type 3 center, mediates a two-electron substrate oxidation (o-diphenols to o-chinones), and turnover of two substrate molecules is coupled to the reduction of O2 to water [34,35]. The non-blue copper oxidases, e.g. galactose oxidase and amine oxidases [27,56-59], perform similar oxidation catalysis at a mononuclear type 2 Cu site, but H2O2 is produced from O2 instead of H2O, in a two-electron reduction. 

Laccases are usually monomers and are considered to be the simplest blue copper oxidases. A fungal genome may express multiple LC isoforms that differ by their substrate specihcity, pH optimum, and redox potentials (Germann et al., 1988 Wahleithner et al., 1996 Xu, 1996 Yaver and... 

The presence of this ESR non-detectable copper has been established in other blue copper oxidases including Rhus laccase (60), ceruloplasmin (98), and cytochrome oxidase (99),... 

The function of the diamagnetic copper in the blue copper oxidases is not clear. Anaerobic titrations of Polyporus laccase with a number of... 

Furthermore, based on earlier calculations (39) for the type 1 copper protein plastocyanin, ligand-field parameters for the blue copper in laccase have been derived. These reports (37,38) also include a structural representation of the type 1 center composed of a flattened tetrahedron (D2d symmetry) with two imidazole side-chains, a cysteine sulfur, and a fourth ligand (which probably is methionine sulfur), bound to the metal ion. Although no such low-temperature experiments have been performed with ascorbate oxidase, one might anticipate similar structural features for the blue type 1 centers. 

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]. 

The blue copper oxidases are similar to cytochrome oxidase in their ability to catalyze reduction of Oj to HjO. Catalysis is centered upon the protein-bound copper ions that can be differentiated into three classes according to their physical, chemical, and functional properties. They are designated Types 1, 2, and 3 copper . In the blue copper proteins (tree and fungal laccases, ceruloplasmin, ascorbate oxidase) these three classes of copper appear in varying amounts the laccases contain the minimum amounts of each (one each of Types 1 and 2 and two Type 3 coppers). 

Laccase, ascorbate oxidase, and ceruloplasmin are the classical members of the multicopper oxidase family also known as blue oxidases. Recently, a small number of bacterial members of this family have been characterized, including CueO from E. coli a spore-coat laccase (CotA) from Bacillus suhtilis and phenoxazinone synthase from Streptomyces antibioticus The catalyzed reaction of these enzymes except for phenoxazinone synthase is given in Equation (11). A comprehensive overview of the broad and active research on blue copper oxidases is presented in Messerschmidt. Recent results have been included in a review on the reduction of dioxygen by copper-containing enzymes. The nature and number of the different copper sites in blue oxidases has been described in the sections about the type-1 copper site and the trinuclear copper cluster. 

Some microbial pathogens can circumvent the defensive response of plants by biotransforming the antimicrobial stilbenoids in a multi-step oxidative detoxification process [106], Research has shown that the pathogenicity of B. cinerea strains is positively correlated with these fungi s production of blue-copper oxidases known as stilbene oxidases or laccases [127,128]. These enzymes are polyphenol oxidases capable of catalyzing the oxidation and polymerization of numerous phenolic substrates [129,130,131,132]. It has been shown that 1 is readily transformed in the presence of B. cinerea culture medium filtrates that contain laccases [107]. Recently, six resveratrol dimers (restrytisols A-C... 

Multicopper Oxidases (Blue Copper Oxidases) Ascorbate Oxidase, Ceruloplasmin, and Laccase. The multicopper oxidases (MCOs) are important enzymes, which are found in many plants (lignin formation), fungi (lignin degradation and detoxification), bacteria, as well as humans (ferroxidase activity) (13). MCOs catalyze the four-electron reduction of O2 to two waters with the electrons coming firom one-electron oxidation of four substrate molecules. The latter are organic reductants for ascorbate oxidase (AO) (32) and laccase (Lc) (130), and a metal ion (ferrous ion) for ceruloplasmin (Cp) (33) (Scheme 9). 

Like other chemical fuel cells, EFCs have cathode-receiving oxidant and anodereceiving reductant or fuel. For most EFCs, O2 is the oxidant of choice because it is freely available and has a high reduction potential, thus maximising the voltage output of the cell. The enzymes commonly used for O2 reduction at cathode are blue copper oxidases such as laccase or bilirubin oxidase. Peroxidases containing iron... 

Laccases are used at the cathode of a BFC to catalyze the four-electron electroreduction of 02(which acts as the terminal electron acceptor) to water. They belong to a group of enzymes called blue copper oxidases," due to the presence of a type 1 (T1) copper site in the enzyme. This copper site acts as the primary electron acceptor and imparts laccases with a blue color. Additional copper ions in type 2 and type 3 (T2/T3) sites form a trinuclear cluster that acts as a binding site for molecular oxygen. Here, the electrons transferred firom the T1 site reduce oxygen to water. Laccases have been the subject of study for several decades, and have found use in fields ranging firom wastewater treatment to the paper industry [19,20], However, it is their DET ability and high redox potential that have led to their use in biosensors and BFCs. 

The mechanism presented above represents a series of stiuctures based on prior knowledge of the laccase system from the hterature and the XAS/FEFF8 analysis presented in this chapter. It is to some extent still unclear in areas (particularly structures II and V), but the aim was to use in situ XAS to elucidate the mechanism of ORR in laccase as it occurs on a BFC cathode. As a result, the mechanism in Figure 15.20 describes the behavior of laccase under the constraints of the experimental conditions for which the measurements were made. It is important to note that many other mechanisms have been proposed. In Particular, the Solomon and Atanassov research groups have been instmmental in providing important and detailed information on the active sites of a variety of blue copper oxidases [42,45,47-50,62,65,68-73]. 

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