Multicopper laccases

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. 

Some proteins may have redox centers with ° s significantly higher than +0.4 V (e.g., the multicopper protein laccase). No general approach is presently available to deal with the high end of the biological redox window, and, in fact, the reduction potentials of these systems (again, e.g., laccases) are frequently poorly defined. 

Copper oxidases Blue oxidases (multicopper oxidases) Laccase Ascorbate oxidase Ceruloplasmin... 

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. 

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. 

Copper has an essential role in a number of enzymes, notably those involved in the catalysis of electron transfer and in the transport of dioxygen and the catalysis of its reactions. The latter topic is discussed in Section 62.1.12. Hemocyanin, the copper-containing dioxygen carrier, is considered in Section 62.1.12.3.8, while the important role of copper in oxidases is exemplified in cytochrome oxidase, the terminal member of the mitochondrial electron-transfer chain (62.1.12.4), the multicopper blue oxidases such as laccase, ascorbate oxidase and ceruloplasmin (62.1.12.6) and the non-blue oxidases (62.12.7). Copper is also involved in the Cu/Zn-superoxide dismutases (62.1.12.8.1) and a number of hydroxylases, such as tyrosinase (62.1.12.11.2) and dopamine-jS-hydroxylase (62.1.12.11.3). Tyrosinase and hemocyanin have similar binuclear copper centres. 

Blue Multicopper Oxidases. These include laccases, ascorbate oxidase, and ceruloplasmin [22,61], which along with cytochrome c oxidase (CcO with Fe and Cu) can couple the one-electron oxidation of substrates (e.g., ascorbate, diamines, monophenols Fe2+ for ceruloplasmin cytochrome c, for CcO) to the full reduction of dioxygen to water (i.e., 02 + 4c + H+ —> 2H20). 

Some proteins contain more than one copper site, and are therefore among the most complicated and least understood of all. The active site known as type 4 is usually composed of a type 2 and a type 3 active site, together forming a trinuclear cluster. In some cases, such proteins also contain at least one type 1 site and are in this case termed multicopper oxidases, or blue oxidases [3], Representatives of this class are laccase (polyphenol oxidase) [7-9], ascorbate oxidase (Figure 5.Id) [10], and ceruloplasmin [11], which catalyze a range of organic oxidation reactions. 

The enzyme catalyses the oxidation of L-ascorbic acid, 4, to dehydroascorbic, 5. The blue protein belongs to the group of blue oxidases together with laccase and ceruloplasmin. These are multicopper enzymes catalysing the electron reduction of molecular oxygen to water with concomitant one electron oxidation of the substrate. 

The multicopper oxidases (laccase, ascorbate oxidase, and ceruloplasmin) catalyze a four-electron reduction of dioxygen to water (285-287). Consistent with the four-electron stoichiometry, the enzymes contain four copper ions. One of the copper ions is type I, causing an intensely blue color of the proteins, thus the enzymes of this family are referred to as blue oxidases. They also contain a monomeric copper site that exhibits normal spectroscopic features, whereas the other two copper... 

The second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

Multicopper blue oxidases are synthesized as a single polypeptide chain, which is composed of three BCB domains in the case of laccases (LC) and ascorbate oxidases (AO) and six such domains in ceruloplasmin (CP) and hephaestin (HP). Structurally they are arranged in a triangular manner. These enzymes, along with heme-copper oxidases (cytochrome c oxidases and quinol-oxidases) and a cyanide-resistant alternative oxidase found in mitochondria of plants and fungi, are the only known enzymes capable of catalyzing four-electron reduction of dioxygen to water. In the... 

Arabidopsis thaliana has three different genes encoding ascorbate oxidases. They display 50-70% sequence identity with one another and only 20-25% identity with the proteins of the LC family. They all have a Met as the axial ligand for blue copper, whereas most plant laccases have Leu or He (see Fig. 9). A multicopper blue oxidase has also been characterized from fungus Acremonium sp. HI-25 and identified as AO because of its... 

Guss et al., 1992 Romero et at., 1994). In stellacyanin, a glutamine Os fulfills this role (Hart et al., 1996 Vila and Fernandez, 1996). In azurin, in addition to the axial methionine, a peptide carhonyl of a glycine acts as weak axial hgand located on the opposite side to Met (Baker et al., 1988 Nar et al., 1991) (Fig. 2). The multicopper oxidase laccase possesses a trigonal Type I site, with no axial hgand (Ducros et al., 1998). However, no NMR studies are available for any protein of the latter class and they are not considered further here. 

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. 

The high-affinity pathway involves oxidation of Fe to Fe by the ferroxidase FET3 and subsequent transport of Fe " " across the plasma membrane by the permease FTRl. FET3p is a member of the family of multicopper oxidases, which include ascorbate oxidase, laccase, and ceruloplasmin (see Chapter 14), and does not become functional until it is loaded with copper intracellularly through the activities of the copper chaperone ATX Ip and the copper transporter CCC2p. It appears that Fe " " produced by FET3 is transferred directly to FTRl, and does not equilibrate with the bulk phase, as is illustrated in Fig. 7.13. This is almost certainly achieved by the classic metabolite-channeling mechanism, a common feature of multifunctional enzymes. 

An important family of multicopper enzymes couple the reduction of O2 to H2O with substrate oxidation. They include ascorbate oxidase, ceruloplasmin, Fet3, hephaestin, and laccase, and contain at least four copper ions. The four Cu ions are distributed between one type 1 blue copper site, one type 2 site, and one type 3 copper site. The blue Type 1 site is usually located some 12—13 A distant from a trinuclear site which has the two Type 3 coppers, linked by a bridging oxygen and one Type 2 copper. We illustrate this class of oxidases with laccase which catalyses the four-electron reduction of O2 to water, coupled with the oxidation of small organic... 

Multicopper blue copper proteins include ascorbate oxidase and laccase. These are metalloenzymes that catalyse the reduction of O2 to H2O (equation 28.9) and, at the same time, an organic substrate (e.g. a phenol) undergoes a one-electron oxidation. The overall scheme can be written in the form of equation 28.10 R undergoes polymerization. 

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