Blue copper proteins multicopper oxidases

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). 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

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. 

The copper centres in the multicopper blue oxidases have been classified into three groups. This classification may be extended to include other copper proteins. 

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 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... 

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... 

The blue multicopper oxidases constitute a heterogeneous family of enzymes from different sources (7). In addition to the well characterized members of this family, ascorbate oxidase (45,46), laccase (47,48), and ceruloplasmin (49,50), all from higher organisms, two other proteins have attracted much recent interest FetSp, which is involved in iron uptake in yeast (51), and CueO, which is required for copper homeostasis in Escherichia coli (52). The characteristic reactivity of these enzymes is the one-electron oxidation of four substrate equivalents coupled to the four-electron reduction of dioxygen to water (1). These processes occur at a catalytic unit constituted by four copper atoms classified according to their spectroscopic properties in... 

Recently several laccases have been crystallized and their three-dimensional crystal structures are now available 13,33-35). Laccase belongs to the class of multicopper-oxidases and contains four copper centers per protein molecule type 1 (Tl) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled dinuclear copper centers 36). It catalyzes the oxidation of electron rich aromatic substrates, usually phenols or aromatic amines, via four single electron oxidation steps concomitant with the four electron reduction of O2 to H2O 15,37). Electron transfer takes place at the Tl site, which is followed by electron transfer to the T2/T3 assembly. This is where the reduction of dioxygen takes place. 

Ceruloplasmin is a blue a-2 glycoprotein of 132 kD. This plasma protein is responsible for the binding of 90 to 95% of the blood plasma copper in vertebrates. In addition to its primary role in copper transport and homeostasis, it possesses a number of additional enzymatic activities (see ref. 179 for a recent review). The complete amino acid sequence of human ceruloplasmin has been determined (42), establishing that this large multicopper oxidase is synthesized as a single chain polypeptide, containing 1046 amino acid residues. 

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