Copper multicopper oxidase

Figure 12.4 Proposed path for the intracellular transfer of Cu(I) by Atxl. Copper destined for incorporation into the vascular multicopper oxidase Fet3 requires both Ctrl and Ccc2. Cytoplasmic Cu(I)-Atxl, but not apo-Atxl, associates with the amino-terminal domain of Ccc2 and Cu(I) is transferred to the latter. (Inset) A proposed mechanism for the exchange of Cu(I) involving two- and three-coordinate Cu-bridged intermediates. The human homologues of Atxl (Hahl), Ccc2 (Menkes and Wilson s proteins) and Fet3 (ceruloplasmin) are likely to employ similar mechanisms. Reprinted with permission from Pufahl et al., 1997. Copyright (1997) American Association for the Advancement of Science.

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. 

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. 

Other Copper Oxidases. A number of additional multicopper oxidases have been detected [22], including phenoxazinone synthase (Table 1). This enzyme catalyzes the overall six-electron oxidative coupling of 2-aminophenols to form 1-aminophenoxazinone, the final step in the bacterial (Streptomyces) biosynthesis of the antineoplastic agent actinomycin. 

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... 

This research was supported by the National Science Foundation (CHE-9217628) for the blue copper studies and by the National Institutes of Health (DK-31450) for the coupled binuclear and multicopper oxidase studies. Edward I. Solomon expresses his sincere appreciation to all his students and collaborators who are listed as co-authors in the references for their commitment and contributions to this science. 

What distinguishes multicopper oxidases from other copper proteins is that they contain one each of these three types of copper site (Solomon and Lowery, 1993 Solomon et al., 1996). Not only does this make them excellent models for all copper proteins, but because they have four redox-active metal ions, they also serve as paradigms for other enzymes that couple a one-electron reductant to a four-electron oxidant, most notably cytochrome c oxidase. Indeed, the three copper sites (and four copper atoms) in the multicopper oxidases play essentially equivalent roles in comparison to the two heme groups and two copper atoms in cytochrome c oxidase. 

One also should be aware of the cohort of proteins that provide copper resistance to bacteria that commonly are encoded by an extrachromoso-mal element. Sequence analysis indicates that one of these proteins is a multicopper oxidase since this member of the group contains the copper liganding motifs highlighted in Fig. 1. However, this member also contains a M/S-rich motif that is thought to be essential to the copper trafficking supported by a copper transporter like Ctrlp Sa. cerevisiae) (Dancis et al., 1994) or the CopA (Cha and Cooksey, 1991) and CopB... 

This putative protein product (gil073083) is termed a copA homo-logue although it does not have the CXXC motifs common to the gene products similar to the copA protein from Escherichia coli that is a known copper-translocating ATPase (Rensing et al., 2000). In summary, bacteria also produce multicopper oxidases, and they potentially could be involved in metal metabolism. However, essentially no research has been reported that in any way tests this possible involvement. 

Fig. 8. Spatial relationship between the three copper sites in a multicopper oxidase. The structure (and residue numbering) is a representation of these sites in hCp. The trinuclear cluster is a near-isosceles triangle, 3.4 A on a side. The notation Cu(2) and Cu(3) is taken from Messerschmidt et al. (1992a) and corresponds to the notation Type 3" and Type 3, respectively, used by Zaitseva et al. (1996). Oxygen is thought to bind between the type 2 copper and Cu(2).

These copper site mutants have been particularly useful in two different types of experiments. First, the T2D protein allows for investigation of the electron transfer to the type 1 Cu(II) in the absence of turnover. This is because in the multicopper oxidase reaction, electron transfer from the type 1 copper—as Cu(I)—to the trinuclear cluster where O2 is reduced requires the type 2 Cu(II) (Solomon and Lowery, 1993 Solomon... 

One recombinant FetSp mutant is unique among multicopper oxidase species and has been particularly informative about the structure of the type 3 binuclear cluster in these species. This is the T1D/T2D double mutant that contains only this type 3 site (Blackburn et al., 2000). EXAFS analysis of this protein contains contributions from electron ejection and scattering from only the type 3 copper atoms and thus provides direct structural information about this cluster. The K-edge XAS spectrum for this mutant in its oxidized and reduced states is shown in Fig. 21. The oxidized sample has a nearly featureless edge with a midpoint energy of 8990 eV typical of tetragonally distorted type 2 Cu(ll) centers, i.e., those with predominantly histidine imidazole coordination. The reduced type 3 cluster exhibited a pronounced shoulder at 8984 eV just below the... 

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