Multicopper oxidases oxidation site

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. 

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. 

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 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... 

The three copper sites which represent a total of four one-electron acceptors place the multicopper oxidases in the small family of enzymes that can couple the oxidation of four equivalents of a one-electron reducing... 

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. 

Cu-binding sites, and this serves as one of the better examples of protein mediated Cu-Cu interaction in the multicopper oxidases. These authors also established that the EPR and immunochemical properties of reconstituted ceruloplasmin were identical with those of native oxidized protein. 

Cu nitrite reductase The Cu-dependent nitrite reductase, which transforms nitrite to nitric oxide in denitrifying bacteria, contains both the type-1 and the type-2 Cu center but lacks the binuclear type-3 site [89]. Yet, its arrangement of Cu atoms within the protein indicates a strong structural relationship with the classical blue multicopper oxidases [85], It catalyzes the single-electron reduction of NO2 to NO and water [Eqs. (5)-(7)] ... 

Trinuclear Cu clusters play a central role in catalytic O2 reduction in the multicopper oxidases [1,76]. These enzymes contain at least four Cu centers that are necessary to catalyze the 4e reduction of O2 to H2O with concomitant le oxidations of various substrates (Fig. 6). The electrons are taken up at the T1 blue copper site and transferred 13 A to the trinuclear copper cluster site that is comprised of a T2 and a T3 copper center where O2 reduction occurs [100]. Reaction of the fully reduced enzyme with O2 involves two 2e intermediate stages, where the first 2e reduction step is rate determining (A 2 x 10 M s ) while the second is fast (k > 350 s ), therefore effectively resulting in a single 4e process [101,102]. 

Figure 6. Copper sites in the resting oxidized form of the multicopper oxidases and associated ground state orbitals.

Figure 5.1 Schematic representations of selected active sites of the copper proteins plastocyanin [56] (type 1, a) galactose oxidase [57] (type 2, b) oxy hemocyanin [58] (type 3, c) ascorbate oxidase [10] (type 4, or multicopper site, d) nitrous oxide reductase [59] (CuA site, e) cytochrome c oxidase [15]...

Many of the coupled two-copper (and some multicopper) sites are to be found in oxidases. Their function is to assist in the four-electron reduction of 02 to water with concomitant oxidation of a substrate. To do this they must first receive and bind the 02 molecule and the peroxo complexes mentioned earlier appear to be models for the way they do this. A cycle of activity for tyrosinase illustrates this, Fig. 17-H-6. 

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... 

---