Multicopper oxidases center types

The goal of our research on the multicopper oxidases has been to determine the spectral features of the type 3 (and type 2) centers, to use these spectral features to define geometric and electronic structural differences relative to hemocyanin and tyrosinase, and to understand how these structural differences contribute to their variation in biological function. The hemocyanins and tyrosinases reversibly bind and activate dioxygen whereas the multicopper oxidases catalyze its four-electron reduction to water. 

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 presence of a single type 2 center in ascorbate oxidase is not consistent with the proposed concept of a quaternary structure composed of two identical subunits afi (25). On the other hand, all the multicopper oxidases described in the literature (5-8) have only one type 2 center per active molecule. Additional copper with type 2 characteristics can be bound by the macromolecule during isolation and purification (19). A close examination of the EPR spectra presented by Lee and Dawson (9) indicates the presence of so-called nonspecific copper with large hyperfine splittings at g. As expected, the ratio A330/A610 is approximately 1.5-2 for these preparations (estimated from Figure 2 in Ref. 9). 

Multicopper oxidases containing a type-1 copper center and a trinuclear copper site 527... 

Multicopper oxidases are typically active in the catalytic one-electron oxidation of a variety of diphenolic, polyphenolic, enediolic, and aminophe-nolic substrates 1,53,166,167). The mechanism of these reactions is complex and, as discussed in Section I, it involves a sequence of four one-electron oxidations of substrate molecules. The radical products of these reactions undergo dismutation, as shown in Scheme 21 for the oxidation of ascorbate to semidehydroascorbate radical 168,169). The substrate binds to the enzymes close to type 1 Cu, whereas the trinuclear cluster is only accessible to dioxygen, or other small molecules. This situation is clearly difficult to reproduce in a model system and for this reason the type of model oxidation reactions that have been studied so far using synthetic trinuclear copper complexes is more related to the activity of type 3 Cu enzymes than multicopper oxidases. Nevertheless, such trinuclear complexes open new perspectives in stereoselective catalysis, because one of the metal centers... 

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 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)] ... 

Blue multicopper oxidases (BMCOs) such as laccase, ceruloplasmin, bilirubin oxidase (BOx), and ascorbate oxidase (AOx) have been extensively investigated as cathodic biocatalysts for DET-based biodevices [44]. BMCOs have a catalytic center consisting of four coppers a type 1 (Tl) Cu site, which accepts electrons from the substrate and from the electrode surface, and a type 2/type 3 (T2/T3) cluster, where O2 is reduced directly to water. High redox potential laccases and BOx, with redox potential up to 780 and 670 mV versus normal hydrogen electrode (NHE), respectively [44,45], can be used to create efficient biocathodes with current densities up to a few mA cm . In 2012, Shleev and coworkers used the DET ability of these enzymes to create several completely DET-based BFCs [42]. The enzymes have also been used in different MET-based approaches [46,47] specifically, Heller and coworkers... 

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). 

The blue copper center in cupredoxins is also found in multidomain, multicopper enzymes " such as ascorbate oxidase (AO), laccase (Lc), human ceruloplasmin (Cp), " and a subfamily of copper-containing nitrite reductase. Nitrite reductase (NiR) catalyzes reduction of nitrite (N(32 ) to nitric oxide (NO), a step in the biological dinitrification cycle. Two distinct types of NIR are known multiheme NiR (see Chapter 8.29) and multicopper NiR. The multicopper... 

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