Redox potentials blue copper oxidases

The redox potential of blue copper oxidases varies from species to species. The high redox potential of around 700 mV in fungal laccase is primarily attributed to nonaxial methionine ligand, a geometry that stabilizes the reduced state. Other factors such as solvent accessibility, dipole orientation, and hydrogen bonding also play an important role. ... 

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. 

Laccases are usually monomers and are considered to be the simplest blue copper oxidases. A fungal genome may express multiple LC isoforms that differ by their substrate specihcity, pH optimum, and redox potentials (Germann et al., 1988 Wahleithner et al., 1996 Xu, 1996 Yaver and... 

In addition to providing a binding site for the intermediate described above, Type 2 copper appears to mediate interactions between the metal components of the enzyme. Thus Type 2 depleted enzyme shows slow electron transfer between the remaining Type 1 and 3 copper species and the reoxidation of the reduced Type 2 depleted enzyme is slow ". Moreover, the redox potential of the Type 3 copper center depends on the redox and ligation state of the Type 2 center and the formation of a Type II copper-OH species apparently inactivates the protein. It appears, therefore, that deeper insight into the function and mechanism of the blue copper oxidases will come through a more fundamental understanding of the role and position of the Type 2 copper. 

Laccases are used at the cathode of a BFC to catalyze the four-electron electroreduction of 02(which acts as the terminal electron acceptor) to water. They belong to a group of enzymes called blue copper oxidases," due to the presence of a type 1 (T1) copper site in the enzyme. This copper site acts as the primary electron acceptor and imparts laccases with a blue color. Additional copper ions in type 2 and type 3 (T2/T3) sites form a trinuclear cluster that acts as a binding site for molecular oxygen. Here, the electrons transferred firom the T1 site reduce oxygen to water. Laccases have been the subject of study for several decades, and have found use in fields ranging firom wastewater treatment to the paper industry [19,20], However, it is their DET ability and high redox potential that have led to their use in biosensors and BFCs. 

Ceruloplasmin is involved in copper storage and transport as well as in iron mobilisation and oxidation. Among the blue oxidases it is unique since it contains, in addition to the usual motif of a type 1 combined with the trinuclear cluster, two other type 1 coppers. Electron transfer occurs, however, only between five of the six copper ions since one of the type 1 centres is not catalytically relevant due to its too high redox potential. The redox potentials of the centres were determined and possible electron transfer pathways among the copper sites were discussed.101... 

Some progress has been made in the characterization of the redox centres. The presence of a potential type 1 blue copper site in subunit I is in accord with EXAFS data that have demonstrated Cu—S interactions, in particular the possibility of two sulfur atoms bound to CuA.1309 It seems possible therefore that CuA is a type 1 copper, typical of copper electron-transfer proteins. The nature of CuB is less certain ESR parameters indicate that CuB is similar to type 3 copper, which occurs pairwise in copper oxidases as the 02-binding site. 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

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). 

Blue (Type 1) copper proteins are found widely in nature. Typical examples are plasiocyaiiin (MW 10,500) and ascorbale oxidase (MW 150,000) which contain one and eight Cu atoms per protein, respectively. The former serves as a component of the electron transfer chain in plant photosynthesis while the latter is an enzyme involved in the oxidation of ascorbic acid. The oxidized form is characterized by intense blue color due to electronic absorption near 600 nm. In addition, blue copper proteins exhibit unusual properties such as extremely small hyperfine splitting constants (0.003 0.009 cm" ) in ESR spectra and rather high redox potential (4-0.2 0.8 V) compared to the Cu(ll)/Cu(I) couple in aqueous solution. 

Christenson A, Shleev S, Mano N, Heller A, Gorton L. Redox potentials of the blue copper sites of bilirubin oxidases. Biochim Biophys Acta Bioenerg 2006 1757 1634-1641. 

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... 

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