Laccase redox potentials

The low specificity of electron-donating substrates is remarkable for laccases. These enzymes have high redox potential, making them able to oxidize a broad range of aromatic compounds (e.g. phenols, polyphenols, methoxy-substituted phenols, aromatic amines, benzenethiols) through the use of oxygen as electron acceptor. Other enzymatic reactions they catalyze include decarboxylations and demethylations [66]. 

Reported redox potentials of laccases are lower than those of non-phenolic compounds, and therefore these enzymes cannot oxidize such substances [7]. However, it has been shown that in the presence of small molecules capable to act as electron transfer mediators, laccases are also able to oxidize non-phenolic structures [68, 69]. As part of their metabolism, WRF can produce several metabolites that play this role of laccase mediators. They include compounds such as /V-hvdi oxvacetan i I ide (NHA), /V-(4-cyanophenyl)acetohydroxamic acid (NCPA), 3-hydroxyanthranilate, syringaldehyde, 2,2 -azino-bis(3-ethylben-zothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), violuric acid, 1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpipperidin-iV-oxide radical and acetovanillone, and by expanding the range of compounds that can be oxidized, their presence enhances the degradation of pollutants [3]. 

Although several works suggested that these enzymes cannot oxidize nonphenolic compounds because the redox potentials of laccases are lower than those of the... 

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. ... 

Different chemical environments surrounding the T1 copper result in different redox potentials. Fungal laccases demonstrate the highest potential, close to the equilibrium potential of oxygen reduction in their respective pH regions (see Table 1). Laccases, however, are anion sensitive, with deactivation involving dissociation of T2 copper from the active site of the enzyme. Alternative copper oxidases such as bilirubin oxidase and ceruloplasmin ° ... 

Palmore et al. first demonstrated the use of ABTS in a biofuel cell cathode, combining it with laccase from Pyricularia oryzae ABTS was dissolved at 2 mM in oxygen-saturated 0.2 M acetate buffer, pH 4, 25 °C. With a glassy carbon working electrode, an open-circuit potential of 0.53 V vs SCE was observed, reflecting the presence of HABTS in low-pH solution. Protonation of ABTS shifts the redox potential to 0.57 V vs SCE. With negligible stirring, current densities of 100 / A/cm were achieved at an electrode potential of 0.4 V vs SCE. 

This electrode is unique in that the bilirubin oxidase is active at neutral pH, whereas the laccase cited above is not, even though the redox potential of laccase is somewhat higher. Additionally, the bilirubin oxidase is much less sensitive to high concentrations of other anions such as chloride and bromide, which deactivate laccase. It was shown that mutations of the coordination sphere of bilirubin oxidase led to an increased redox potential of the enzyme, which increased current density and reduced current decay to 5%/day over 6 days at 300 rpm. The latter improvement was attributed to improved electrostatic attraction between the enzyme and the redox polymer. An electrode made with high-purity bilirubin oxidase and this redox polymer has recently been shown to outperform a planar platinum electrode in terms of activation potential and current density of oxygen reduction. ... 

The fuel cell described above exhibited three key flaws. First, the anode redox mediator operates at a redox potential well above that of glucose oxidase, raising the operating potential of the anode and lowering the achievable cell potential. Second, the cell operates at pH 5, near-optimal for the laccase electrode but suboptimal for the current-limiting glucose... 

The produchon of oxidase enzymes in native strains is often not constitutive, since these enzymes are only required under specific conditions, such as nutrient stress. This imposes limitations on their application in continuous bioprocesses and one way of dealing with this issue is to develop improved expression systems. Laccase from Trametes sp. C30, which in its native strain is inducible and only weakly expressed, has been cloned and expressed in yeast to provide a functional recombinant enzyme with the same high activity and low redox potential as the native enzyme [68]. 

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. 

Operation of the latter mechanism has also been invoked for the oxidation of X-substituted benzyl alcohols with TEMPO and the enzyme laccase becanse the redox potential of the enzyme (0.78 is adeqnate for the oxidation of TEMPO to oxoammonium ion (0.8 Strangely enough, no linear correlation of the log A x/ h ratios... 

Visible MCD spectra of plastocyanin, azurin, Rhus vernicifera laccase, ascorbate oxidase and ceruloplasmin are similar on a per copper basis, but show differences from those of stellacyanin and fungal laccase. This is of interest in view of the absence of methionine from the coordination sphere of copper in stellacyanin, and the very high redox potential of fungal laccase.925... 

Laccase contains one type 1 Cu and one type 2 Cu in addition to the type 3 pair. The copper can be reversibly removed from the type 2 site (to give T2D-laccase). Reconstitution may be accomplished by adding CuS04 or Cu1 under anaerobic conditions.958 Loss of type 2 copper has little effect on the redox potentials of the type 1 and type 3 copper, or on the electron-transfer reactivity of the type 1 copper. It appears that type 2 Cu is a substrate-binding site in the reduction pathway for the blue copper.959... 

Cu(II) is intramolecular. The effect of fluoride on the reduction rate is consistent with both a direct involvement of type 2 Cu(II) in the reduction or an indirect effect mediated via a change in conformation or in redox potential of the type 1 Cu(II). The type 2 copper ion could be the primary electron-accepting site of the laccase molecule, as has been proposed for the reduction of the enzyme by hydroquinone (36), the first-order process observed being therefore the electron transfer from type 2 Cu to type 1 Cu(II). The particaption of type 3 Cu(II) instead of type 2 Cu(II) is not excluded, but no associated change of its absorption band at 330 nm could be observed during the redox cycle described for the 614-nm band. 

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... 

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 redox potential of the Tl Cu-site has been determined using potentiometric titrations with redox mediators for a large number of different laccases and varies between 410 mV vs. NHE for Rhus vernicifera [67] and 790 mV for laccases from Polyporus versicolor and Coriolus hirsutus [244,251]. The T2 and T3 sites have higher potentials [251]. 

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