Laccase reoxidation

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

On adding dioxygen to the fully reduced laccase of the lacquer tree Rhus vemicifera, the type-1 Cu and the type-3 Cu-pair were oxidized in the ms range and an optical intermediate was observed at 360 nm At liquid helium temperatures an EPR signal was observed, which was tentatively interpreted as due to O ", as a result of its very short relaxation time and of the increase of its linewidth when the reduced laccase of the fungus Polyporus versicolor was treated with 0 A similar paramagnetic oxygen intermediate was also observed with the laccase of another lacquer tree Rhus succedanea and with ceruloplasmin. The decay of the intermediate at 25 °C (tj = 1 s at pH 5.5 with R. succedanea laccase) was accompanied by the reoxidation of the type-2 Cu >. One would expect, however, such an intermediate to be extremely reactive (See Sect. 3.3), while it was stable in tree laccase depleted of type-2 Cu(II)... 

The reduction of laccase by O J obtained by pulse radiolysis in the presence of Oj was followed by the decrease of the absorbance at 614 nm of type-1 Cu. Only a very partial reduction was observed (up to 7%). The binding of F anions to type-2 Cu lowered the reduction and the reoxidation rates... 

Figure 10. Rhus laccase—reduction by 02 and reoxidation by oxygen observed at 614 nm. Solution contained 1.5 X 10 5M enzyme, 1.2 X 10 3 M oxygen, 1% tert-butanol, pH 6.9, 25°. Upper trace ...

In the presence of molecular oxygen, and only then, the type 1 copper is reoxidized in a first-order process. The type 1 copper is generally thought to be deeply embedded inside the protein, therefore a direct interaction between oxygen and this copper site is improbable. This, together with the fact that no laccase molecule contained more than one reduction equivalent suggests the following ... 

Figure 12 shows the titration data in the form of a Nemst plot of the 330-nm absorbance against the 614-nm absorbance, including values up to full reoxidation of the type 1 chromophore. The slope n of the straight line is 1. This is in contrast to the value obtained from titrations of oxidized laccase with hydroquinone as reductant, which gave n = 2... 

The reoxidation studies on laccase and ascorbate oxidase are listed in Table IX. The reoxidation of the type-1 copper and of the trinuclear copper site occurs at a rate of 5 x 10 M" sec" both for tree laccase 134) and for ascorbate oxidase 135). During reoxidation with H2O2, an 02 " intermediate is formed in several minutes, which is documented for tree laccase by changes in the CD spectrum 136) and for ascorbate oxidase in the formation of an absorption band at 350 nm... 

In studies on the anaerobic reduction of tree laccase by hydroquinone and ascorbate (49), the existence of a plateau phase at low substrate concentration was reported for the reaction of the type 1 copper. This observation was explained in terms of an intramolecular reoxidation by the type 3 copper pair. A similar plateau phase is a dominant feature of the reduction of both chromophores of ascorbate oxidase by reductate (Figure 7). However, the plateau phase is only observed in the presence of "contaminating dioxygen rigorous removal of these dioxygen traces removes the plateau phase at all wavelengths. The reaction of reduced ascorbate oxidase with dioxygen is very rapid, k = 5 X 10 at pH... 

Hexacyanoferrate(III) was added to the samples in order to oxidize reductive interferents such as ascorbic acid, and the hexacyanofer-rate(II) formed was reoxidized by a laccase-catalyzed reaction. This approach appears to be particularly useful for samples with extreme concentrations of oxidizable compounds, such as urine. 

Finally, it is of interest to compare intramolecular ET in hCp with the corresponding processes in laccase and ascorbate oxidase. As in hCp, pulse radiolytically produced RSSR radicals are also the primary reaction products in tree laccase (73), and the reduction equivalents are further transferred to the TFCu ) center in an intramolecular process. The rate of Tl(Cu ) reoxidation by intramolecular ET to T2/T3 takes place unimolecularly with a rate constant of 2s at room temperature, similar to that observed in hCp (2.9 s ), which is hardly surprising, since the stmctural arrangements of the T1-T2/3 sites in these two proteins are quite similar (the driving forces also are comparable). The situation in ascorbate oxidase, however, was found to be more complex (Section... 

Glucose measurements in urine and fermentation samples by means of GOD electrodes based on hydrogen peroxide detection usually suffer from interferences by anodically ox-idizable compounds. These can be oxidized in the measuring solution by reaction with hexa-cyanoferrate(III). However, the hexacyanoferrate(lI) ion formed is also oxidizable at an electrode potential of +600 mV. In order to prevent the hexacyanoferrate(II) from reaching the electrode, laccase has been co-immobilized with GOD in the sensor membrane [352]. Thus, the mediator is reoxidized in a laccase-catalyzed reaction with consumption of oxygen. The system is capable of shielding the electrode from ascorbic acid at concentrations up to 2 mmol/L. 

There are various alternatives for reoxidizing the hydroxylamine back to TEMPO to complete the catalytic cycle. It can be oxidized by dioxygen, laccase or the oxoammonium cation. The active oxidant is the same as that in the TEMPO catalyzed oxidations of alcohols with hypochlorite (or other single oxygen donors), a method which is widely used in the oxidation of a broad range of alcohols using low catalyst loadings (1 mol % or less) (59). 

The redox properties can be divided among those involved with its reduction by potential substrates of the enzyme, its reoxidation by molecular oxygen, and by electron transfer to and from other redox centers within the molecule. Type 1 Cu + is reduced rapidly by a variety of both one-electron and potential two-electron substrates via a one electron process 63, 64). A likely reason for this ease of reduction of Type 1 Cu2+ ion lies with its exceptionally high redox potential (0.77 V at pH 6.2, Table 2). Introduction of molecular oxygen to reduced laccase results in a rapid reappearance of the blue color characteristic of Type 1 Cu2+. The question of intramolecular electron transfer occurring during reoxidation will be considered below. 

With the exception of a study carried out with a partially characterized multicopper oxidase isolated from tea leaves (85), there has been very little detailed work concerned with the steady state kinetic behavior of laccases. Early work on the transient kinetics indicated, however, that (1) enzyme bound Cu + was reduced by substrate and reoxidized by O2, and (2) substrate was oxidized in one-electron steps to give an intermediate free radical in the case of the two electron donating substrates such as quinol and ascorbic acid. The evidence obtained suggested that free radicals decayed via a non-enzymatic disproportionation reaction rather than by a further reduction of the enzyme (86—88). In the case of substrates such as ferrocyanide only one electron can be donated to the enzyme from each substrate molecule. It was clear then that the enzjmie was acting to couple the one-electron oxidation of substrate to the four-electron reduction of oxygen via redox cycles involving Cu. 

Fig. 5. Time course of the approach to the steady state in the system laccase, ferrocyanide, and oxygen at pH 5.4. The reduction in laccase (bottom frame) and ferricyanide formation (upper frame). The concentration of laccase was 8.3 //M, the concentration of ferrocyanide was 2.5, 5.0, 12.5, 25, 125, 500 and 1000 //M and the concentration of oxygen was 275 fiM.. Note that with the higher concentrations of ferrocyanide Type I Cu + is initially reduced beyond the steady state level and significant reoxidation occurs during the steady state. Also note the distinct lag in product formation subsequent to the initial burst and prior to the zero-order condition. This is most evident in the traces corresponding to 25 and 50 //M ferrocyanide. [Taken from McUmstrom, Finaszi-Agro, and AntoninaRef. (94)]...

Holwerda and Gray (97) proposed a mechanism for the reduction process involving a central role of Type 2 Cu2+ as the initijil point at which electrons enter and are subsequently distributed to the other electron acceptors. This interpretation would seem to be supported by the very recent observation of Branden and Reinhammar (98) that the Type 2 ion of Poljqjorus laccase is reduced and subsequently reoxidized in a very short time period. The authors also emphasize the parallel behavior of the T q)e 1 and Type 3 centers it is particularly striking that under a variety of conditions the rates of Type 1 and 3 reductions are very similar. Indeed, when Cr2+ was used as the reductant, a similar observation was made (99). Perhaps Cr2+ reduces the enzyme via a bridging ligand (H2O ) between it and Type 2 Cu2+. 

The conditions used for these experiments indicate that a greater effect can be created by metering in the substrate. Oxygen is required in these incubations to reoxidize the laccase. It might also react with laccase generated radicals and propagate oxygen free radicals that could be additional reactants. 

In 2011, alcohol dehydrogenase (ADH) was used as a model enzyme coupled with poly (MG) for NADH reoxidation in the construction of a three-dimensional BFC with ethanol as fuel [103]. In combination with an air-breathing/gas difhision cathode (using laccase as an oxygen reduction enzyme), a BFC was fabricated that was able to successfully exploit ethanol oxidation by an NAD -dependent ADH, immobilized by entrapment in a multiwaUed CNT (MWCNT)/chitosan matrix [106]. The feasibility and reproducibUity of the resulting BFC were demonstrated in 2008 with a series of standardized multilaboratory experiments [96]. 

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