Oxygen intermediate, laccase

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)... 

Figure 1. Experimental (left) and simulated (right) EPR spectra of the laccase-oxygen intermediate obtained with 1602 and1702, respectively.

Figure 22. Comparison of oxygen intermediates. A Electronic absorption spectra of the peroxy-intermediate in laccase versus oxyhemocyanin and oxytyrosinase. B Proposed structural differences between peroxide binding in oxyhemocyanin and oxytyrosinase relative to the end-on bound hydroperoxide intermediate at the trinuclear copper cluster in laccase.

The presumed H202 intermediate appears to decompose by l-e steps, as a paramagnetic intermediate is formed with laccase. With 170 this has been shown to represent an oxygen radical, possibly Or, having unusual relaxation properties. 

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. 

The effect of the distance between the active center and the electrode on the reaction rate has been studied using as an example the electrocatalysis of the oxygen reduction reaction by laccase adsorbed on soot. Variation in the distance between the active center and the electroconductive substrate was achieved by inserting an intermediate monolayer of lipid molecules flatly and vertically oriented cholesterol molecules and vertically oriented lecithin molecules (scheme in Figure 36). In this case, the conditions of obtaining compact lipid monolayers were fulfilled. The subsequent setting of laccase did not lead to their desorption. 

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