Reactions of nitric oxide with tree and fungal laccase

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Abstract
The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal [Polyporus versicolor] and tree [Rhus vernicifera] laccase, as well as with tree laccase depleted in type 2 Cu, are reported. The products of the reactions were determined by NMR and mass spectroscopy; the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All 3 Cu sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 Cu. NO similarly reduces all of the Cu sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 Cu becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 Cu, suggesting that it involves intramolecular electron transfer from the type 1 Cu to NO bound to the type 2 Cu. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced Cu of a half-oxidized type 3 Cu site.