1H NMR of native and azide-inhibited laccase from Rhus vernicifera

The H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time.These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in thehigh frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differencesbetween the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structuralproperties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over1 the T1 copper ligands. Overall, these data demonstrate that H NMR can be fruitfully applied to characterize the electronic properties ofthe metal sites of blue oxidases at room temperature.