Interaction of Rhus laccase with dioxygen and its reduction intermediates.

Reduction-oxidation of native Rhus vernicifem laccase produces specific long-lived spectral changes in the near UV and visible region. These can be attained 1) in aerobic reduction experiments, 2) from partially and fully reduced laccase molecules reacting with 0 2 , 3) by reacting peroxylaccase with 2 reduction eq, and 4) upon reoxidation of fully reduced laccase by 2 mol eq of HzOz. The spectral changes are interpreted as arising from an enzyme form, named C state which, although fully oxidized, is different from the native state and is most probably of catalytic importance. The formation of the C state is proposed to involve changes in the coordination geometry of at least one copper site. The changes in the near W spectrum occurring upon conversion of the native oxidized enzyme into the C state are related to the type 3 site, while those in the visible spectrum suggest perturbation of the type 1 chromophore. We conclude that laccase can interact with O2 in any one of its reduced states, whether containing 1,2 , 3, or 4 electron eq. The reaction of Rhus laccase, containing 2 reduction eq, with O2 leads to the formation of a species which has spectroscopic and chemical properties closely related to those of the peroxy compound obtained by reacting native oxidized laccase with HzOz and is therefore identified with it. A long-lived transient species with an extra absorbance extending from 300 to 700 nm and peaking in the near W region is rapidly formed upon reaction of O2 with partially reduced enzyme molecules (reduced either in the absence or in the presence of Oz). It is suggested that it originates from laccase molecules containing 3 reduction eq. In the absence of external electron donors, it decays slowly in a monomolecular reaction (kl = 4 X s-’, 20°C, pH 7.0). The turnover of laccase with ascorbate as substrate can be monitored by conventional absorption spectroscopy (0.25 l l l ~ 0 2 , pH 7,20°C). The establishment of the steady state phase, starting from native oxidized laccase, is accompanied by an absorbance increase in the 330 to 360 nm region and by a small and concentrationindependent decrease in the intensity of the type 1 Cu(II) band at 615 nm. These difference spectra were resolved into components which could be assigned to catalytic intermediates of the enzyme. Apparently, the first reduction-oxidation cycle during the pre-steady state phase converts native enzyme molecules into the C state. At low reductant concentration, this species is presumably the predominant enzyme form present dur-