Magnetic studies of the trinuclear center in laccase and ascorbate oxidase approached by EPR spectroscopy and magnetic susceptibility measurerments

The trinuclear centers in Rhus vernicifera laccase and Cucumis sativas ascornate oxidase have been studied by EPR spectroscopy and magnetic susceptobility measurements over the wide range of 5K to 300K. The EPR spectra showesd that type 2 copper receives increaseing tetrahedral distortion with raising temperature. magnetic susceptibilities of laccase showed that both of type 1 and type 2 coppers are almost fully paramagnetic since the antiferromagnetic interaction between typpe 3 coppers is extremely strong from 5K to 300K. On the other hand, the effective magnetic moment of ascorbate oxidase is contributed by ca. 1.7Cu2+ even below ca. 100K, since type 2 Cu is partly in the redused form. The effective magnetic moment continuously increased with raising temperature because the antiferromagnetic interaction between type 3 coppers of not as srong as in the case of laccase. The simulation of the SQUID measurement results suggested that the conformational change of the ascorbate oxidase molecule caused the temperature dependence of the antiferromagnetic interaction. The tpe 2 Cu EPR signals in laccase and ascorbate oxidase were conspicuously broadened with rasing temperature because of the increasing contribbution of the triplet state by type 3 Cu's and/or of the rapod relaxation whith finally led to only ca. 30% detection of the type 3 Cu's to be EPR detectable. SQUID measurements indicated that only one- and two-azide bound forms. The binding mode of azide to the trinuclear center was discussed based on some models.