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

The trinuclear centers in Rhus vernicifera laccade and Cucumis sativus ascorbate oxidase have been studied by EPR spectroscopy and magneic susceptibility measurements over the wide range of 5K to 300K. The EPR spectra showd that type 2 copper receives increasing tetrahedral distortion with raising temperature. Magnetic suscetibilities of laccase showed that both of type 2 copers are almost fully paramagnetic since the antiferromagnetic interaction between type 3 coppers is not as strong from 5K to 300K. On the other hand, the efffective magnetic moment of ascornate oxidase is contributed by ca. 1.7 Cu2+ even below ca. 100K, since type 2 Cu is partly in the aniferromagnetic interaction between type 3 coppers is not as strong 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 type 2 Cu EPR signals in laccase and ascorbate oxidase were conspicuously broadened with rasing temperature because of the increasing contribution of the triplet state by typw3 Cu's and/or of the rapod relation which finally led to only ca. 30% detection of the type 2 Cu signals at room temperature. The stepwise binding of azide to the trinuclear center made one of type 3 Cu's to be EPR detectable. SQUID measurements indicated that only one Cu in the trinuclear center is paramagnetic and other two Cu's are antiferromagnetically coupled for both of the one- and two-azide bound forms. The binding mode of azide to the trinuclear center was discussed based on some models.