EPR and magnetic susceptibility studies of the trinuclear copper center innative and azide-reacted zucchini ascorbate oxidase

The magnetic properties of the trinuclear center composed of type 2 Cu and a pair of type 3 Cu atoms in zucchini ascorbate oxidase have been studied b y EPR spectroscopy and magnetic susceptibility measurements from cryogenic temperature to room temperature. The EPR spectra showed that the A(z) value of the type 2 Cu signal decreased with increasing temperature concomitantl y with an appreciable broadening above 150 K, differing from the type 1 Cu signal. While the EPR spectrum can be simulated for the 1:0.3-0.4 ratio of type 1 Cu and type 2 Cu per subunit, the total amount of the EPR detectable Cu2+ was not 1.3-1.4 but 1.7 above 150 K and 1.3 at room temperature. The magnetic moment determined by SQUID measurements was 2.4 BM above 200 K, th e value for 1.5 free spins per subunit. However, the magnetic moment increa sed above 200 K, reaching 3.2 BM at room temperature, the value for 2.3 fre e spins. This decrease in the EPR detectable amount of Cu2+ and the increas e in magnetic moment with increasing temperature are due to the contributio n of the triplet state by the antiferromagneticially coupled type 3 Cu atom s. The binding modes for the one- and two-azide bound forms to the trinucle ar Cu center are discussed based on the temperature-dependent EPR and magne tic susceptibility data. (C) 1999 Elsevier Science Inc. All rights reserved .