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

Abstract

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 by 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 concomitantlywith 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 Cu 2+ 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, the value for 1.5 free spins per subunit. However, the magnetic moment increased above 200 K, reaching 3.2 BM at room temperature, the value for 2.3 free spins. This decrease in the EPR detectable amount of Cu 2+ and the increase in magnetic moment with increasing temperature are due to the contribution of the triplet state by the antiferromagneticially coupled type 3 Cu atoms. The binding modes for the one- and two-azide bound forms to the trinuclear Cu center are discussed based on the temperature-dependent EPR and magnetic susceptibility data.