Magnetic susceptibility study of the laccase‐peroxide derivative

Laccases are copper containing oxidases in which the 4 copper ions are bound in 3 distinct sites. Two of these are EPR detectable (type 1 and type 2) while the third site (type 3) most probably consists of an antiferromagnetically coupled Cu(II)-Cu(lI) pair [ 11. The magnetic susceptibility of native oxidized Rhus lactase can be quantitatively accounted for by the EPR detectable copper [2-41. Thus, formulation of the type 3 site as a copper pair requires a lower limit for the coupling, expressed by the exchange integral, J of 250-300 cm-’ [3,4]. We have demonstrated the formation of a high affinity peroxy-lactase complex upon reacting native oxidized enzyme with H202 [5]. Primarily on the basis of the absorption and CD spectra it was proposed that the peroxide binds to the copper pair of the type 3 site [6]. We have shown spectral similarities between the peroxy-lactase complex and intermediates formed during the reoxidation of reduced lactase with O2 [7]. These observations have led us to extend the study of the type 3 site, since the interaction with dioxygen or its derivatives might affect the exchange coupling of this copper pair. Therefore the magnetic properties of peroxy-lactase was measured by means of high resolution NMR technique. Indeed we have observed an increase in the magnetic susceptibility upon peroxide binding which is assigned to a decrease in the antiferromagnetic coupling of the type 3 copper pair.