A paramagnetic intermediate in the reduction of oxygen by reduced laccase

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1. Introduction The four-electron reduction of molecular oxygen in the catalytic reaction of lactase, like that of cyto- chrome c oxidase, poses great mechanistic problems [ 1,2] . With lactase, in particular, a mechanism involv- ing four consecutive one-electron steps would appear unlikely, since the high oxidation-reduction poten- tials of the redox sites of the enzymes [3] would lead to a very high, positive free-energy change for the formation of the superoxide radical. This fact, together with the presence of a co-operative two- electron acceptor in the enzyme, has led to the sug- gestion [2] that the reaction of reduced lactase with oxygen comprises consecutive two-electron steps. Spectroscopic evidence for an intermediate in the reaction of reduced lactase with oxygen has been presented earlier [4]. It was tentatively proposed that this species is HZ02 (or one of its ions) bound to Type 2 Cu2’. However, recent experiments [5]with tree as well as fungal lactase indicate, that when the fully reduced enzymes react with oxygen, both the Type 1 copper and the two-electron acceptor become rapidly re-oxidized, while the Type 2 copper remains reduced. The intermediate so formed has optical properties similar to that described previously [4]. The e.p.r. spectrum at 77 K showed only the presence of Type 1 Cu”. As 02 is an even-electron system and three electrons had been transferred from reduced enzyme-sites, the absence of another e.p.r. signal appeared inconsistent with the kinetic results. This prompted an e.p.r. study at lower temperatures, leading to the discovery of the paramagnetic inter-