FOUR-ELECTRON REDUCTION OF DIOXYGEN AT THE TRINUCLEAR COPPER CLUSTER IN MULTICOPPER OXIDASES

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The reactions of the reduced laccase and bilirubin oxidase with dioxygen were studied using mainly absorption and EPR spectra at a cryogenic temperature and also using stopped flow spectroscopy and SQUID magnetic measurement. The hydroxyl or oxyl radical bound to the trinuclear Cu site was detected as a reaction intermediate, three-electron reduced form of dioxygen, . Decays of this radical species were highly pH dependent on pH, indicating that the process was gated by the protonation to be water finally. A peroxide intermediate corresponding to the three electron reduced form of dioxygen could not be detected because diffusion of dioxygen to the trinuclear Cu site was rate-determining step and the electron transfer from type 1 Cu to the putative peroxide intermediate was very rapid. However, the peroxide intermediate could be detected by reaction dioxygen to the CuCl-acted type 2 Cu-depleted laccase under a strict anaerobic condition. Based on the successful detection and characterization of the two reaction intermediates the general reaction mechanism how dioxygen is four-electron reduced by multicopper oxidases was proposed.