Systematic Perturbation of the Trinuclear Copper Cluster in the Multicopper Oxidases: The Role of Active Site Asymmetry in its Reduction of O2 to H2O

The multicopper oxidase Fet3p catalyzes the four-electron reduction of dioxygen to water, coupled to the one-electron oxidation of four equivalents of substrate. To carry out this process, the enzyme utilizes four Cu atoms: a type 1, a type 2, and a coupled binuclear, type 3 site. Substrates are oxidized at the T1 Cu, which rapidly transfers electrons, 13 A away, to a trinuclear copper cluster composed of the T2 and T3 sites, where dioxygen is reduced to water in two sequential 2e - steps. This study focuses on two variants of Fet3p, H126Q and H483Q, that perturb the two T3 Cu's, T3α and T3β, respectively. The variants have been isolated in both holo and type 1 depleted (T1 D) forms, T1 DT3αQ and T1 DT3βQ, and their trinuclear copper clusters have been characterized in their oxidized and educed states. While the variants are only mildly perturbed relative to T1D in the resting oxidized state, in contrast to T1D they are both found to have lost a ligand in their reduced states. Importantly, T1DT3αQ reacts with O 2 , but T1DT3βQ does not. Thus loss of a ligand at T3β, but not at T3α, turns off O 2 reactivity, indicating that T3β and T2 are required for the 2e - reduction of O 2 to form the peroxide intermediate (PI), whereas T3α remains reduced. This is supported by the spectroscopic features of PI in T1DT3αQ, which are identical to T1D PI. This selective redox activity of one edge of the trinuclear cluster demonstrates its asymmetry in O 2 reactivity. The structural origin of this asymmetry between the T3α and T3β is discussed, as is its contribution to reactivity.