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 angstrom 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, T3 alpha and T3 beta, respectively. The variants have been isolated in both holo and type 1 depleted (T1D) forms, T1DT3 alpha Q and T1DT3 beta Q, and their trinuclear copper clusters have been characterized in their oxidized and reduced 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, T1DT3 alpha Q reacts with O-2, but T1DT3 beta Q does not. Thus loss of a ligand at T3 beta, but not at T3 alpha, turns off O-2 reactivity, indicating that T3 beta and T2 are required for the 2e(-) reduction of O-2 to form the peroxide intermediate (PI), whereas T3 alpha remains reduced. This is supported by the spectroscopic features of PI in T1DT3 alpha Q, 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 T3 alpha and T3 beta is discussed, as is its contribution to reactivity.