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

Abstract

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 Å 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 (T1D) forms, T1DT3αQ and T1DT3βQ, 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, 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.

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states

“…1a; Augustine et al, 2010 Lee, George et al, 2002;Palmer et al, 2002;. It has previously been demonstrated that protons are not involved in all steps of the reduction of O 2 at the TNC (Augustine et al, 2007) ) is symmetrically located between the binuclear T3Cu-T3 0 Cu cluster (Augustine et al, 2010). Thus, after the two two-electron transfers have taken place, the NI displays four catalytic Cu ions in the cupric state and forms the RS by releasing one H 2 O molecule ( Fig.…”

“…1) starting from the fully Cu reduced (FR) enzyme with four catalytic Cu ions in the cuprous state ( Fig. 1a; Augustine et al, 2010 Lee, George et al, 2002;Palmer et al, 2002;. It has previously been demonstrated that protons are not involved in all steps of the reduction of O 2 at the TNC (Augustine et al, 2007) ) is symmetrically located between the binuclear T3Cu-T3 0 Cu cluster (Augustine et al, 2010).…”

“…II -Cys-His-T3Cu II electron-transfer pathway to the TNC (Augustine et al, 2010). However, O 2 reduction at the TNC could also be observed in the crystalline state without substrate oxidation, since electrons and protons are released into the crystal by the radiolysis of water molecules during X-ray data collection (Hakulinen et al, 2006;Macedo et al, 2009;Garman, 2010).…”

“…In fact, under catalytic conditions the cycle is completed upon reduction of the NI by a total of four electrons and the release of 2H 2 O molecules by the addition of three protons, regenerating the FR enzyme for the next enzyme cycle without decaying to the RS. Nevertheless, because of the lack of sufficient electrons to form the FR enzyme, the NI necessarily decays to the RS (Augustine et al, 2010). Indeed, the RS is rather common in a large number of crystallographic structures of MCOs deposited in the PDB (~15 MCO structures), since the catalytic cycle for O 2 reduction in the crystalline state using the protons and electrons released by X-rays is rather inefficient (Hakulinen et al, 2006;Kjaergaard et al, 2012).…”

“…Indeed, the RS is rather common in a large number of crystallographic structures of MCOs deposited in the PDB (~15 MCO structures), since the catalytic cycle for O 2 reduction in the crystalline state using the protons and electrons released by X-rays is rather inefficient (Hakulinen et al, 2006;Kjaergaard et al, 2012). Thus, since the FR enzyme is the first state of the catalytic cycle for O 2 reduction of MCOs (Augustine et al, 2010), and reacts immediately with O 2 to form the PI, the structural stabilization of the O 2 state (Fig. 1e) observed in the structure of M. albomyces laccase at low absorbed X-ray dose (Hakulinen et al, 2006) seems to be a process that is exclusive to the crystalline state as a result of the deficiency of electrons to generate the FR enzyme at low doses.…”