Spectroscopic Characterization and O₂ Reactivity of the Trinuclear Cu Cluster of Mutants of the Multicopper Oxidase Fet3p

Abstract

Fet3p is a multicopper oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O(2) to H(2)O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O(2) reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O(2), the type 1 Cu Cys(484) was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His(126)Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His(126)Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O(2) to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.

O2 Reduction to H2O by the multicopper oxidases

“…27,28 (Scheme 1, top) Derivatives of the native enzyme have been prepared where the T1 is either eliminated 29,30 (replacement of the Cys ligand of the T1 with a Ser to generate a type 1 depleted or T1D form) or replaced by a redox innocent mercuric ion (the T1Hg derivative). 31,32 These have valid TNCs which, when reduced, react with O 2 with essentially the same rate constant as the native enzyme.…”

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

“…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).…”

Four-electron Reduction of Dioxygen by a Multicopper Oxidase, CueO, and Roles of Asp112 and Glu506 Located Adjacent to the Trinuclear Copper Center

“…One of them, intermediate I, could be trapped by the following modified multicopper oxidases so as to interrupt the electron transfer from the type I copper: a plant laccase whose type I copper was substituted with mercury (13); a mixed valent laccase in which the type I copper was oxidized, but the trinuclear copper center was reduced (14); and a Cys 3 Ser mutant of bilirubin oxidase (15) and Fet3p (16) whose type I copper center became vacant. Although the trinuclear copper center must be fully reduced to produce intermediate I, it has been considered to be a twoelectron reduced form and, therefore, also called the peroxide intermediate (13,16). Another reaction intermediate, II, also called the native intermediate, has been detected at the final stage of a single turnover (15,(17)(18)(19) (13)(14)(15)(16)(17)(18)(19).…”

“…Although the trinuclear copper center must be fully reduced to produce intermediate I, it has been considered to be a twoelectron reduced form and, therefore, also called the peroxide intermediate (13,16). Another reaction intermediate, II, also called the native intermediate, has been detected at the final stage of a single turnover (15,(17)(18)(19) (13)(14)(15)(16)(17)(18)(19).…”