Fet3p is a multicopper oxidase that contains four Cu ions: one type 1, one type 2, and a coupled binuclear type 3 site. The type 2 and type 3 centers form a trinuclear cluster that is the active site for O(2) reduction to H(2)O. When the type 1 Cu is depleted (C484S mutation), the reaction of the reduced trinuclear cluster with O(2) generates a peroxide intermediate. Kinetic studies of the decay of the peroxide intermediate suggest that a carboxyl residue (D94 in Fet3p) assists the reductive cleavage of the O-O bond at low pH. Mutations at the D94 residue (D94A, D94N, and D94E) have been studied to evaluate its role in the decay of the peroxide intermediate. Spectroscopic studies show that the D94 mutations affect the geometric and electronic structure of the trinuclear cluster in a way that is consistent with the hydrogen bond connectivity of D94. While the D94E mutation does not affect the initial reaction of the cluster with O(2), the D94A mutation causes larger structural changes that render the trinuclear cluster unreactive toward O(2), demonstrating a structural role for the D94 residue. The decay of the peroxide intermediate is markedly affected by the D94E mutation, confirming the involvement of D94 in this reaction. The D94 residue appears to activate a proton of the type 2 Cu(+)-bound water for participation in the transition state. These studies provide new insight into the role of D94 and proton involvement in the reductive cleavage of the O-O bond.

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.

The multicopper oxidases are a family of enzymes that couple the reduction of O(2) to H(2)O with the oxidation of a range of substrates. Saccharomyces cerevisiae Fet3p and human ceruloplasmin (hCp) are members of this family that exhibit ferroxidase activity. Their high specificity for Fe(II) has been attributed to the existence of a binding site for iron. In this study, mutations at the E185 and Y354 residues, which are putative ligands for iron in Fet3p, have been generated and characterized. The effects of these mutations on the electronic structure of the T1 Cu site have been assessed, and the reactivities of this site toward 1,4-hydroquinone (a weak binding substrate) and Fe(II) have been evaluated and interpreted in terms of the semiclassical Marcus theory for electron transfer. The electronic and geometric structure of the Fe(II) substrate bound to Fet3p and hCp has been studied for the first time, using variable-temperature variable field magnetic circular dichroism (VTVH MCD) spectroscopy. The iron binding sites in Fet3p and hCp appear to be very similar in nature, and their contributions to the ferroxidase activity of these proteins have been analyzed. It is found that these iron binding sites play a major role in tuning the reduction potential of iron to provide a large driving force for the ferroxidase reaction, while still supporting the delivery of the Fe(III) product to the acceptor protein. Finally, the analysis of possible electron-transfer (ET) pathways from the protein-bound Fe(II) to the T1 Cu site indicates that the E185 residue not only plays a role in iron binding, but also provides the dominant ET pathway to the T1 Cu site.