Spectroscopic, kinetic, and mutational studies of the multicopper oxidases: Insight into the mechanism of oxygen reduction to water

The multicopper oxidase (MCO) family of enzymes, which includes Fet3p and R.v. laccase, catalyzes the four-electron reduction of O2 to H2O, coupled to the one-electron oxidation of four equivalents of substrate. To carry out this process these enzymes require a minimum of four Cu atoms, a type 1 site (T1), a type 2 site (T2), and a coupled binuclear, type 3 site (T3). The T1 is the site of substrate oxidation and also shuttles electrons over >13 A, via the T1-CysHis-T3 electron transfer (ET) pathway, to the T2 and T3, which form a trinuclear Cu cluster (TNC) where O2 is reduced to H2O.;Variants of two carboxylate residues, E487 near the two T3 Cu's (T3A and T3B) and D94 near the T2 and one of the T3 Cu's (T3B), in the second coordination sphere of the TNC of Fet3p have been isolated. All variants have intact TNC's and different impacts upon reactivity, thus allowing the role of each residue in the reduction of O2 to be elucidated. E487 is responsible for transferring a proton during reductive cleavage of the O-O bond and for an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. D94 plays a key role in the reaction of the reduced TNC with O2 and drives ET from the T2 to cleave the O-O bond by deprotonating the T2 water ligand.;Two variants of Fet3p have been isolated with a T3 Cu His ligand mutated to Gln, H126Q (T3A') at the T3A and H483Q (T3B') at the T3B. These variants have valid and similarly perturbed TNC's, but only T3A' reacts with O 2. Spectroscopic characterization of the resting oxidized and reduced forms of both variants and the peroxide intermediate in T3A' has shown that the TNC is asymmetric, with reduction of O2 to O2 2- occurring selectively on its T3BT2 edge. This is a result of the structural activation of T3B to react with O2, and the negative charge of D94, which increases the driving force for O2 reduction by the T3B and T2 Cu's. In T3B' the T3B Cu is perturbed such that it is longer activated, accounting for its lack of O2 reactivity. The role of TNC asymmetry throughout the MCO catalytic cycle is also discussed.;The His residues in T1-Cys-His-T3 ET pathway in Fet3p have been mutated to evaluate how a perturbation of the TNC impacts the T1. A combination of spectroscopic methods, in particular resonance Raman (rR), show that as the His-T3 interaction is weakened, the covalency of the Cys S-T1 Cu bond increases and the reduction potential of the T1 decreases. Similar studies were carried out on a number of well defined derivatives of R.v. laccase, including the native intermediate (which can be trapped for spectroscopic study). The covalency of the T1 Cu-S bond and the T1 reduction potential are not affected in these derivatives relative to the resting oxidized enzyme; however, there are changes to the T1 rR spectrum indicative of perturbations to the protein backbone near the T1. This provides a possible mechanism for regulating T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.;Fet3p couples the reduction of O2 to H2O to oxidation of substrate Fe(II) to Fe(III). Variants of three carboxylate residues in its proposed Fe(II) binding site have been prepared in order to elucidate the contribution of these residues to substrate reactivity. Analysis of single and multiple turnover kinetic data indicates that all three help to tune the reduction potential of enzyme bound Fe(II) below that of aqueous Fe(II), thus providing extra driving force for its oxidation. Furthermore, two of the residues are experimentally shown to be critical components of the directed ET pathway from bound Fe(II) to the T1, and to be necessary for Fet3p's Fe(II) specificity.