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Multicopper oxidases (MCOs) carry out the most energy efficient reduction of O2 to H2O known, i.e., with the lowest overpotential. This four-electron process requires an electron mediating type 1 (T1) Cu site and an oxygen reducing trinuclear Cu cluster (TNC), consisting of a binuclear type 3 (T3)- and a mononuclear type 2 (T2) Cu center. The rate-determining step in O2 reduction is the first two-electron transfer from one of the T3 Cu's (T3β) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3β Cu variants of the Fet3p, where a first shell His ligand is mutated to Glu or Gln. This converts the fast two-electron reaction of the wild-type (WT) enzyme to a slow one-electron oxidation of the TNC. Both variants initially react to form a common T3β Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3β Cu in the reduced TNCs in the two variants, in contrast to the trigonal arrangement observed in the WT enzyme. This structural perturbation is found to significantly alter the electronic structure of the reduced TNC, which is no longer capable of rapidly transferring two electrons to the two perpendicular half occupied π*-orbitals of O2, in contrast to the WT enzyme. This study provides new insight into the geometric and electronic structure requirements of a fully functional TNC for the rate determining two-electron reduction of O2 in the MCOs.

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) which oxidizes Fe(2+) to Fe(3+). The electronic structure of the different copper centers in this family of enzymes has been extensively studied and discussed for years with a particular focus on the exchange coupling regime in the trinuclear cluster (TNC). Using NMR spectroscopy we have quantified the exchange coupling constant in the type 3 center in a fully metalated oxidase; this value in Fet3p is significantly higher than that reported for proteins containing isolated type 3 centers as tyrosinase. We also provide evidence of exchange coupling between the type 2 and the type 3 Cu(2+) ions, which supports the crystallographic evidence of dioxygen binding to the TNC. This work provides the foundation for the application of NMR to these complex systems.

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 A 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, T3alpha and T3beta, respectively. The variants have been isolated in both holo and type 1 depleted (T1D) forms, T1DT3alphaQ and T1DT3betaQ, 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, T1DT3alphaQ reacts with O(2), but T1DT3betaQ does not. Thus loss of a ligand at T3beta, but not at T3alpha, turns off O(2) reactivity, indicating that T3beta and T2 are required for the 2e(-) reduction of O(2) to form the peroxide intermediate (PI), whereas T3alpha remains reduced. This is supported by the spectroscopic features of PI in T1DT3alphaQ, 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 T3alpha and T3beta is discussed, as is its contribution to reactivity.

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like beta-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (T(m)) of 45 degrees C, 62 degrees C, and 72 degrees C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (T(m) of 50 degrees C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by approximately 5 degrees C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (T(m) 50-55 degrees C; 1 K/min), followed by unfolding of domains 1 and 3 ( approximately 65-70 degrees C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.

Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(ll) to Fe(lll). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(lll) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(ll) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase. [ABSTRACT FROM AUTHOR]

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 hole and type 1 depleted (T1D) forms, T1DT3αQ and T1DT3/tQ, 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 O2, but T1DT3βQ does not. Thus loss of a ligand at T3β, but not at T3α, turns off O2 reactivity, indicating that T3β and T2 are required for the 2e- reduction of O2 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 O2 reactivity. The structural origin of this asymmetry between the T3α and T3β is discussed, as is its contribution to reactivity. [ABSTRACT FROM AUTHOR]