The copper-iron connection in biology: structure of the metallo-oxidase Fet3p.
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ABSTRACT: Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O(2) to H(2)O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) 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(II) 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.
Project description:Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.
Project description:Eukaryotic cells, whether free-living or organismal, rely on metallo-reductases to process environmental ferric iron and cupric copper prior to uptake. In addition, some free-living eukaryotes (e.g. fungi and algae) couple ferri-reduction to ferro-oxidation, a process catalyzed by a small cohort of multi-copper oxidases; in these organisms, the ferric iron product is a ligand for cell iron uptake via a ferric iron permease. In addition to their support of iron uptake in lower eukaryotes, ferroxidases support ferrous iron efflux in Chordata; in this process the release of the ferrous iron from the efflux transporter is catalyzed by its ferroxidation. Last, ferroxidases also catalyze the oxidation of cuprous copper and, as metallo-oxidases, mirror the dual activity of the metallo-reductases. This Perspective examines the teleos of the yin-yang of this redox cycling of iron and copper in their metabolism.
Project description: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 (T3?) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3? 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 T3? Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3? 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 ?*-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.
Project description:The multicopper oxidase Fet3p couples four 1e(-) oxidations of substrate to the 4e(-) reduction of O2 to H2O. Fet3p uses four Cu atoms to accomplish this reaction: the type 1, type 2, and coupled binuclear type 3 sites. The type 2 and type 3 sites together form a trinuclear Cu cluster (TNC) which is the site of O2 reduction. This study focuses on mutants of two residues, E487 and D94, which lie in the second coordination sphere of the TNC and defines the role that each plays in the structural integrity of the TNC, its reactivity with O2, and in the directional movement of protons during reductive cleavage of the O-O bond. The E487D, E487A, and D94E mutants have been studied in the holo and type 1 depleted (T1D) forms. Residue E487, located near the T3 center, is found to be responsible for donation of a proton during the reductive cleavage of the O-O bond in the peroxide intermediate and an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. Residue D94, near the T2 site, plays a key role in the reaction of the reduced TNC with O2 and drives electron transfer from the T2 Cu to cleave the O-O bond by deprotonating the T2 Cu water ligand. A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC.
Project description:Iron plays an essential role in normal biological processes: The generation of cellular energy, oxygen transport, DNA synthesis and repair are all processes that require iron-coordinated proteins, either as elemental iron, heme or iron-sulfur clusters. As a transition metal with two major biological oxidation states, iron is also a critical intermediate in the generation of reactive oxygen species that can damage cellular structures and contribute to both aging and cancer. In this review, we focus on experimental and epidemiologic evidence that links iron and cancer, as well as strategies that have been proposed to either reduce or increase cellular iron for cancer therapy.
Project description:Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.
Project description: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.
Project description:In an effort to further probe metal binding to metallo-beta-lactamase L1 (mbetal L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin-spin coupling. Cu-L1 exhibited higher k(cat) (96 s(-1)) and K(m) (224 microM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equiv of Ni and 0.3 equiv of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by (1)H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. (1)H NMR spectra demonstrate that Ni(II) binds in the Zn(2) site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn(2) site modulates substrate preference in mbetal L1. These studies demonstrate that the Zn(2) site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with beta-lactam-containing antibiotics.
Project description:The effect of a cueO mutation on global gene expression in E. coli has been analysed using whole genome oligonucleotide microarrays. A cueO mutant lacks a periplasmic multi-copper oxidase, an enzyme that is believed to be involved in the oxidation of cuprous and ferrous ions as well as phenolic compounds in the periplasm. Deletion renders the cell sensitive to copper and this toxicity is likely associated with increased oxidative stress in a cueO mutant. Keywords: genetic modification
Project description: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.