Ferroxidase (Fet3) and Permease (Ftr1) in Iron Uptake

Summary

Principal Investigator: DANIEL KOSMAN
Affiliation: University at Buffalo
Country: USA
Abstract: DESCRIPTION (provided by applicant): Multicopper oxidases (MCOs) couple the 4e- reduction of dioxygen to 2H2O with the oxidation of 4 equivalents of a 1-electron donor. A sub-family of these ubiquitous enzymes possesses specificity towards FeII that makes them essential to iron homeostasis in their respective organisms. Our hypothesis is that these ferroxidases function by channeling their FeIII product to a down-stream partner in their iron metabolic pathways. This program has delineated the structure and function in the Fet3p, Ftr1p high-affinity Fe-uptake complex in the Saccharomyces cerevisiae (Sc) plasma membrane (PM). The two fundamental questions addressed in this work are: 1) what structural motifs confer on an MCO this specificity for FeII as substrate;and 2) how is the Fet3p ferroxidase reaction coupled kinetically and physically to the membrane permeation of FeIII by Ftr1p. In this application we propose three specific aims. In Aim I we will test our hypothesis of what structural motifs define a ferroxidase, specifically that a cohort of carboxylate side chains maximize three factors that determine e- transfer from FeII: FeII binding, FeII redox potential and electronic matrix coupling of the FeII and the type 1 CuII (T1Cu) in the ferroxidase. We will do this by interconverting laccase and ferroxidase enzymes based on our design principles. In addition, we will test our hypothesis that another cohort of carboxylate side chains stabilizes the increasing negative charge on dioxygen as it is reduced in 2, 2e- steps at the trinuclear cluster (TNC). Last we will test our hypothesis that the coordination changes associated with O2 reduction at the TNC trigger e- transfer from the T1 Cu via the canonical MCO His-Cys-His motif that connects the two. In Aim II we propose to test our hypothesis that Fox1 in Chlamydomonas reinhardtii (Cr) is a human ceruloplasmin-like ferroxidase. We will characterize the Fox1 protein, demonstrating that it has the ferroxidase-specificity motifs that support both FeII oxidation and FeIII trafficking in a Fox1, Ftr1 complex in the Cr PM. We will construct mutants of both Fox1 and Ftr1 that we predict will be sensitive to a FeIII-chelator acting as a metabolite trap in Fe-trafficking between the two proteins in Fe-uptake;we have used this classic test of channeling in the Sc Fet3, Ftr1p complex. We propose that the Fe-trafficking between Fox1 and Ftr1 in Cr provides a realistic model of the putative FeIII-trafficking between hCp and transferrin. In Aim III, we will test our hypothesis that the two human fungal pathogens, Candida albicans and Cryptococcus neoformans express an equivalent PM Fet, Ftr high-affinity Fe-uptake complex. We will quantify the 59Fe-uptake kinetics via these complexes both in situ and in recombinant form in Sc. Targeting specific ferroxidase and Fe-trafficking residues in the Ca and Cn proteins, we will test our hypothesis that these mutants exhibit the channeling defect exhibited by the Sc homologues. We propose that strains expressing these channeling mutants will exhibit a reduced virulence in vitro and in vivo. Last, using these chelator-sensitive clones, the NCI diversity collection will be mined for compounds with the potential as inhibitors of Fet, Ftr Fe-uptake in these fungal pathogens. PUBLIC HEALTH RELEVANCE: All oxygen-utilizing organisms from fungi to humans require the activity of a copper oxidase enzyme - a multicopper oxidase - to manage their metabolism of the essential nutrient, iron. Fungi from baker's yeast to the human pathogens C. albicans and C. neoformans use these enzymes to acquire the iron they need to thrive and survive. The goal of this research is to take a snap-shot of how these enzymes work and then to use this knowledge to block their trafficking of iron as way of suppressing the virulence of pathogenic fungi in both otherwise healthy and immunocompromised patients.
Funding Period: ----------------1999 - ---------------2012-
more information: NIH RePORT

Top Publications

  1. ncbi An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane
    E Y Kwok
    Department of Biochemistry, The University at Buffalo, 140 Farber Hall, Buffalo, NY 14214, USA
    J Inorg Biochem 100:1053-60. 2006
  2. pmc In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site
    Erik Sedlak
    Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA
    Proc Natl Acad Sci U S A 105:19258-63. 2008
  3. pmc Iron source preference and regulation of iron uptake in Cryptococcus neoformans
    Won Hee Jung
    The Michael Smith Laboratories, Department of Microbiology and Immunology, and Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada
    PLoS Pathog 4:e45. 2008
  4. doi Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites
    Anthony J Augustine
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    Biochemistry 47:2036-45. 2008
  5. pmc Spectroscopic and kinetic studies of perturbed trinuclear copper clusters: the role of protons in reductive cleavage of the O-O bond in the multicopper oxidase Fet3p
    Anthony J Augustine
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    J Am Chem Soc 129:13118-26. 2007
  6. ncbi Shall we dance? How a multicopper oxidase chooses its electron transfer partner
    Liliana Quintanar
    Centro de Investigacion y de Estudios Avanzados, Mexico, D F, Mexico
    Acc Chem Res 40:445-52. 2007
  7. ncbi Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae
    Christopher S Stoj
    Department of Biochemistry, School of Medicine and Biomedical Sciences, The University at Buffalo, Buffalo, New York 14214, USA
    J Biol Chem 282:7862-8. 2007
  8. ncbi Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p
    Christopher S Stoj
    Department of Biochemistry, School of Medicine and Biological Sciences, State University of New York, Buffalo, New York 14214, USA
    Biochemistry 45:12741-9. 2006
  9. ncbi Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane
    Ernest Y Kwok
    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA
    Biochemistry 45:6317-27. 2006
  10. ncbi Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae
    Arvinder Singh
    Department of Biochemistry, School of Medicine and Biomedical Sciences, The University at Buffalo, Buffalo, New York 14214, USA
    J Biol Chem 281:13355-64. 2006

Scientific Experts

  • DANIEL KOSMAN
  • Anthony J Augustine
  • Lynn Ziegler
  • Edward I Solomon
  • Christopher S Stoj
  • Alaina Terzulli
  • Erik Sedlak
  • Liliana Quintanar
  • Arvinder Singh
  • Ryan C McCarthy
  • María Eugenia Zaballa
  • Alaina J Terzulli
  • Britt Hedman
  • Keith O Hodgson
  • Won Hee Jung
  • E Y Kwok
  • Ernest Y Kwok
  • Scott Severance
  • Ruchi Gaur
  • Ryan McCarthy
  • Christian Kjaergaard
  • Alejandro J Vila
  • Munzarin Qayyum
  • Anita Sham
  • Satoshi Fujii
  • Pernilla Wittung-Stafshede
  • Tianshun Lian
  • Ritimukta Sarangi
  • James W Kronstad
  • Mads Emil Kragh
  • Barry D Liboiron
  • Christopher Stoj
  • P John Hart
  • Alexander B Taylor
  • William Wiltsie
  • C S Stoj
  • S Severance
  • Navjot Kaur
  • Lynn Zeigler

Detail Information

Publications20

  1. ncbi An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane
    E Y Kwok
    Department of Biochemistry, The University at Buffalo, 140 Farber Hall, Buffalo, NY 14214, USA
    J Inorg Biochem 100:1053-60. 2006
    ..The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p...
  2. pmc In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site
    Erik Sedlak
    Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA
    Proc Natl Acad Sci U S A 105:19258-63. 2008
    ..Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family...
  3. pmc Iron source preference and regulation of iron uptake in Cryptococcus neoformans
    Won Hee Jung
    The Michael Smith Laboratories, Department of Microbiology and Immunology, and Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada
    PLoS Pathog 4:e45. 2008
    ..Overall, these results indicate that C. neoformans utilizes iron sources within the host (e.g., holo-transferrin) that require Cft1 and a reductive iron uptake system...
  4. doi Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites
    Anthony J Augustine
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    Biochemistry 47:2036-45. 2008
    ..These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover...
  5. pmc Spectroscopic and kinetic studies of perturbed trinuclear copper clusters: the role of protons in reductive cleavage of the O-O bond in the multicopper oxidase Fet3p
    Anthony J Augustine
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    J Am Chem Soc 129:13118-26. 2007
    ..A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC...
  6. ncbi Shall we dance? How a multicopper oxidase chooses its electron transfer partner
    Liliana Quintanar
    Centro de Investigacion y de Estudios Avanzados, Mexico, D F, Mexico
    Acc Chem Res 40:445-52. 2007
    ....
  7. ncbi Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae
    Christopher S Stoj
    Department of Biochemistry, School of Medicine and Biomedical Sciences, The University at Buffalo, Buffalo, New York 14214, USA
    J Biol Chem 282:7862-8. 2007
    ..These kinetic defects render the Fet3pM345A unable to support wild type cellular copper resistance, suggesting that there is a finely tuned copper redox balance at the yeast plasma membrane...
  8. ncbi Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p
    Christopher S Stoj
    Department of Biochemistry, School of Medicine and Biological Sciences, State University of New York, Buffalo, New York 14214, USA
    Biochemistry 45:12741-9. 2006
    ..Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one...
  9. ncbi Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane
    Ernest Y Kwok
    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA
    Biochemistry 45:6317-27. 2006
    ..By analogy to the Fe(III) trafficking that leads to the mineralization of the ferritin core, we propose that ferric iron channeling is a conserved feature of iron homeostasis in aerobic organisms...
  10. ncbi Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae
    Arvinder Singh
    Department of Biochemistry, School of Medicine and Biomedical Sciences, The University at Buffalo, Buffalo, New York 14214, USA
    J Biol Chem 281:13355-64. 2006
    ..The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM...
  11. pmc Mechanistic analysis of iron accumulation by endothelial cells of the BBB
    Ryan C McCarthy
    Department of Biochemistry, SUNY University at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY, USA
    Biometals 25:665-75. 2012
    ..These results are the first to demonstrate multiple mechanism(s) of TBI and NTBI reduction and uptake by endothelial cells (EC) of the BBB...
  12. pmc Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans
    Lynn Ziegler
    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA
    Mol Microbiol 81:473-85. 2011
    ..albicans Fe-uptake proteins, we demonstrate that they support a mechanism of Fe-trafficking that involves channelling of the CaFet34-generated Fe³⁺ directly to CaFtr1 for transport into the cytoplasm...
  13. pmc NMR study of the exchange coupling in the trinuclear cluster of the multicopper oxidase Fet3p
    María Eugenia Zaballa
    IBR Instituto de Biologia Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
    J Am Chem Soc 132:11191-6. 2010
    ..This work provides the foundation for the application of NMR to these complex systems...
  14. pmc Core glycan in the yeast multicopper ferroxidase, Fet3p: a case study of N-linked glycosylation, protein maturation, and stability
    Lynn Ziegler
    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214 3000, USA
    Protein Sci 19:1739-50. 2010
    ....
  15. pmc Systematic perturbation of the trinuclear copper cluster in the multicopper oxidases: the role of active site asymmetry in its reduction of O2 to H2O
    Anthony J Augustine
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    J Am Chem Soc 132:6057-67. 2010
    ..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...
  16. pmc Analysis of the high-affinity iron uptake system at the Chlamydomonas reinhardtii plasma membrane
    Alaina Terzulli
    Department of Biochemistry, The University at Buffalo, 140 Farber Hall, 3435 Main St, Buffalo, NY 14214, USA
    Eukaryot Cell 9:815-26. 2010
    ..Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase...
  17. ncbi Multicopper oxidases: a workshop on copper coordination chemistry, electron transfer, and metallophysiology
    Daniel J Kosman
    Department of Biochemistry, The University at Buffalo, NY 14214, USA
    J Biol Inorg Chem 15:15-28. 2010
    ..These enzymes, in particular the ferroxidases found in all fungi and metazoans, play critical roles in the metal metabolism of the expressing organism...
  18. pmc The Fox1 ferroxidase of Chlamydomonas reinhardtii: a new multicopper oxidase structural paradigm
    Alaina J Terzulli
    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA
    J Biol Inorg Chem 14:315-25. 2009
    ..These diverse structures suggest alternative mechanisms for intramolecular electron transfer and require a new trajectory for the evolution of the MCO superfamily...