Molecular Recognition in Biological Systems and Bioinformatics

The Fet3 ferroxidase enzyme in the fungal plasma membrane. This multicopper oxidase produces the ferric iron that is transported into the fungal cytoplasm via the permease, Ftr1. This is high-affinity iron accumulation system utilized by the human pathogens, Candida albicans and Cryptococcus neoformans.

Protein structure and function; bioinorganic chemistry; biophysical methods; oxidative stress.

2008-2013 DK53820-A1 (09-13)
Fet3p (Ferroxidase) and Ftr1p (Permease) in Iron Uptake in Yeast ($1,250,000 TDC requested, final budget not yet determined)

Eukaryotic iron metabolism involves two processes: redox cycling and trafficking. The transport of 'free' iron across eukaryotic plasma and some intracellular membranes is a paradigm of this metabolism. Thus, uptake of environmental Fe³⁺ involves first its reduction by a plasma membrane ferrireductase. The Fe²⁺ produced can be substrate for a multicopper oxidase - a ferroxidase - that couples the reduction of O₂ to the production of 4Fe³⁺. This ferric iron is then ligand for an iron permease that transports the iron across the plasma membrane. High affinity iron uptake in the yeast, Saccharomyces cerevisiae, exhibits all of these features. The metalloreductase, Fre1p, produces the Fe²⁺ that is substrate for ferroxidation by Fet3p, a ceruloplasmin ortholog, with permeation facilitated by Ftr1p. In yeast, as in the intestinal epithelium, the ferroxidation and permeation steps are coupled in the strict metabolic sense: permeation requires ferroxidation. This coupling suggests a primary hypothesis of this research: in the Fet3p, Ftr1p system the ferric iron product of the Fet3p ferroxidase reaction is channeled to Ftr1p for subsequent transmembrane trafficking. A template for this model is the movement of iron into and out of the ferritin (Ft) core. This hypothesis requires that both Fet3p and Ftr1p possess amino acid residues that participate in this channeling process, in addition to those structural motifs required for ferroxidation and permeation per se. There also may be motifs associated with the coupling of these two processes. The objective of this research is a full and detailed structure-function analysis of the Fet3p, Ftr1 system using biochemical, biophysical, genetic and cell biology approaches. These include: kinetic, spectral and crystallographic studies of wild type and mutant Fet3 proteins; iron uptake kinetic analysis of Ftr1p iron trafficking mutants; biochemical, genetic and fluorescence analysis of the physical and functional interaction between Fet3p and Ftr1p; and kinetic and electrophysiologic analysis of the coupling of ferroxidation and uptake. This structure-function characterization of the Fet3p, Ftr1p system will provide significant new understanding of eukaryotic iron trafficking.

2007-2011 RO1DK77826 (01-04)
Managing Ionic Iron: Architecture and Mechanism in Cell Iron Metabolism (745,000 TDC)

The goal of this application is to identify the components in the cytoplasm of Saccharomyces cerevisiae that are involved in the trafficking of ionic iron to sites of utilization for the metallation of apo-Fe enzymes and for storage within the yeast vacuole. A primary hypothesis to be tested is that these components are confined within contiguous conformation spaces so as to support a mechanism of direct transfer of Fe from one to another component. Dr. Kosman is the PI for this project.

2007-2009 R21 RR024178 (01-02)
Production of Recombinant Eukaryotic Ferroxidases as Protein Therapeutics (275,000 TDC)

The two goals of this application are to develop a robust system for the production of research quantities of the human ferroxidase ceruloplasmin and to engineer it and the yeast ferroxidase Fet3p into pharmacokinetically stable and physiologically effective ferroxidase replacement drugs.

• Isothermal calorimetry
• Oxygen electrode

S. cerevisiae, C. albicans, C. neoformans

Ferroxidases, iron permeases, metalloreductases: Fet, Ftr, Fre

Managing redox active metal ions - Fe and Cu - neurodegeneration

Fluorescence, FRET, kinetics, spectroscopy

Basic lines

*Wang, T.-P., *Severance, S., Quintanar, L., Solomon, E. I., and Kosman, D. J. 2003. Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase, Fet3p, from Saccharomyces cerevisiae. J. Biol. Inorg. Chem., 8:611-620 (this manuscript was in press at the time of the last review)
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12684851

Shi, X., *Stoj, C., *Romeo, A., Kosman, D. J., and Zhu, Z. 2003. Fre1p Cu²⁺ reduction and Fet3p Cu¹⁺ oxidation modulate copper toxicity in Saccharomyces cerevisiae. J. Biol. Chem., 278:50309-50315.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12954629

*Stoj, C., and Kosman, D. J. 2003. Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function. FEBS Lett., 554:422-426
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14623105

*Severance, S., *Chakraborty, S., and Kosman, D. J. 2004. The Ftr1p iron permease in the yeast plasma membrane: orientation, topology, and structure-function relationships. Biochem. J. 380:487-496
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14992688

Quintanar, L., Gebhard, M., *Wang, T.-P., Kosman, D. J., and Solomon, E. I. 2004. Ferrous binding to the multicopper oxidases Saccharomyces cerevisiae Fet3p and human ceruloplasmin: contributions to ferroxidase activity. J. Amer. Chem. Soc. 126:6579-6589
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15161286

Quintanar, L., *Stoj, C. S., *Wang, T.-P., Kosman, D. J. and Solomon, E. I. 2005. The role of aspartate 94 in the decay of the peroxide intermediate in the multicopper oxidase Fet3p. Biochemistry 44:6081-6091
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15835897

Taylor, A. B., *Stoj, C. S., *Ziegler, L., Kosman, D. J., and Hart, P. J. 2005. The copper-iron connection in biology: Structure of the metallo-oxidase Fet3p. Proc. Natl. Acad. Sci. USA 102:15459-15464
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16230618

*Stoj, C. S., and Kosman, D. J. 2005. Copper Oxidases. In Encyclopedia of Inorganic Chemistry, Vol. II, King, B., ed., John Wiley, pp. 1134-1159

*Kwok, E. Y. and Kosman, D. J. 2006. Iron in yeast: mechanisms involved in homeostasis. Top. Curr. Genet., 14:59-99

*Kwok, E. Y., *Stoj, C. S., *Severance, S., Kosman, D. J. 2006. An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane. J. Inorg. Biochem. 100:1053-1060
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16387364

*Singh, A., *Severance, S., *Kaur, N., *Wiltsie, W., Kosman, D. J. 2006. Assembly, activation and trafficking of the Fet3p, Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae. J. Biol. Chem. 281:13355-13364
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16522632

*Kwok, E. Y., *Severance, S., Kosman, D. J. 2006. Evidence for iron channeling in the Fet3p, Ftr1p high affinity iron uptake complex in the yeast plasma membrane. Biochemistry 45:6317-6327
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16700543

*Stoj, C. S., Augustine, T. J., *Zeigler, L., Solomon, E. I., and Kosman, D. J. 2006. The structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p. Biochemistry 45, 12741-12749.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17042492

*Stoj, C. S., Augustine, T. J., Solomon, E. I., and Kosman, D. J. 2007. Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae. J. Biol. Chem. 282:7862-7868.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12954629

Quintanar, L., *Stoj, C. S., Taylor, A. B., Hart, P. J., Kosman, D. J., and Solomon, E. I. 2007. Shall we dance? How a multicopper oxidase chooses its electron transfer partner. Acc. Chem. Res. 40:445-452.