Iron Trafficking and Regulation in Biological Systems
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Project Summary/The PI requests support of his research program in Iron Trafficking and Regulation in BiologicalSystems. Using powerful biophysical, bioanalytical, and mathematical tools, the PI and hiscoworkers propose to characterize and quantify the major iron species in isolated organelles,whole cells and organs from a wide diversity of biological systems. MÃ¶ssbauer spectroscopy willbe the primary biophysical tool, with EPR and UV-vis spectroscopies playing supporting roles.Various genetic strains of budding yeast and human cells will be enriched in 57Fe. Mitochondria,cytosol, vacuoles and other organelles will be isolated. The major Fe species in these cellularcomponents will be probed. Ordinary-differential-equations-based mathematical models will bedeveloped to analyze the results obtained and to generate a systemâ€™s level description of Fetrafficking and regulation. No other group worldwide investigates Fe in this way. Yeast strainsare being investigated in which Mrs3/4, high-affinity Fe importers located on the mitochondrialinner membrane, have been deleted and overexpressed. Other Fe-associated yeast strains, aswell as human Jurkat cells in which mitoferrins (homologs of Mrs3/4) and ferritin are knocked-down, will also be investigated. The Fe content of Escherichia coli and other prokaryotes will besimilarly explored. The central bioanalytical tool will be a liquid chromatography system locatedin a refrigerated anaerobic glove box that is linked to an on-line inductively coupled plasmamass spectrometer AND to an on-line electrospray ionization mass spectrometer. This LC-(ICP)-ESI-MS system will be unique worldwide. It will be used to characterize dozens of labilelow-molecular-mass (LMM) metal complexes that the PI and his group have discovered inorganelles, cytosol, blood plasma, and in E. coli. A LMM Fe complex in mitochondria that has amass of ca. 580 Da (called Fe580) is actively under investigation. Fe580 is most probably used asfeedstock for iron-sulfur cluster (ISC) assembly and for the iron-insertion step of hemebiosynthesis. The composition of a LMM sulfur-containing species called X-S will also beinvestigated. X-S is exported from mitochondria in association with ISC assembly, and it may beused to assemble cytosolic ISCs and to regulate Fe trafficking. A LMM Fe species in bloodplasma called â€œnon-transferrin-bound ironâ€ or NTBI will be investigated using pigs into which afeeding tube and sampling catheters have been surgically implanted. NTBI damages organs inpatients with Fe-overload diseases such as hemochromatosis. Various strains of mice with Fe-associated diseases will be investigated to determine the form of Fe that accumulates in theirorgans. The effect of hypoxia on Fe accumulation will also be examined.