Summary (Paul Alan Lindahl, PI) This MIRA renewal focuses on the cell biology of iron and to a more limited extent on copper. Transition metals have exceptional properties that render them indispensable for life, but they are also dangerous to the cell, such that trafficking must be tightly regulated. The PI is developing innovative and powerful approaches to fill huge gaps in understanding transition metal ion trafficking and regulation, especially in mitochondria which are iron and copper “traffic hubs”, and in the cytosolic Labile Fe Pool (LFeP) which accepts nutrient iron and distributes them to ~ 100 client apo-proteins in yeast cells. The chemical identity of the LFeP remains unestablished due to its inherent lability. To investigate such pools, the PI and his coworkers employ a novel custom liquid chromatography system in a refrigerated anaerobic glove box interfaced to an inductively-coupled plasma mass spectrometer (ICP-MS). Mössbauer (MB) spectroscopy is used to characterize the iron content of 57Fe-enriched cells, organelles, mouse organs, and blood plasma. Differential equations-based mathematical models are designed and developed to help understand the kinetics and mechanism of Fe trafficking in growing yeast cells. Few groups use any one of these innovative tools and no other lab worldwide uses all of them. This affords the PI a unique opportunity to solve critical problems in this field. In the past 5 years, with NIH MIRA support, the PI has published 23 peer-reviewed papers. Moving forward, the Lindahl lab will continue to investigate labile metal pools in biological systems using these approaches, coupled with electrospray ionization mass spectrometry (ESI-MS). Innovative chromatographic methods will be developed to minimize the lability of metal complexes. Most studies will use yeast cells, but labile metal pools in mammalian cells will also be investigated. How the LFeP changes with different genetic strains, metabolic conditions, and nutrient levels will be assessed. The LFeP in intact yeast cells will be detected and characterized by MB spectroscopy. The Fe/S species (known as X-S) that is exported from mitochondria into the cytosol will be identified. The LFeP in mitochondria will be reinvestigated using improved methods. Sophisticated and realistic mathematical models will be developed to simulate the kinetics of Fe trafficking and regulation. Whether non-transferrin-bound iron (NTBI), found in iron- overload diseases, is a high-molecular-mass FeIII aggregate or an FeIII citrate complex will be determined. Copper homeostasis and the mechanism of copper trafficking from cytosol to mitochondria will be probed, focusing on the role of metallothionein Cup1 in homeostasis, and on Cox17 and small nonproteinaceous CuLMM complexes as candidate trafficking species. Low-molecular mass CuLMM complex(es) will be isolated and identified by ESI-MS.