Jhurry, Nema (2013-08). Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae. Doctoral Dissertation.
The speciation of iron in intact Jurkat cells and their isolated mitochondria was assessed using biophysical methods. [Fe4S4]^(2+) clusters, low-spin (LS) Fe^(II) heme centers, non-heme high-spin (NHHS) FeII species, ferritin-like material and FeIII oxyhydroxide nanoparticles were detected, via Mossbauer, in intact Jurkat cells and their isolated mitochondria. EPR spectroscopy was used to quantify Fe-containing species in the respiratory complexes. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Results were collectively assessed to estimate the first "ironome" profile of a human cell. The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and Fe^(III) citrate (FC), and of isolated mitochondria therefrom, was characterized. On average, only 400 +- 100 Fe's loaded per ferritin complex, regardless of the medium Fe concentration. The extent of nanoparticle formation scaled nonlinearly with the concentration of FC in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticles Fe, converting them into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage, and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Fermenting Saccharomyces cerevisiae cells grown with varying [Fe] were also studied. The high-affinity Fe import pathway was active only in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400 - 450 uM Fe. In these cells the concentration of nonmitochondrial NHHS Fe^(II) declined 3-fold, relative to in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe^(III) increased to a limiting cellular concentration of ~ 300 uM. Isolated mitochondria contained more NHHS Fe^(II) ions and substantial amounts of Fe^(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change of nutrient Fe levels.