Allison Cockrell - Mössbauer, EPR, and Modeling Study of Iron Trafficking and Regulation in Δccc1 and CCC1-up Saccharomyces cerevisiae

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      Allison L.   Cockrell, Sean P. McCormick, Michael J. Moore,   Mrinmoy Chakrabarti, and Paul A. Lindahl (2014) Biochemistry 53  (18), 2626 – 2940


        Strains lacking and overexpressing the vacuolar iron (Fe)   importer CCC1 were   characterized using Mössbauer and EPR spectroscopies. Vacuolar Fe   import is impeded in Δccc1 cells   and enhanced in CCC1-up cells,   causing vacuolar Fe in these strains to decline and accumulate,   respectively, relative to WT cells. Cytosolic Fe levels should   behave oppositely. The Fe content of Δccc1 cells   grown under low-Fe conditions was similar to that in WT cells.   Most Fe was mitochondrial with some nonheme high spin (NHHS)   FeII present. Δccc1 cells   grown with increasing Fe concentration in the medium contained   less total Fe, less vacuolar HS FeIII,   and more NHHS FeII than   in comparable WT cells. As the Fe concentration in the growth   medium increased, the concentration of HS FeIII in Δccc1 cells   increased to just 60% of WT levels, while NHHS   FeII increased   to twice WT levels, suggesting that the NHHS   FeII was   cytosolic.Δccc1 cells   suffered more oxidative damage than WT cells, suggesting that the   accumulated NHHS FeII promoted   Fenton chemistry. The Fe concentration in CCC1-up cells   was higher than in WT cells; the extra Fe was present as NHHS   FeII and   FeIII and   as FeIII oxyhydroxide   nanoparticles. These cells contained less mitochondrial Fe and   exhibited less ROS damage than Δccc1 cells. CCC1-up cells   were adenine-deficient on minimal medium; supplementing with   adenine caused a decline of NHHS FeII suggesting   that some of the NHHS FeII that   accumulated in these cells was associated with adenine deficiency   rather than the overexpression of CCC1.   A mathematical model was developed that simulated changes in Fe   distributions. Simulations suggested that only a modest   proportion of the observed NHHS FeIIin   both strains was the cytosolic form of Fe that is sensed by the   Fe import regulatory system. The remainder is probably generated   by the reduction of the vacuolar NHHS FeIII species.

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