Mössbauer and EPR detection of iron trafficking kinetics and possibly labile iron pools in whole Saccharomyces cerevisiae cells

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The kinetics of iron trafficking in whole respiring Saccharomyces cerevisiae cells were investigated using Mössbauer and EPR spectroscopies. The Mössbauer-active isotope 57Fe was added to cells growing under iron-limited conditions; cells were analyzed at different times post iron addition. Spectroscopic changes suggested that the added 57Fe initially entered the labile iron pool, and then distributed to vacuoles and mitochondria. The first spectroscopic feature observed, ∼3min after adding 57Fe plus a 5 to 15min processing dead time, was a quadrupole doublet typical of nonheme high-spin FeII. This feature likely arose from labile FeII pools in the cell. At later times (15-150min), magnetic features due to S=5/2 FeIII developed; these likely arose from FeIII in vacuoles. Corresponding EPR spectra were dominated by a g=4.3 signal from the S=5/2 FeIII ions that increased in intensity over time. Developing at a similar rate was a quadrupole doublet typical of S=0 [Fe4S4]2+ clusters and low-spin FeII hemes; such centers are mainly in mitochondria, cytosol, and nuclei. Development of these features was simulated using a published mathematical model, and simulations compared qualitatively well with observations. In the five sets of experiments presented, all spectroscopic features developed within the doubling time of the cells, implying that the detected iron trafficking species are physiologically relevant. These spectroscopy-based experiments allow the endogenous labile iron pool within growing cells to be detected without damaging or altering the pool, as definitely occurs using chelator-probe detection and possibly occurs using chromatographic separations.