Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae

An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.