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Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 µM 57Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. Mössbauer spectra of isolated vacuoles were dominated by two spectral features; a mononuclear magnetically isolated high-spin (HS) FeIII species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands, and superparamagnetic FeIII oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a gave ~ 4.3 signal typical of HS FeIII with E/D ~ 1/3. Chemical reduction of the HS FeIII species was possible, affording a Mössbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 µM. Mössbauer analysis of FeIII polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, FeIII polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. FeIII polyphosphate could also be reduced to the FeII state, affording similar Mössbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the FeIII species in vacuoles, but they suggest a complex closely related to FeIII polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.

Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 μM Fe, distributed primarily into nonheme high-spin (NHHS) FeII species and mitochondrial Fe. Most NHHS FeII was not located in mitochondria, and their function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe4S4]2+ clusters, low- and high-spin hemes, S=½ [Fe2S2]1+ clusters, NHHS FeII species, and [Fe2S2]2+ clusters. The presence of [Fe2S2]2+ clusters was unprecedented; their presence in previous samples was obscured by the spectroscopic signature of FeIII nanoparticles which were absent 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. The majority of Mn ions in WT yeast cells was EPR-active MnII and not located in mitochondria or vacuoles. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400 – 450 μM Fe. In these cells the concentration of nonmitochondrial NHHS FeII declined 3-fold, relative to in Fe-deficient cells, whereas the concentration of vacuolar NHHS FeIII increased to a limiting cellular concentration of ~ 300 μM. Isolated mitochondria contained more NHHS FeII ions and substantial amounts of FeIII nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change of nutrient Fe levels. The ability to limit Fe import prevents cells from overloading with Fe.

The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with 57Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K Mössbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe4S4]2+ clusters and low-spin (LS) FeII heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be FeIII oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) FeII species. A portion of these species may constitute the “labile iron pool” (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. Electron paramagnetic resonance spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS FeIII heme and non-heme species. The basal heme redox state of mitochondria within cells was reduced; this redox poise was unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b, and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using inductively coupled plasma mass spectrometry. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells  the first “ironome” profile of a human cell.

Liquid chromatography was used with an on-line inductively coupled plasma mass spectrometer to detect low-molecular-mass (LMM) transition metal complexes in mitochondria isolated from fermenting yeast cells, human Jurkat cells, and mouse brain and liver. These complexes constituted 20 – 40% of total mitochondrial Mn, Fe, Zn, and Cu ions. The major LMM Mn complex in yeast mitochondria had a mass of ca. 1100 Da and a concentration of ~ 2 μM. Mammalian mitochondria contained a second Mn species with a mass of ca. 2000 Da at a comparable concentration. The major Fe complex in mitochondria isolated from exponentially growing yeast cells had a mass of ca. 580 Da; the concentration of Fe580 in mitochondria was ca. 100 μM. When mitochondria were isolated from fermenting cells in post-exponential phase, the mass of the dominant LMM Fe complex was ca. 1100 Da. Upon incubation, the intensity of Fe1100 declined and Fe580 increased, suggesting that the two are interrelated. Mammalian mitochondria contained Fe580 and 2 other Fe species (Fe2000 and Fe1100) at concentrations of ca. 50 μM each. The dominant LMM Zn species in mitochondria had a mass of ca. 1200 Da and a concentration of ca. 110 μM. Mammalian mitochondria contained a second major LMM Zn species at 1500 Da. The dominant LMM Cu species in yeast mitochondria had a mass of ca. 5000 Da and a concentration in yeast mitochondria of ca. 16 μM; Cu5000 was not observed in mammalian mitochondria. The dominant Co species in mitochondria, Co1200, had a concentration of 20 nM and was probably a cobalamin. Mammalian but not yeast mitochondria contained a LMM Mo species, Mo730, at ca. 1 μM concentration. Increasing Mn, Fe, Cu, and Zn concentrations 10 fold in the medium increased the concentration of the same element in the corresponding isolated mitochondria. Treatment with metal chelators confirmed that these LMM species were labile. The dominant S species at 1100 Da was not free GSH or GSSG.