Mössbauer and EPR Study of Iron in Vacuoles from Fermenting Saccharomyces cerevisiae

Abstract

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.

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

“…This signal arose from a mononuclear, NHHS Fe III S = 5/2 center with rhombic symmetry ( E / D ∼ 1/3), 9 the same species that afforded the sextet in Figure 1A. The associated EPR concentration was ∼150 μM (Table 1).…”

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2

“…4), particularly because the vacuoles might be expected to retain sufficient iron stores under these conditions. Mutants with defective vacuolar acidification have lower levels of iron-binding polyphosphates in the vacuole, which ultimately compromises iron storage (18,52,59), but we did not expect to see an immediate collapse of these stores.…”

“…Indeed, P FIT2 -GFP transcription significantly decreased at all concentrations (0.5, 1, and 2 mM) of supplemented iron (Fig. 5C) but was not restored to wild-type levels, possibly resulting from the inability of the vma mutant to properly import, utilize, or store iron (52,53). Interestingly, expression of the P TSA2 -GFP construct was reduced to wild-type levels when the vma2⌬ cells were supplemented with 2 mM iron (Fig.…”

Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis

“…Ferric nanoparticles have been detected in mitochondria [60, 67–69] and in vacuoles [66, 70]. Fe K-edge EXAFS and electron microscopy performed on mitochondria-containing NP indicate a pentacoordination by oxygen atoms for the Fe III ion (Fig.…”

“…Depending on the batch of vacuoles, the major contribution to the Mössbauer spectra is either analogous to that of mitochondrial ferric nanoparticles or to that of magnetically noninteracting high-spin Fe III ions. Based on the Mössbauer signatures of 57 Fe polyphosphate prepared in different acetate/acetic acid-buffered media, it is proposed that pH plays a critical role [70]. All the studies performed evidenced that the accumulation of iron in the cytosol is prevented because of the potential damages that Fe II ions can cause.…”