Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) --> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O(2) in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe(3+) (g = 4.3) and aqueous Mn(2+) ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe(3+) hemes were observed, probably arising from cytochrome c peroxidase and the a(3):Cu(b) site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe(2)S(2)](+) cluster of succinate dehydrogenase, the [Fe(2)S(2)](+) cluster of the Rieske protein of cytochrome bc (1), and the [Fe(3)S(4)](+) cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g (ave) = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe(4)S(4) clusters (60-85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O(2), was a quadrupole doublet with DeltaE (Q) = 1.15 mm/s and delta = 0.45 mm/s, assigned to [Fe(4)S(4)](2+) clusters. Substantial high-spin non-heme Fe(2+) (up to 20%) and Fe(3+) (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.

The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined by an integrative biophysical approach involving Mössbauer and electronic absorption spectroscopies, EPR and ICP-MS. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, was similar to that of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, respiration-related Fe-containing proteins were diminished ca. 3-fold, while nonheme HS Fe II species, nonheme mononuclear HS Fe III , and Fe III nanoparticles dominated. These changes were rationalized by a model in which the pool of nonheme HS Fe II ions serves as feedstock for Fe/S cluster and heme biosynthesis. The absolute concentrations of respirationrelated protein complexes were estimated.Mitochondria are cellular organelles that play critical roles in cellular physiology. Respiration and oxidative phosphorylation occur in these organelles, as do heme biosynthesis and iron/sulfur cluster assembly. As such, mitochondria are "hubs" of cellular iron trafficking (3). The Fe II ions used for these processes are imported by Mrs3p and Mrs4p, high-affinity transporters on the IM (3). Once in the matrix, these ions are delivered to Fe/S scaffold proteins and ferrochelatase (3). Many of these Fe/S and heme centers are inserted into respiratory complexes. Succinate dehydrogenase contains one [ Table S1, Protein and metal concentrations in isolated mitochondria; Figure S1, Electronic absorption spectra of heme-containing proteins; Figure S2, Protection of cytochrome c from protease degradation in isolated mitochondria; Figure S3, Mössbauer spectra of a respiring mitochondrial batch not shown in Figure 2 but used in constructing Table 1; Figure S4, Electronic absorption spectra of respiring mitochondria suspensions; Table S2, Concentrations of each heme component determined for individual mitochondrial samples; Figure S5, 10 K EPR spectra of mitochondria batches not shown in Figure 4 but used in the construction of Table 1; Figure S6, electronic absorption spectra of different batches of fermenting mitochondria. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 6. Mitochondrial dysfunction is associated with various diseases, including aging, cancer, heart disease, anemia and neurodegeneration (7-9). As cells age, there is a decline in Fe/S cluster biogenesis and mitochondrial membrane potential, leading with higher probability to a cellular crisis associated with loss of mitochondrial DNA, the instability and hypermutability of nuclear DNA, and cancer (10 Experimental ProceduresTwenty five L cultures of W303-1B cells were grown on min...

Mössbauer spectroscopy was used to detect pools of Fe in mitochondria from fermenting yeast cells, including those consisting of nonheme high-spin (HS) Fe II species, Fe III nanoparticles, and mononuclear HS Fe III species. At issue was whether these species were located within mitochondria or were exterior to it. None could be removed by washing mitochondria extensively with ethylene glycol tetraacetic acid or bathophenanthroline sulfonate (BPS), Fe II chelators that do not appear to penetrate mitochondrial membranes. However, when mitochondrial samples were sonicated, BPS coordinated the Fe II species, forming a low-spin Fe II complex. This treatment also diminished both Fe III species, suggesting that all of these Fe species are encapsulated by mitochondrial membranes and are protected from chelation until membranes are disrupted. 1,10-phenanthroline (phen) is chemically similar to BPS but is membrane soluble; it coordinated nonheme HS Fe II in unsonicated mitochondria. Further, the HS Fe III species and nanoparticles were not reduced by dithionite until the detergent deoxycholate was added to disrupt membranes. There was no correlation between the percentage of nonheme HS Fe II species in mitochondrial samples and the level of contaminating proteins. These results collectively indicate that the observed Fe species are contained within mitochondria. Mössbauer spectra of whole cells were dominated by HS Fe III features; the remainder displayed spectral features typical of isolated mitochondria, suggesting that the Fe in fermenting yeast cells can be coarsely divided into two categories: mitochondrial Fe and (mostly) HS Fe III ions in one or more non-mitochondrial locations.Iron serves critical roles in cell biology, generally involving catalytic and redox processes. This transition metal is found in many prosthetic groups, including hemes and iron sulfur clusters. These groups typically serve as enzyme active sites and redox centers. Dysfunction in cellular iron metabolism has been implicated in aging and in the pathogenesis of diseases involving reactive oxygen species (1). Clearly, cells need iron but they must handle it carefully to avoid † This study was supported by the National Institutes of Health grants GM084266, (PAL), EB-001475 (EM), T32GM008523 (GPHH and JGM) and the Robert A. Welch Foundation (A1170, PAL).

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Plant secondary metabolites can be used as an alternative way to control phytopathogenic fungi. The knowledge of the action mechanism of these compounds can contribute to design modified molecules with higher antifungal activity.