EPR and Mossbauer spectroscopy of intact mitochondria isolated from Yah1p-depleted Saccharomyces cerevisiae

Yahlp, an [Fe2S2]-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 Yahlp was depleted confirms that Yahlp 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 yfh1 Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe3+ 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 Fe2+ 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 Fe2+ -> Fe3+ 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 Yahlp-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.