Chapter 15 Isolation of Saccharomyces cerevisiae mitochondria for Mössbauer, EPR, and electronic absorption spectroscopic analyses.

Methods are presented to aid in the study of iron metabolism in isolated mitochondria. The "iron-ome" of mitochondria, including the type and concentration of all Fe-containing species in the organelle, is evaluated by integrating the results of four spectroscopic methods, including Mössbauer spectroscopy, electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. Although this systems biology approach only allows groups of Fe centers to be assessed, rather than individual species, it affords new and useful information. There are many considerations in executing this approach, and this chapter focuses on the practical methods that we have developed for this purpose. First, large quantities of mitochondria are required, and so published isolation methods must be scaled up. Second, mitochondria are isolated under strict anaerobic conditions to allow control of redox state and to protect O(2)-sensitive Fe-containing proteins from degradation. Third, the importance of packing mitochondria for both spectroscopic and analytical characterizations is developed. By measuring the volume of packed samples and the percentage of mitochondria contained within that volume, absolute Fe and protein concentrations within the organelle can be obtained. Packing samples into spectroscopy holders also affords maximal signal intensities, which are critical for these studies. Custom inserts designed for this purpose are described. Also described are the designs of a 25-L glass bioreactor, a mechanical cell homogenizer, a device for inserting short EPR tubes into the standard Oxford Instruments EPR cryostat, and a device for transferring samples from Mössbauer holders to EPR tubes while maintaining samples at liquid N(2) temperatures. A brief summary of what we have learned by use of these methods is included.

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

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.

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae.

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.