Original Paper

JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 7, pp 1029-1053

First online:

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

  • Brandon N. HudderAffiliated withDepartment of Chemistry, Texas A&M University
  • , Jessica Garber MoralesAffiliated withDepartment of Chemistry, Texas A&M University
  • , Audria StubnaAffiliated withDepartment of Chemistry, Carnegie Mellon University
  • , Eckard MünckAffiliated withDepartment of Chemistry, Carnegie Mellon University
  • , Michael P. HendrichAffiliated withDepartment of Chemistry, Carnegie Mellon University
  • , Paul A. LindahlAffiliated withDepartment of Chemistry, Texas A&M UniversityDepartment of Biochemistry and Biophysics, Texas A&M University Email author 

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Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O2 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 Fe3+ (g = 4.3) and aqueous Mn2+ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe3+ hemes were observed, probably arising from cytochrome c peroxidase and the a3:Cub 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 [Fe2S2]+ cluster of succinate dehydrogenase, the [Fe2S2]+ cluster of the Rieske protein of cytochrome bc 1, and the [Fe3S4]+ 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 Fe4S4 clusters (60–85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O2, was a quadrupole doublet with ΔE Q = 1.15 mm/s and δ = 0.45 mm/s, assigned to [Fe4S4]2+ clusters. Substantial high-spin non-heme Fe2+ (up to 20%) and Fe3+ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.


Iron Sulfur Cluster assembly Heme biosynthesis Non-heme