JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 7, pp 1029–1053

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

  • Brandon N. Hudder
  • Jessica Garber Morales
  • Audria Stubna
  • Eckard Münck
  • Michael P. Hendrich
  • Paul A. Lindahl
Original Paper

DOI: 10.1007/s00775-007-0275-1

Cite this article as:
Hudder, B.N., Morales, J.G., Stubna, A. et al. J Biol Inorg Chem (2007) 12: 1029. doi:10.1007/s00775-007-0275-1


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 bc1, 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 gave = 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 ΔEQ = 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.


IronSulfurCluster assemblyHeme biosynthesisNon-heme



Coenzyme Q




Ethylenediaminetetraacetic acid


Ethylenebis(oxyethylenenitrilo)tetraacetic acid


Electron paramagnetic resonance


Electron transfer flavoprotein


N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid


Inner membrane


Intermembrane space


Normal hydrogen electrode


Outer membrane

SH buffer

0.6 M sorbitol/20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid buffer pH 7.4

SP buffer

1.2 M sorbitol/20 mM potassium phosphate buffer pH 7.4

Supplementary material

Copyright information

© SBIC 2007

Authors and Affiliations

  • Brandon N. Hudder
    • 1
  • Jessica Garber Morales
    • 1
  • Audria Stubna
    • 2
  • Eckard Münck
    • 2
  • Michael P. Hendrich
    • 2
  • Paul A. Lindahl
    • 1
    • 3
  1. 1.Department of ChemistryTexas A&M UniversityCollege StationUSA
  2. 2.Department of ChemistryCarnegie Mellon UniversityPittsburghUSA
  3. 3.Department of Biochemistry and BiophysicsTexas A&M UniversityCollege StationUSA