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

Authors

  • Brandon N. Hudder
    • Department of ChemistryTexas A&M University
  • Jessica Garber Morales
    • Department of ChemistryTexas A&M University
  • Audria Stubna
    • Department of ChemistryCarnegie Mellon University
  • Eckard Münck
    • Department of ChemistryCarnegie Mellon University
  • Michael P. Hendrich
    • Department of ChemistryCarnegie Mellon University
    • Department of ChemistryTexas A&M University
    • Department of Biochemistry and BiophysicsTexas A&M University
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

Abstract

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.

Keywords

IronSulfurCluster assemblyHeme biosynthesisNon-heme

Abbreviations

CoQ

Coenzyme Q

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

EGTA

Ethylenebis(oxyethylenenitrilo)tetraacetic acid

EPR

Electron paramagnetic resonance

ETF

Electron transfer flavoprotein

HEPES

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

IM

Inner membrane

IMS

Intermembrane space

NHE

Normal hydrogen electrode

OM

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