Journal of Bioenergetics and Biomembranes

, Volume 37, Issue 1, pp 1–15 | Cite as

A Possible Site of Superoxide Generation in the Complex I Segment of Rat Heart Mitochondria

  • S. Tsuyoshi Ohnishi
  • Tomoko Ohnishi
  • Shikibu Muranaka
  • Hirofumi Fujita
  • Hiroko Kimura
  • Koichi Uemura
  • Ken-ichi Yoshida
  • Kozo Utsumi


We searched for possible sites of superoxide generation in the complex I segment of the respiratory chain by studying both forward and reverse electron transfer reactions in isolated rat heart mitochondria. Superoxide production was monitored by measuring the release of hydrogen peroxide from mitochondria with a fluorescence spectrophotometer using the Amplex red/horseradish peroxidase system. In the forward electron transfer, a slow superoxide production in the presence of glutamate and malate was enhanced by both rotenone and piericidin A (specific inhibitors at the end of the complex I respiratory chain). Both diphenileneiodonium and ethoxyformic anhydride (inhibitors for respiratory components located upstream of the respiratory chain) inhibited the enhancement by rotenone and piericidin A.

In contrast, in reverse electron transfer driven by ATP, both diphenileneiodonium and ethoxyformic anhydride enhanced the superoxide production. Piericidin A also increased superoxide production. Rotenone increased it only in the presence of piericidin A. Our results suggest that the major site of superoxide generation is not flavin, but protein-associated ubisemiquinones which are spin-coupled with iron-sulfer cluster N2.


Heart mitochondria complex I superoxide fluorescence assay of hydrogen peroxide iron-sulfur cluster N2 ubiquinone ubisemiquinone 



bovine serum albumin




3,3′-dipropyl-2, 2′-thiodicarbocyanine iodide


dimethyl sulfoxide






ethylenediaminetetraacetic acid


ethoxyformic anhydride (also known as diethyl pyrocarbonate)


ethylene glycol bis(β-aminoethyl ether)


flavin adenine dinucleotide


flavin mononucleotide




reduced ubiquinone

Qi and Qo sites

two ubiquinone binding sites in the complex III segment of the respiratory chain


reactive oxygen species


superoxide dismutase




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Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • S. Tsuyoshi Ohnishi
    • 1
    • 6
  • Tomoko Ohnishi
    • 2
  • Shikibu Muranaka
    • 3
  • Hirofumi Fujita
    • 3
  • Hiroko Kimura
    • 4
  • Koichi Uemura
    • 5
  • Ken-ichi Yoshida
    • 5
  • Kozo Utsumi
    • 3
  1. 1.Philadelphia Biomedical Research InstituteKing of Prussia
  2. 2.The Johnson Research Foundation and Dept. of Biochemistry and BiophysicsUniversity of PennsylvaniaPhiladelphia
  3. 3.Institute of Medical ScienceKurashiki Medical CenterKurashiki, OkayamaJapan
  4. 4.Department of Forensic MedicineJuntendo University School of MedicineTokyoJapan
  5. 5.Department of Forensic MedicineSchool of Medicine, the University of TokyoTokyoJapan
  6. 6.Philadelphia Biomedical Research InstituteRadnor

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