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

Research output: Contribution to journalArticlepeer-review

112 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)1-15
Number of pages15
JournalJournal of Bioenergetics and Biomembranes
Issue number1
Publication statusPublished - Feb 2005
Externally publishedYes


  • Complex I
  • Fluorescence assay of hydrogen peroxide
  • Heart mitochondria
  • Iron-sulfur cluster N2
  • Superoxide
  • Ubiquinone
  • Ubisemiquinone

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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