title: A possible site of superoxide generation in the complex I segment of rat heart mitochondria.
abstract: 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-sulfur cluster N2.
authors.affiliation: Philadelphia Biomedical Research Institute, King of Prussia, PA 19406, USA. stohnishi@aol.com
authors.forename: S Tsuyoshi
authors.initials: ST
authors.lastname: Ohnishi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Shikibu
authors.initials: S
authors.lastname: Muranaka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Fujita
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kimura
authors.forename: Koichi
authors.initials: K
authors.lastname: Uemura
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshida
authors.forename: Kozo
authors.initials: K
authors.lastname: Utsumi
mesh_terms: D000818:Animals; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D007700:Kinetics; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D051381:Rats; D000073739:Strobilurins; D013481:Superoxides / Q000378:metabolism*
chemical_list: D005231:Fatty Acids, Unsaturated; D008689:Methacrylates; D000073739:Strobilurins; D013481:Superoxides; C008556:mucidin; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III