A Possible Site of Superoxide Generation in the Complex I Segment of Rat Heart Mitochondria
- Cite this article as:
- Ohnishi, S.T., Ohnishi, T., Muranaka, S. et al. J Bioenerg Biomembr (2005) 37: 1. doi:10.1007/s10863-005-4117-y
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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.
KeywordsHeart mitochondriacomplex Isuperoxidefluorescence assay of hydrogen peroxideiron-sulfur cluster N2ubiquinoneubisemiquinone
bovine serum albumin
3,3′-dipropyl-2, 2′-thiodicarbocyanine iodide
ethoxyformic anhydride (also known as diethyl pyrocarbonate)
ethylene glycol bis(β-aminoethyl ether)
flavin adenine dinucleotide
- Qi and Qo sites
two ubiquinone binding sites in the complex III segment of the respiratory chain
reactive oxygen species