The Architecture of the Qo Site of Cytochrome bc1 Complex Probed by Superoxide Production
- Washington State University
- 8392
- BATTELLE (PACIFIC NW LAB)
Although several X-ray structures have been solved for the mitochondrial cytochrome (cyt) bc1 complex, none yet shows the position of substrate, ubiquinol, in the quinol oxidase (Qo) site. In the present study, the interaction of molecular oxygen with the reactive intermediate Qo semiquinone is used to probe Qo site. It has been known for some time that partial turnover of the cyt bc1 complex in the presence of antimycin A, a Qi site inhibitor, results in accumulation of a semiquinone at the Qo site, which can reduce O2 to superoxide (O2?-). It was more recently shown that myxothiazol, which binds close to the cyt bL heme in the proximal Qo niche, also induces O2?- production. In this work we show that, in addition to myxothiazol, a number of other proximal Qo inhibitors (including E-b-methoxyacrylate-stilbene, mucidin and famoxadone) also induce O2?- production in isolated yeast cyt bc1 complex, at about 50% the Vmax observed in the presence of antimycin A. We propose that proximal Qo site inhibitors induce O2?- production because they allow formation, but not oxidation of the semiquinone at the distal niche of the Qo site pocket. The apparent Km for ubiquinol at the Qo site in the presence of Qo proximal inhibitors suggests that the distal niche of the Qo pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures these results suggest substrate ubiquinol binds in essentially the same position as stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin, and ubiquinol can bind simultaneously to the Qo site with virtually no steric hindrance, whereas progressively bulkier inhibitors show increasing overlap. The fact that partial turnover of the Qo site is possible even with bound proximal Qo site inhibitors is consistent with the participation of two separate functional Qo binding niches, occupied simultaneously or sequentially.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US), Environmental Molecular Sciences Lab. (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15005590
- Report Number(s):
- PNWD-SA-5911; 2358; TRN: US200323%%500
- Journal Information:
- Biochemistry, Vol. 42, Issue 21; Other Information: PBD: 3 Jun 2003
- Country of Publication:
- United States
- Language:
- English
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