The Inhibitor DBMIB Provides Insight into the Functional Architecture of the Qo Site in the Cytochrome b(6)f Complex
Previously, we showed that two equivalents of the quinone analog, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), could occupy the Qo site of the cytochrome (cyt) b6f complex simultaneously. In this work, study of electron paramagnetic resonance (EPR) spectra from oriented cyt b6f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Qo site. With a single DBMIB at the Qo site, the ISP is oriented with the 2Fe2S cluster toward cyt f, which is similar to the orientation of the ISP in the x-ray crystal structure of the cyt b6f complex from thermophilic cyanobacteria, Mastigocladus laminosus, in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc1 complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called ''proximal niche,'' near the cyt bL heme. These data suggest that the high affinity DBMIB site is at the proximal niche Qo pocket. With 2 equivalents or more of DBMIB bound, the Rieske ISP is in a position that resembles the ISPB position of chicken mitochondrial cyt bc1 complex in the presence of stigmatellin and Chlamydamonas reinhardtii cyt b6f complex in the presence of tridecyl-stigmatellin (TDS), which suggests that the low affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations where single binding was expected, did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, where double occupancy was expected, increased the extent of cyt f oxidation to levels similar to cyt f oxidation in the presence of saturating stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe2S cluster versus the physical orientations based on single crystal studies of the cyt bc1 complex suggest that the soluble ISP domain of spinach cyt b6f complex can rotate by at least 53o, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the x-ray crystal structures of the chicken mitochondrial cyt bc1 complex, and M. laminosus and C. reinhardtii cyt b6f complexes.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US), Environmental Molecular Sciences Laboratory (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15008816
- Report Number(s):
- PNWD-SA-6399; 1379; 2358; TRN: US200427%%243
- Journal Information:
- Biochemistry, Vol. 43, Issue 24; Other Information: PBD: 22 Jun 2004
- Country of Publication:
- United States
- Language:
- English
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