Cytochrome bc1 Complex [2Fe-2S] Cluster and Its Interaction with Ubiquinone and Ubihydroquinone at the Qo Site: A Double-Occupancy Qo Site Model

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The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Qpool) amounts to about 80% of the total ubiquinone and has an Em7 value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Qpoolcyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Q pool in different redox states, by the presence of inhibitors specific for the Qo site (myxothiazol and stigmatellin) and Qi site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Qo site structure. Evidence suggests that the Qo site can accommodate two ubiquinone molecules. One (designated gM ) is bound relatively strongly and is second only to the ubiquinone of the gA site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Qo site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at gx at 1.783 (Q) and gx at 1.777 (QH2); titration of the line-shape change reveals an Em7 value of approximately 95 mV. The other molecule (gow ) is bound more weakly, in the same range as the ubiquinone of the Qb site of the reaction center. Again, the affinities of the Q form (gx at 1.800) and QH2 form (gx at 1.777) are nearly equal, and the Eml value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Qo site double-occupancy model is considered that builds on the previous model based on Qo site mutants [Robertson, D. E., Daldal, F., & Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2Fe-2S] cluster ligands of the R. capsulatus cyt bcx complex are histidines [Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bcx complex completes a full enzymatic turnover without necessary exchange of ubiquinone with the Qpool. © 1992, American Chemical Society. All rights reserved.

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