Protein−Protein Interactions between Cytochrome b and the Fe-S Protein Subunits during QH2 Oxidation and Large-Scale Domain Movement in the bc1 Complex

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Abstract
The ubihydroquinone:cytochrome (cyt) c oxidoreductase, or bc1 complex, and its homologue the b6f complex are key components of respiratory and photosynthetic electron transport chains as they contribute to the generation of an electrochemical gradient used by the ATP synthase to produce ATP. The bc1 complex has two catalytic domains, ubihydroquinone oxidation (Qo) and ubiquinone reduction (Qi) sites, that are located on each side of the membrane. The key to the energetic efficiency of this enzyme relies upon the occurrence of a unique electron bifurcation reaction at its Qo site. Recently, several lines of evidence have converged to establish that in the bc1 complex the extrinsic domain of the Fe-S subunit that contains a [2Fe2S] metal cluster moves during catalysis to shuttle electrons between the Qo site and c1 heme. While this step is required for electron bifurcation, available data also suggest that the movement might be controlled to ensure maximal energetic efficiency [Darrouzet et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4567−4572]. To gain insight into the plausible control mechanism, we used a biochemical genetic approach to define the different regions of the bc1 complex that might interact with each other. Previously, we found that a mutation located at position L286 of the ef loop of Rhodobacter capsulatus cyt b could alleviate movement impairment resulting from a mutation in the hinge region, linking the [2Fe2S] cluster domain to the membrane anchor of the Fe-S subunit. Here we report that various substitutions at position 288 on the opposite side of the ef loop also impair Qo site catalysis. In particular, we note that while most of the substitutions affect only QH2 oxidation, yet others like T288S also hinder the rate of the movement of the Fe-S subunit. Thus, position 288 of cyt b appears to be important for both the QH2 oxidation and the movement of the Fe-S subunit. Moreover, we found that, upon substitution of T288 by other amino acids, additional compensatory mutations located at the [2Fe2S] cluster or the hinge domains of the Fe-S subunit, or on the cd loop of cyt b, arise readily to alleviate these defects. These studies indicate that intimate protein−protein interactions occur between cyt b and the Fe-S subunits to sustain fast movement and efficient QH2 oxidation and highlight the critical dual role the ef loop of cyt b to fine-tune the docking and movement of the Fe-S subunit during Qo site catalysis.