Probing the Role of the Fe−S Subunit Hinge Region during Qo Site Catalysis in Rhodobacter capsulatus bc1 Complex

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Abstract
The ubihydroquinone:cytochrome c oxidoreductase, or bc1 complex, functions according to a mechanism known as the modified Q cycle. Recent crystallographic data have revealed that the extrinsic domain containing the [2Fe2S] cluster of the Fe−S subunit of this enzyme occupies different positions in various crystal forms, suggesting that this subunit may move during ubihydroquinone oxidation. As in these structures the hydrophobic membrane anchor of the Fe−S subunit remains at the same position, the movement of the [2Fe2S] cluster domain would require conformational changes of the hinge region linking its membrane anchor to its extrinsic domain. To probe the role of the hinge region, Rhodobacter capsulatusbc1 complex was used as a model, and various mutations altering the hinge region amino acid sequence, length, and flexibility were obtained. The effects of these modifications on the bc1 complex function and assembly were investigated in detail. These studies demonstrated that the nature of the amino acid residues located in the hinge region (positions 43−49) of R. capsulatus Fe−S subunit was not essential per se for the function of the bc1 complex. Mutants with a shorter hinge (up to five amino acid residues deletion) yielded functional bc1 complexes, but contained substoichiometric amounts of the Fe−S subunit. Moreover, mutants with increased rigidity or flexibility of the hinge region altered both the function and the assembly or the steady-state stability of the bc1 complex. In particular, the extrinsic domain of the Fe−S subunit of a mutant containing six proline residues in the hinge region was shown to be locked in a position similar to that seen in the presence of stigmatellin. Interestingly, the latter mutant readily overcomes this functional defect by accumulating an additional mutation which shortens the length of the hinge. These findings indicate that the hinge region of the Fe−S subunit of bacterial bc1 complexes has a remarkable structural plasticity.