Characterization of Mutations in the Cytochrome b Subunit of the bc₁ Complex of Rhodobacter sphaeroides That Affect the Quinone Reductase Site (Q_c)

The cytochrome b subunit of the bc₁ complex contains two heme components, cytochrome b_L and cytochrome b_H, and is the locus of both a quinol oxidizing site (Q_o or Q_z) and a quinone reducing site (Q_i or Q_c). The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO. In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis. These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Q_c-directed inhibitors. The site-directed mutants all exhibit a normal rate of reduction of cytochrome b_h, suggesting a fully functional quinol oxidizing site. However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome b_H Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc₁ complex. In both cases, the cause of the defect is the lack of reoxidation of cytochrome bn by ubiquinone. This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome b_H to the Q_c-site. This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.