Site-Directed Mutations of Conserved Residues of the Rieske Iron-Sulfur Subunit of the Cytochrome bc₁ Complex of Rhodobacter sphaeroides Blocking or Impairing Quinol Oxidation

Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinolxytochrome c₂ oxidoreductase (bc₁ complex) of Rhodobacter sphaeroides have been constructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolated and characterized in yeast by Gatti et al. [Gatti, D. L., Meinhardt, S. W., Ohnishi, T., & Tzagoloff, A. (1989) J. Mol. Biol. 205, 421–435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinolxytochrome c₂ oxidoreductase on flash activation of the photosynthetic chain. The impaired catalysis in this mutant complex is localized to the quinol oxidizing site. The apparent second-order rate constant for reduction of cytochrome b_H via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with its apparent activation barrier being increased relative to that of the wild-type. Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact. Reversion properties of these strains are consistent with the mutated residues being essential. Membranes of the cytochrome c₁ mutant CQ228stop [Konishi, K., Van Doren, S. R., Kramer, D. M., Crofts, A. R., & Gennis, R. B. (1991) J. Biol. Chem. 266, 14270–14276], with the soluble domain of cytochrome c₁ released from the cytoplasmic membrane to the periplasm, retain a crippled complex which contains a relatively unperturbed 2Fe-2S center and cytochrome b titrating in the same range as cytochrome b_H, but with a broader a band and a peak shifted to the red (λ_max at 563 nm). The complex binds antimycin and stigmatellin in the absence of both membrane-bound cytochrome c₁ and any center with the properties of the low-potential cytochrome b heme. Hence, the essential architecture of the 2Fe-2S cluster, as reported by ERR spectroscopy and by stigmatellin binding is independent of the cytochrome c₁ subunit.