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

Steven R. Van Doren, Robert B. Gennis, Bianca Barquera, Antony R. Crofts

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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 bH 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 bH, but with a broader a band and a peak shifted to the red (λmax 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.

Original languageEnglish (US)
Pages (from-to)8083-8091
Number of pages9
Issue number32
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry

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