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, Blanca Barquera and Antony R. Crofts

Biophysics Division and Departments of Chemistry and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801


Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome 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, 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 ubiquinol:cytochrome 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 contained 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 (max at 563 nm). The complex bound antimycin and stigmatellin in the absence both of membrane-bound cytochrome c1, and of any center with the properties of the low potential cytochrome b heme. Hence, the essential architecture of the 2Fe-2S cluster, as reported by EPR spectroscopy and by stigmatellin binding, are independent of the cytochrome c1 subunit.