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 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 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Biophysics Division, University of Illinois, Urbana-Champaign 61801.
S R
SR
Van Doren
R B
RB
Gennis
B
B
Barquera
A R
AR
Crofts
D000595:Amino Acid Sequence; D001665:Binding Sites; D003575:Cytochromes c1 / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D013816:Thermodynamics
D006873:Hydroquinones; D007506:Iron-Sulfur Proteins; D003575:Cytochromes c1; D014450:Electron Transport Complex III