SITE-DIRECTED MUTATIONS OF CONSERVED RESIDUES OF THE RIESKE IRON-SULFUR SUBUNIT OF THE CYTOCHROME-BC(1) COMPLEX OF RHODOBACTER-SPHAEROIDES BLOCKING OR IMPAIRING QUINOL OXIDATION

Site-directed mutations of conserved residues in the domain binding th e 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides have been cons tructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG 133D), which mimicked a mutati on previously isolated and characterized in yeast by Gatti et al. [Gat ti, D. L., Meinhardt, S. W., Ohnishi, T., & Tzagoloff, A. (1989) J. Mo l. Biol. 205, 421-435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinol:cytochrome c2 oxidoreducta se on flash activation of the photosynthetic chain. The impaired catal ysis 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 bein g increased relative to that of the wild-type. Substitutions for the c ysteines and a histidine which are conserved in the putative 2Fe-2S bi nding domain of the Rieske subunit selectively knock out the 2Fe-2S cl uster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact. Reversion properties of the se strains are consistent with the mutated residues being essential. M embranes 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 rele ased from the cytoplasmic membrane to the periplasm, retain a crippled complex which contains a relatively unperturbed 2Fe-2S center and cyt ochrome b titrating in the same range as cytochrome b(H), but with a b roader a band and a peak shifted to the red (lambda(max) at 563 nm). T he complex binds antimycin and stigmatellin in the absence of both mem brane-bound cytochrome c1 and any center with the properties of the lo w-potential cytochrome b heme. Hence, the essential architecture of th e 2Fe-2S cluster, as reported by EPR spectroscopy and by stigmatellin binding is independent of the cytochrome c1 subunit.