title: Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cyt b providing resistance to Qo-inhibitors of the bc1 complex from Rhodobacter capsulatus.
abstract: The substitutions M1401, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc1 complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Qo) inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc1 complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Qo site. In particular, the presence of a methyl group on the beta-carbon (Ile and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc1 complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of the bc1 complex indicating that this position affects its assembly or stability in vivo. These findings are discussed in terms of the contributions of these residues to inhibitor recognition and quinol oxidation at the Qo site of the bc1 complex.
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia 19104.
authors.forename: M K
authors.initials: MK
authors.lastname: Tokito
authors.forename: F
authors.initials: F
authors.lastname: Daldal
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D003062:Codon; D003573:Cytochrome b Group; D017931:DNA Primers; D004269:DNA, Bacterial; D006873:Hydroquinones; D011994:Recombinant Proteins; D014450:Electron Transport Complex III