title: Site-directed mutagenesis of arginine-114 and tryptophan-129 in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides: two highly conserved residues predicted to be near the cytoplasmic surface of putative transmembrane helices B and C.
abstract: The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.
authors.affiliation: School of Life Sciences, University of Illinois, Urbana 61801.
authors.forename: B
authors.initials: B
authors.lastname: Hacker
authors.forename: B
authors.initials: B
authors.lastname: Barquera
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
authors.forename: A R
authors.initials: AR
authors.lastname: Crofts
mesh_terms: D000595:Amino Acid Sequence; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004563:Electrochemistry; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
chemical_list: D003573:Cytochrome b Group; D017931:DNA Primers; C032456:antimycin; D000968:Antimycin A; D014364:Tryptophan; D001120:Arginine; D014450:Electron Transport Complex III