title: The role of charged amino acids in the alpha1-beta4 loop of the iron-sulfur protein of the cytochrome bc1 complex of yeast mitochondria.
abstract: Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138-153 might be involved in the assembly of this protein into the cytochrome bc1 complex. To determine which specific residues might be involved in the assembly process, charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type. Activity of the cytochrome bc1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c1 content, determined by immunoblotting, was comparable with that of wild-type cells. However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein. The efficiency of assembly in vitro compared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc1 complex.
authors.affiliation: Department of Biochemistry, West Virginia University School of Medicine, Morgantown, West Virginia 26506, USA.
authors.forename: V H
authors.initials: VH
authors.lastname: Obungu
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: D S
authors.initials: DS
authors.lastname: Beattie
mesh_terms: D001692:Biological Transport; D002451:Cell Compartmentation; D014450:Electron Transport Complex III / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000648:ultrastructure; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000378:metabolism; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000254:growth & development*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
chemical_list: D005656:Fungal Proteins; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D009245:NADH Dehydrogenase; D014450:Electron Transport Complex III