The role of various domains of the iron-sulfur protein in the assembly and activity of the cytochrome bc1 complex of yeast mitochondria.
Assembly studies in vitro of deletion mutants of the iron-sulfur protein into the cytochrome bc1 complex revealed that mutants localized in the extramembranous regions of the protein were not assembled into the complex in contrast to the efficient assembly of mutants in the membrane-spanning region. Charged amino acids located in the extramembranous alpha1-beta4 loop and the alpha1 helix were mutated and expressed in yeast cells lacking the gene for the iron-sulfur protein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 as well as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron-sulfur protein suggesting that these amino acids are required to maintain protein stability. By contrast, no loss of iron-sulfur protein accompanied the 30-50% loss of bc1 complex activity in mutants of three conserved alanine residues, A86, A90, and A92, suggesting that these residues may be involved in the proposed movement of the flexible tether of the iron-sulfur protein during catalysis.
Beattie
D S
DS
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142, USA. dbeattie2@wvvhsc1.hsc.wvv.edy
Wang
Y
Y
Obungu
V H
VH
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D017353:Gene Deletion; D006418:Heme / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D009038:Motion; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D005656:Fungal Proteins; D007506:Iron-Sulfur Proteins; D011522:Protons; C037989:Rieske iron-sulfur protein; D014451:Ubiquinone; D006418:Heme; D014450:Electron Transport Complex III; C003741:ubiquinol