The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase.
The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.
Canadian Institutes of Health Research Group in the Molecular Biology of Membrane Proteins, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
K S
KS
Oyedotun
B D
BD
Lemire
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D042963:Electron Transport Complex II; D005800:Genes, Fungal; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009097:Multienzyme Complexes; D011809:Quinones; D011994:Recombinant Proteins; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D019297:2,4-Dinitrophenol; C018527:dinoseb