The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain.
The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.
Oyedotun
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The Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alta. T6G 2H7, Canada.
Lemire
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D003573:Cytochrome b Group / Q000737:chemistry; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D014451:Ubiquinone / Q000737:chemistry
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D014451:Ubiquinone; C017850:cytochrome b562, E coli; D013385:Succinate Dehydrogenase