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J. Biol. Chem., Vol. 276, Issue 20, 16936-16943, May 18, 2001
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From the Canadian Institutes of Health Research Group in the
Molecular Biology of Membrane Proteins, Department of Biochemistry,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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.
The Quinone-binding Sites of the Saccharomyces
cerevisiae Succinate-ubiquinone Oxidoreductase*
*
This work was supported by Canadian Institutes of Health
Research Grant MT-15290.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 780-492-4853;
Fax: 780-492-0886; E-mail: bernard.lemire@ualberta.ca.
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