Molecular Studies of the Succinate:ubiquinone Oxidoreductase of Escherichia Coli
Vibat, Cecile Rose Tandoc
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https://hdl.handle.net/2142/72360
Description
Title
Molecular Studies of the Succinate:ubiquinone Oxidoreductase of Escherichia Coli
Author(s)
Vibat, Cecile Rose Tandoc
Issue Date
1993
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Abstract
Succinate:ubiquinone oxidoreductase (Complex II) is a membrane-bound enzyme present in aerobically grown bacteria and in the mitochondria of eukaryotes. It functions as succinate dehydrogenase in the citric acid cycle and transfers reducing equivalents to ubiquinone in the aerobic electron transport chain. Complex II of Escherichia coli is homologous to its mammalian counterparts in subunit composition, structure, and function. It is composed of two large hydrophilic subunits (SDHA and SDHB) and two small hydrophobic subunits (SDHC and SDHD). The hydrophilic subunits are responsible for succinate dehydrogenase activity and contain the flavin and iron-sulfur redox centers. The small hydrophobic peptides contain cytochrome $b\sb{556},$ anchor the hydrophilic domain to the membrane, and allow electrons to be transferred to ubiquinone in the aerobic respiratory chain. The role of the b heme has eluded investigators for years. This work focuses on elucidating the function, location, and axial ligation of the heme in cytochrome $b\sb{556}$ of E. coli Complex II. Near-infrared MCD and EPR suggest bis-histidine coordination of the low spin heme. Site-directed mutagenesis was used to alter the histidine residues in the membrane-spanning subunits in an attempt to localize the ferric heme ligands. Biochemical characterization of the mutants indicates that the heme is not required for assembly or catalytic activity of the complex and suggest three possible candidates for the two ligands. Expression of the hydrophobic subunits from plasmid vectors implies that both SDHC and SDHD participate in heme ligation. This study provides strong evidence that suggests: (1) SDHC and SDHD are the minimum polypeptide components of cytochrome $b\sb{556},$ (2) the heme b bridges both subunits with the heme-iron coordinated to His84 in SDHC and His71 in SDHD, and (3) the heme in cytochrome $b\sb{556}$ is not critical for enzymatic activity or the assembly of membrane bound Complex II of E. coli.
Also described in this work are: (1) a simple protocol for the overproduction of Complex II in E. coli membranes and the rapid purification of stable enzyme, (2) a procedure for generating chromosomal replacements in E. coli K-12, and (3) efforts to obtain antibodies directed against the hydrophobic subunits of Complex II from E. coli.
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