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PMID:2246242

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Citation

Westenberg, DJ, Gunsalus, RP, Ackrell, BA and Cecchini, G (1990) Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli. J. Biol. Chem. 265:19560-7

Abstract

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.

Links

PubMed

Keywords

Amino Acid Sequence; Catalysis; Cell Membrane/enzymology; Cloning, Molecular; Electron Transport; Escherichia coli/enzymology; Fumarates/metabolism; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthols/metabolism; Plasmids; Structure-Activity Relationship; Succinate Dehydrogenase/chemistry; Succinate Dehydrogenase/genetics; Succinate Dehydrogenase/metabolism; Terpenes/metabolism; Transformation, Bacterial

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:FRDC

enables

GO:0102040: fumarate reductase (menaquinone)

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:FRDD

enables

GO:0102040: fumarate reductase (menaquinone)

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:FRDC

enables

GO:0008177: succinate dehydrogenase (ubiquinone) activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

Notes

See also

References

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