title: Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.
abstract: Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(III) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.
authors.affiliation: Department of Chemistry, University of Georgia, Athens 30602.
authors.forename: M T
authors.initials: MT
authors.lastname: Werth
authors.forename: G
authors.initials: G
authors.lastname: Cecchini
authors.forename: A
authors.initials: A
authors.lastname: Manodori
authors.forename: B A
authors.initials: BA
authors.lastname: Ackrell
authors.forename: I
authors.initials: I
authors.lastname: Schröder
authors.forename: R P
authors.initials: RP
authors.lastname: Gunsalus
authors.forename: M K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008024:Ligands; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; D008024:Ligands; D013385:Succinate Dehydrogenase; D003545:Cysteine