Consequences of removal of a molybdenum ligand (DmsA-Ser-176) of Escherichia coli dimethyl sulfoxide reductase.
We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli dimethyl sulfoxide (Me2SO) reductase (DmsABC). Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me2SO and in vitro benzyl viologen:Me2SO oxidoreductase activity. EPR spectroscopy reveals changes in the line shape and the gav of the Mo(V) signals of the S176A and S176C enzymes. The midpoint potentials (Em,7) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are -15 and -175 mV. The Em,7 of the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide. Titration of the S176C mutant produced several overlapping Mo(V) species occurring at Eh > -150 mV, suggesting heterogeneity in the molybdenum environment. A Mo(V) spectrum was not visible in S176H membranes poised between -435 to 350 mV or oxidized with 200 microM ferricyanide. No differences were detected in the EPR spectra of the reduced [4Fe-4S] clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced [4Fe-4S] clusters.
Department of Biochemistry and the Medical Research Council Group in the Molecular Biology of Membranes, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. joel.weiner@ualberta.ca
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D012694:Serine; D008982:Molybdenum; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase