Electron paramagnetic resonance spectroscopic characterization of dimethyl sulfoxide reductase of Escherichia coli.
The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme. Iron-sulfur clusters of the [4Fe-4S] type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C. The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule. The dithionite-reduced clusters were reoxidized by DMSO or TMAO. The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E. coli nitrate reductase. The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor. In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased. Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur. These results are in agreement with predictions from amino acid sequence comparisons, that the molybdenum center is located in DmsA, while four iron-sulfur clusters are in DmsB. The midpoint potentials of the molybdenum and iron-sulfur clusters in the various preparations were determined by mediator titrations. The iron-sulfur signals could be best fitted by four clusters, with midpoint potentials spread between -50 and -330 mV. The midpoint potentials of the iron-sulfur clusters and Mo(V) species were pH dependent. In addition, all potentials became less negative in the presence of the detergent Triton X-100. Observation of relaxation enhancement of the Mo(V) species by the reduced [4Fe-4S] clusters indicated that the centers are in proximity within the protein.
Cammack
R
R
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Weiner
J H
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D010088:Oxidoreductases; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C045664:dimethyl sulfoxide reductase