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.