Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154, and Cys158 are replaced individually by Ser have been constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters have been investigated. The Cys154Ser and Cys158Ser FrdB mutations result in enzymes with negligible activity that have largely dissociated from the cytoplasmic membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters. In contrast the Cys148Ser and Cys151Ser FrdB mutations result in functional membrane bound enzymes that are able to support growth under anaerobic and aerobic conditions. EPR studies of these mutants indicate that all three of the constituent Fe-S clusters are assembled, and the redox and spectroscopic properties of the [2Fe-2S] and [3Fe-4S] clusters are unchanged compared to the wild-type enzyme. In both mutants the [4Fe-4S] cluster is assembled with one non-cysteinyl ligand, and the available data suggest serinate coordination. The physicochemical consequences are perturbation of the intercluster spin interaction between the S = 1/2 [4Fe-4S]+ and S = 2 [3Fe-FS]0 clusters and a 60-mV decrease in redox potential for the [4Fe-FS]2+,+ cluster in the FrdB Cys148Ser mutant, and a S = 1/2 to S = 3/2 spin state conversion for the [4Fe-4S]+ cluster and a 72-mV decrease in redox potential for the [4Fe-4S]2+,+ cluster in the FrdB Cys151Ser mutant. Taken together with the previous FrdB Cys to Ser mutagenesis results [Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965-8969; Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703-2712], the results provide strong support for the proposal that all three clusters are located in the FrdB subunit with Cys57, Cys62, Cys65, and Cys77 ligating the [2Fe-2S] cluster, Cys148, Cys151, Cys154, and Cys214 ligating the [4Fe-4S] cluster, and Cys158, Cys204, and Cys210 ligating the [3Fe-4S] cluster. The role of the low potential [4Fe-4S] cluster in mediating electron transfer from menaquinol to the FAD active site is discussed in light of these mutagenesis results.