TOPOLOGICAL CHARACTERIZATION OF ESCHERICHIA-COLI DMSO REDUCTASE BY ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY OF AN ENGINEERED [3FE-4S] CLUSTER

We have applied the technique of distance estimations using the exogen ous paramagnetic probe dysprosium(III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase ( DmsABC) in situ in cytoplasmic membrane and whole cell preparations. T he electron transfer subunit (DmsB) of this enzyme contains four [4Fe- 4S] clusters and has complex EPR properties making it unsuitable for s tudies utilizing exogenous paramagnetic probes. We have utilized a mut ant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been c hanged to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) B iochemistry 30,8296-8305]. This mutant (DmsB-C102S) has a single magne tically isolated EPR visible [3Fe-4S] cluster in its fully oxidized st ate, making it suitable for studies using DyEDTA as paramagnetic probe . We have studied the effect of DyEDTA on the microwave power saturati on properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhan ces the spin relaxation of the [3Fe-4S] signal in everted membrane ves icles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] clust er of the DmsB-C102S mutant is located on the inside of the cytoplasmi c membrane. We have estimated the distance of this center from the sur face of the DmsAB dimer to be approximately 21 angstrom, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA eff ect using a myoglobin nitroxide standard.