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 exogenous 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. The electron transfer subunit (DmsB) of this enzyme contains four [4Fe-4S] clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes. We have utilized a mutant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been changed to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) Biochemistry 30, 8296-8305]. This mutant (DmsB-C102S) has a single magnetically isolated EPR visible [3Fe-4S] cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe. We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhances the spin relaxation of the [3Fe-4S] signal in everted membrane vesicles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane. We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.
Department of Biochemistry, University of Alberta, Edmonton, Canada.
R A
RA
Rothery
J H
JH
Weiner
D002462:Cell Membrane / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003593:Cytoplasm / Q000737:chemistry; D004419:Dysprosium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D008872:Microwaves; D016296:Mutagenesis; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D013113:Spin Labels; D004419:Dysprosium; D004492:Edetic Acid; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase