Ligand interactions of the ArsC arsenate reductase.
ArsC encoded by Escherichia coli plasmid R773 catalyzes the reduction of arsenate to arsenite. The enzymatic reaction requires reduced glutathione and glutaredoxin. In this study a direct association between ArsC and glutaredoxin was demonstrated. An arsC gene with six histidine codons added to the 5' end of the gene was constructed, and the resulting ArsC enyzme was shown to be functional. Interaction of the histidine-tagged ArsC and glutaredoxin was examined by Ni2+ affinity chromatography. The association required the presence of reduced glutathione and either the substrate arsenate or a competitive inhibitor, phosphate or sulfate. A free thiolate on glutathione was not required. A tryptophan residue was introduced into ArsC at the 11th position, immediately adjacent to the active site Cys-12. Trp-11 fluorescence was quenched upon addition of arsenate. Addition of reduced glutathione after arsenate resulted in a rapid increase in fluorescence followed by a slower decay of the signal. These spectroscopic signals were specific for arsenate and reduced glutathione; neither competitive inhibitors nor non-thiol glutathione analogs produced this effect. Cys-12 thiolate was also required. Thus the intrinsic fluorescence of Trp-11 provides a useful probe to investigate the mechanism of this novel reductase.
Liu
J
J
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D053501:Arsenite Transporting ATPases; D001426:Bacterial Proteins / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000737:chemistry; D016623:Ion Pumps; D008024:Ligands; D009097:Multienzyme Complexes; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D001426:Bacterial Proteins; D004220:Disulfides; D016623:Ion Pumps; D008024:Ligands; D009097:Multienzyme Complexes; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D005978:Glutathione