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 gluta thione and glutaredoxin. In this study a direct association between Ar sC and glutaredoxin was demonstrated. An arsC gene with six histidine codons added to the 5' end of the gene was constructed, and the result ing ArsC enyzme was shown to be functional. Interaction of the histidi ne-tagged ArsC and glutaredoxin was examined by Ni2+ affinity chromato graphy. The association required the presence of reduced glutathione a nd either the substrate arsenate or a competitive inhibitor, phosphate or sulfate. A free thiolate on glutathione was not required, A trypto phan residue was introduced into ArsC at the 11th position, immediatel y adjacent to tl-re active site Cys-la, Trp-ll fluorescence was quench ed upon addition of arsenate, Addition of reduced glutathione after ar senate resulted in a rapid increase in fluorescence followed by a slow er decay of the signal. These spectroscopic signals were specific for arsenate and reduced glutathione; neither competitive inhibitors nor n on-thiol glutathione analogs produced this effect, Cys-12 thiolate was also required, Thus the intrinsic fluorescence of Trp-ll provides a u seful probe to investigate the mechanism of this novel reductase.