PROPERTIES OF THE ARSENATE REDUCTASE OF PLASMID R773

Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon c atalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resi stance to arsenate is conferred by the product of the arsC gene. In th is report, purified ArsC protein was shown to catalyze reduction of ar senate to arsenite. The enzymatic activity of the ArsC protein require d glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron don ors. A spectrophotometric assay was devised in which arsenate reductio n was coupled to NADPH oxidation. The results obtained with the couple d assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (K-m = 8 mM); other oxyanions including phosphate, sulfate, and antim onate were not reduced. Phosphate and sulfate were weak inhibitors, wh ile the product, arsenite, was a stronger inhibitor (K-i = 0.1 mM). Ar senate reductase activity exhibited a pH optimum of 6.3-6.8. These res ults indicate that the ArsC protein is a novel reductase, and elucidat ion of its enzymatic mechanism should be of interest.