Arsenate resistance in E.coti conferred by the ars operon of plasmid R773 requires the product of the arsC gene. The region 88-96 in arsC gene is highly conserved in all known analogous of ArsC protein. By site directed mutagenesis codon for Arg94 was changed for Ser codon. Cells bearing arsC gene with the alteration R94S were arsenate sensitive and purified R94S ArsC protein was completely inactive. The role of Arg94 in catalytic activity of ArsC arsenate reductase was investigated. ArsC has no tryptophan residues. By site directed mutagenesis tryptophans were individually introduced around Arg94 in the positions for Q87, H88, I90, L91, N93, I96 and V97. Cells expressing arsC genes with mutations in codons for I90, L91 and N93 were arsenate sensitive and purified corresponding ArsC proteins were inactive. Q87W and H88W ArsC proteins had the same arsenate reductase activity as a wild type ArsC, but no changes in tryptophan fluorescence were observed after addition of substrate (AsV) or product (AsIII) of the reaction. I96W and V97W ArsC proteins were partially active. The fluorescence of I96W ArsC decreased after arsenite and arsenite + GSH , but not arsenate and arsenate + GSII addition. These results suggest that the highly conserved region I90 - V97 in ArsC is likely responsible for the binding of the product of the arsenate reduction. Supported by USPHS Grant CA54141.
