The ars operon of the E. coli plasmid R773 encodes resistance to arsenate and arsenite. The arsC gene product, ArsC, is reduces arsenate (As(V)) to arsenite (As(III)). Cysl2 is required for catalytic activity. The reductase has a pH optimum of about 6.5, considerably lower than that of cysteinyl residues in protein, which are usually in the range of 8.5-9. We have postulated that there is a basic residue near Cysl2 that forms a stable ion pair, lowering the pKa of Cysl2. ArsC has two histidine residues, His8 and His88, both of which were individually altered to other residues by site directed mutagensis. Mutations in the codon for His88 had no effect on arsenate resistance in vivo or on reductase activity in vitro. Cells expressing 4 different mutations in the codon for His8 were arsenate sensitive, and the corresponding purified proteins were inactive. Titration of Cysl2 measured either with DTNB or from the absorption of the thiolate anion at 240 nm yielded a pKa =6.9 for Cysl2. In the absence of His8 the pKa of Cysl2 was determined to be 9.5. The pKa of His8 was determined by modification with di ethyl pyrocarbonate. ArsC with Cysl2 had a pKa of 6.5; reductase with a C12S substitution yielded a pKa of 6.7 for His8. These results suggest that Hîs8 and Cysl2 form an ion pair which results in lowering of the pKa of the active site cysteine.
