HIS-8 LOWERS THE PK(A) OF THE ESSENTIAL CYS-12 RESIDUE OF THE ARSC ARSENATE REDUCTASE OF PLASMID R773

The 141-residue ArsC arsenate reductase of plasmid R773 has an essenti al cysteine residue, Cys-12. The pK(a) of Cys-12 was determined to be 6.4, compared with a pK(a) of 8.3 for free cysteine. The possibility o f the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymatic activity was rapidly inactivated by the histid ine-modifying reagent diethylpyrocarbonate. The codons for the two his tidine residues in ArsC, His-8 and His-88, were changed by site direct ed mutagenesis. Cells expressing arsC(H88R), arsC(H88S), arsC(H88W), o r arsC(H88V) genes retained arsenate resistance, and the purified prot eins had wild type level of reductase activity, Cells expressing arSC( H8P), arsC(H8S), arsC(H8G), or arsC(H8R), genes were each sensitive to arsenate, and the purified H8P, H8G, and H8R proteins each lacked enz ymatic activity, Using the single histidine proteins it was shown that both histidines react with dethylpyrocarbonate but that only reaction with His-8 resulted in inactivation. The pK(a) value of Cys-12 was de termined to be 6.3 in the H8R enzyme and 8.3 in the H8G enzyme. These results indicate that His-8 is essential for catalytic activity and th at a positively charged residue is required at position 8 to lower the pK(a) of the cysteine thiolate at position 12.