The ArsA ATPase is the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. ArsD is a metallochaperone that delivers As(III) to ArsA, increasing its affinity for As(III), thus conferring resistance to environmental concentrations of arsenic. R773 ArsD is a homodimer with three vicinal cysteine pairs, Cys12-Cys13, Cys112-Cys113, and Cys119-Cys120, in each subunit. Each vicinal pair binds As(III) or Sb(III). Alignment of the primary sequence of homologues of ArsD indicates that only the first vicinal cysteine pair, Cys12-Cys13, and an additional cysteine, Cys18, are conserved. The effect of cysteine-to-alanine substitutions and truncations were examined. By yeast two-hybrid analysis, nearly all of the ArsD mutants were able to interact with wild type ArsD, indicating that the mutations do not interfere with dimerization. ArsD mutants with alanines substituting for Cys112, Cys113, Cys119, or Cys120 individually or in pairs or truncations lacking the vicinal pairs retained ability to interact with ArsA and to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys 12, Cys13, or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. We propose that ArsD residues Cys12, Cys13, and Cys 18, but not Cys112, Cys113, Cys119, or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.
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