THE ROLE OF ARSENIC THIOL INTERACTIONS IN METALLOREGULATION OF THE ARS OPERON

The ars operon of the Escherichia coil plasmid R773 that confers arsen ical and antimonial resistance is negatively regulated by the ArsR rep ressor, ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordinat ion between As(III) and the two cysteine thiolates, However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coord inate. A site-directed mutagenic approach was employed in a search for a third arsenic ligand, ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-ars DNA complexes could not be dissoci ated by inducers, Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the prote in, had no effect on the inducibility of the operon. While these resul ts indicated that neither the thiolate of Cys-37 nor the hydroxyl oxyg en of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand. Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations, Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo, In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex, Thus, tw o ArsR-arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide aff inity resin. ArsR proteins containing any two of the three cysteine re sidues Cys-32, Cys-34, and Cys-37 bound to the resin, Alteration of an y two of the three resulted in loss of binding, Arsenic x-ray absorpti on spectroscopy of ArsR treated stoichiometrically with arsenite confi rmed the average arsenic coordination as AsS3. These results suggest t hat all three cysteine thiolates are arsenic ligands, but binding to o nly two, the Cys-32 and Cys-34 thiolates, is required to produce the c onformational change that results in release of the repressor from the DNA and induction.