The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys7, Cys11, Cys58, and Cys60. It is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. To examine whether Cys7 and Cys11 are spatially proximate to Cys58 and Cys60 of the same subunit or of the other subunit, homodimers with the same cysteine mutation in each subunit and heterodimers containing different cysteine mutations in the two subunits were reacted with 4,6-bis(bromomethyl)-3,7- dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, which cross-links thiol groups that are within 3-6 A of each other. Cys7 or Cys11 cross-linked only with Cys58 or Cys60 on the other subunit. The data demonstrate that Cys7 and Cys11 from one monomer are within 3-6 Å of either Cys58 or in the other monomer. The results of this study strongly indicate that each of the two Cd(II) binding sites in the CadC homodimer is composed of Cys7 and Cys11 from one monomer and Cys58 and Cys60 from the other monomer.
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