Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC

Staphylococcus aueus pI258 CadC is an extrachromosomally encoded metalloreg ulatory repressor protein from the ArsR superfamily which negatively regula tes the expression of the cad operon in a metal-dependent fashion. The meta lloregulatory hypothesis holds that direct binding of thiophilic divalent c ations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulate s the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA b inding properties of wild-type CadC. The results of analytical ultracentrif ugation experiments suggest that both apo- and Cd-1-CadC are stable or weak ly dissociable homodimers characterized by a K-dimer = 3.0 x 10(6) M-1 (pH 7.0, 0.20 M NaCl, 25.0 degreesC) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the s toichiometry of Cd(II) and Pb(II) binding is approximate to0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms re veal that Cd(II) binds very tightly, with K-Cd = 4.3 (+/-1.8) x 10(12) M-1. The results of UV-Vis and X-ray absorption spectroscopy of the Cd-1 comple x are consistent with a tetrathiolate (S-4) complex formed by four cysteine Ligands. The Cd-113 NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S-3(N,O) or unusual upfield-shifted S-4 complex. T he Pb(II) complex reveals two prominent absorption bands at 350 nm (is an e lement of = 4000 M-1 cm(-1)) and 250 nm (is an element of = 41 000 M-1 cm(- 1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligo nucleotide containing the cad O/P upon binding CadC and analyzed using a di ssociable CadC dimer binding model reveals that apo-CadC forms a high-affin ity complex [K-a = (1.1 +/- 0.3) x 10(9) M-1; pH 7.0, 0.40 M NaCl, 25 degre esC], the affinity of which is reduced approximate to 300-fold upon the bin ding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.