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  • Title: Ratiometric pulsed alkylation mass spectrometry as a probe of thiolate reactivity in different metalloderivatives of Staphylococcus aureus pI258 CadC.
    Author: Apuy JL, Busenlehner LS, Russell DH, Giedroc DP.
    Journal: Biochemistry; 2004 Apr 06; 43(13):3824-34. PubMed ID: 15049689.
    Abstract:
    The coordination structure and reactivities of metal ligands in metal-sensing metalloregulatory coordination complexes may well dictate their biological properties. Here, we use the technique of ratiometric pulsed alkylation mass spectrometry (rPA-MS) to probe the structure and reactivities of metal coordination complexes formed by different metalloderivatives of Staphylococcus aureus plasmid pI258-encoded CadC, the metal-regulated transcriptional repressor of the cad operon. The cad operon provides resistance to large thiophilic heavy metal pollutants including Cd(II), Pb(II), and Bi(III). Two cysteines, an invariant Cys7 and a conserved Cys11, separated by three amino acids near the N-terminus of each subunit within dimeric CadC, donate two of the four coordination bonds to Cd(II) and Bi(III); in contrast, Cys11, but not Cys7, is excluded from the trigonal Pb(II) complex. rPA-MS reveals that Cys7 is strongly protected from alkylation in all metal complexes, Pb(II) being most effective, reducing k(app)(C7)by approximately 1000-fold relative to apo-CadC; in contrast, the reactivity of Cys11 is indistinguishable from that of apo-CadC, consistent with an S(3) coordination complex. Only in the tetrathiolate complexes formed by Cd(II) and Bi(III) is the reactivity of Cys11 appreciably reduced, but only by >or=10-fold. These data suggest that the Cys11-S(-)-metal coordination bond or that side of the coordination chelate in the trigonal Pb(II) complex defines a "weak point" in the chelate and thus might provide an entry site for potential metal ligand exchange reactions important for metal resistance in vivo. In contrast, Cys7 forms a tight coordination bond with all inducing metals, consistent with its role as a critical allosteric ligand in the metalloregulation of the operator/promoter binding.
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