The coordination structure and reactivities of metal ligands in metal-sensing metalloregulatorycoordination complexes may well dictate their biological properties. Here, we use the technique ofratiometric pulsed alkylation mass spectrometry (rPA-MS) to probe the structure and reactivities of metalcoordination complexes formed by different metalloderivatives of
Staphylococcus aureas plasmid pI258-encoded CadC, the metal-regulated transcriptional repressor of the
cad operon. The
cad operon providesresistance 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 eachsubunit 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 stronglyprotected from alkylation in all metal complexes, Pb(II) being most effective, reducing
by ~1000-fold relative to apo-CadC; in contrast, the reactivity of Cys11 is indistinguishable from that of apo-CadC,consistent with an
S3 coordination complex. Only in the tetrathiolate complexes formed by Cd(II) andBi(III) is the reactivity of Cys11 appreciably reduced, but only by
10-fold. These data suggest that theCys11-S
--metal coordination bond or that side of the coordination chelate in the trigonal Pb(II) complexdefines a "weak point" in the chelate and thus might provide an entry site for potential metal ligandexchange reactions important for metal resistance in vivo. In contrast, Cys7 forms a tight coordinationbond with all inducing metals, consistent with its role as a critical allosteric ligand in the metalloregulationof the operator/promoter binding.