Lead Fingers:  Pb2+Binding to Structural Zinc-Binding Domains Determined Directly by Monitoring Lead−Thiolate Charge-Transfer Bands

Here, we report that lead−thiolate charge-transfer bands (250−400 nm) can be used to monitor lead binding to cysteine-rich sites in proteins and report the application of this technique to determine the thermodynamics of lead binding to a series of structural zinc-binding domains. These studies reveal that Pb 2+ binds tightly to structural zinc-binding domains with dissociation constants that range from K d Pb = 10 - 9 to 10 - 14 M, depending on the number of cysteine residues in the metal-binding site. Competition experiments with Zn 2+ lead to two striking conclusions: first, the two metals rapidly equilibrate, and second, the ratio of Pb 2+ to Zn 2+ bound to a particular site is determined by the relative affinities of the two metals for that site, rather than being under kinetic control. We conclude that Pb 2+ should be able to compete effectively with Zn 2+ for Cys 4 sites under physiological conditions. Despite the fact that Pb 2+ binds tightly to cysteine-rich structural zinc sites, circular dichroism and 1 H NMR studies reveal that Pb 2+ does not stabilize the correct fold of the peptides.