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

J. Am. Chem. Soc., 121 (29), 6850 -6855, 1999. 10.1021/ja990899o S0002-7863(99)00899-9
Web Release Date: July 9, 1999

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

John C. Payne, Marc A. ter Horst, and Hilary Arnold Godwin*

Contribution from the Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113

Received March 19, 1999

Abstract:

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 Pb²⁺ 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 Zn²⁺ lead to two striking conclusions: first, the two metals rapidly equilibrate, and second, the ratio of Pb²⁺ to Zn²⁺ 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 Pb²⁺ should be able to compete effectively with Zn²⁺ for Cys₄ sites under physiological conditions. Despite the fact that Pb²⁺ binds tightly to cysteine-rich structural zinc sites, circular dichroism and ¹H NMR studies reveal that Pb²⁺ does not stabilize the correct fold of the peptides.

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