Study of coordination chemistry, thermodynamics, and kinetics of metal binding to zinc-binding peptides

Lead poisoning is the most common environmental disease in the United States, but the molecular mechanism(s) of lead's action in the body are not well understood. To understand how a toxic metal such as lead can disrupt biological systems, it is essential to understand the basic physical and inorganic chemistry of metal binding. Studies of the rates and affinities of metal ions for protein sites, and the coordination environment of the bound metal, are the key to understanding the speciation, transport, and exchange of metals in cells.; In this study, the kinetics of lead binding to a structural zinc-binding domain from HIV nucleocapsid protein (HIV-CCHC) were examined directly using stopped-flow absorption spectroscopy to monitor changes in lead-thiolate charge-transfer bands. Lead binds to this domain on a millisecond timescale by a mechanism involving the formation of a stable intermediate. The intramolecular rearrangement of this intermediate is the rate-limiting step of the process. Additionally, the rates of Pb-Zn exchange on this peptide were examined. Pb-Zn exchange occurs by an associative mechanism; and although the exchange reaction is slower than simple lead binding by several orders of magnitude, metal substitution occurs on a physiologically relevant timescale (seconds).; X-ray absorption spectroscopy (XAS) was used to examine the Pb coordination environment in cysteine-rich structural zinc sites. These studies showed that Pb prefers a trigonal PbS3 coordination even when four sulfurs are available for binding. Pseudotetrahedral geometry, with the Pb lone pair in the fourth position, must be significantly stabilized over a distorted coordination environment. XAS studies of a Pb-S small molecule complex also showed that lead has a tendency to avoid distorted four-coordinate geometries; Pb(II) iso-maleonitriledithiolate forms a Pb-Pb dimer with bridging sulfur atoms in order to be five-coordinate pseudooctahedral rather than four-coordinate distorted.; Finally, a new apparatus for conducting rigorous, automated metal-protein affinity studies in an inert atmosphere is described. Detailed studies of Co binding to HIV-CCHC under a variety of buffer, pH, and chelating conditions were conducted using this method in order to point out the potential pitfalls that may arise in such studies.