Reexamination of lead(II) coordination preferences in sulfur-rich sites: implications for a critical mechanism of lead poisoning.

Recent studies suggest that the developmental toxicity associated with childhood lead poisoning may be attributable to interactions of Pb(II) with proteins containing thiol-rich structural zinc-binding sites. Here, we report detailed structural studies of Pb(II) in such sites, providing critical insights into the mechanism by which lead alters the activity of these proteins. X-ray absorption spectroscopy of Pb(II) bound to structural zinc-binding peptides reveals that Pb(II) binds in a three-coordinate Pb(II)-S(3) mode, while Zn(II) is known to bind in a four-coordinate mode in these proteins. This Pb(II)-S(3) coordination in peptides is consistent with a trigonal pyramidal Pb(II)-S(3) model compound previously reported by Bridgewater and Parkin, but it differs from many other reports in the small molecule literature which have suggested Pb(II)-S(4) as a preferred coordination mode for lead. Reexamination of the published structures of these "Pb(II)-S(4)" compounds reveals that, in almost all cases, the coordination number of Pb is actually 5, 6, or 8. The results reported herein combined with this new review of published structures suggest that lead prefers to avoid four-coordination in sulfur-rich sites, binding instead as trigonal pyramidal Pb(II)-S(3) or as Pb(II)-S(5-8). In the case of structural zinc-binding protein sites, the observation that lead binds in a three-coordinate mode, and in a geometry that is fundamentally different from the natural coordination of zinc in these sites, explains why lead disrupts the structure of these peptides and thus provides the first detailed molecular understanding of the developmental toxicity of lead.

High metal concentrations are required for self-association of synaptotagmin II.

Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.

Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors.

Zinc binding to the two Cys(4) sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co(2+) as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys(4) structural zinc-binding sites found in the DBD of human estrogen receptor alpha (hERalpha-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co(2+) to the two proteins was determined relative to the binding affinity of Co(2+) to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co(2+) from CP-1, the dissociation constants of cobalt from hERalpha-DBD were calculated: K(d1)(Co) = 2.2 (+/- 1.0) x 10(-7) M and K(d2)(Co) = 6.1 (+/- 1.5) x 10(-7) M. Similarly, the dissociation constants of Co(2+) from GR-DBD were calculated: K(d1)(Co) = 4.1 (+/- 0.6) x 10(-7) M and K(d2)(Co) = 1.7 (+/- 0.3) x 10(-7) M. Metal-binding studies conducted in which Zn(2+) displaces Co(2+) from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn(2+) binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co(2+) does: the stoichiometric dissociation constants are K(d1)(Zn) = 1 (+/- 1) x 10(-10) M and K(d2)(Zn) = 5 (+/- 1) x 10(-10) M for hERalpha-DBD and K(d1)(Zn) = 2 (+/- 1) x 10(-10) M and K(d2)(Zn) = 3 (+/- 1) x 10(-10) M for GR-DBD. These affinities are comparable to those observed for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERalpha-DBD and GR-DBD do not interact.

Spectropotentiometric analysis of metal binding to structural zinc-binding sites: accounting quantitatively for pH and metal ion buffering effects.

Studies of the metal-binding affinity of protein sites are ubiquitous in bioinorganic chemistry and are valuable for the information that they can provide about metal speciation and exchange in biological systems. The potential for error in these studies is high, however, since many competing equilibria are present in solution and must be taken into consideration. Here, we report a new spectropotentiometric titration apparatus that allows pH and UV-vis absorption to be monitored simultaneously on small samples under inert atmosphere. In addition, we explain how data obtained from the complex equilibria can be combined with tabulated information about the protonation and metal-binding constants for common buffers to provide detailed, quantitative information about metal-protein interactions. Application of this approach to the investigation of metal binding to structural zinc-binding domains and common pitfalls encountered when performing these experiments are also discussed. We have used this approach to reevaluate the metal-binding constants of the N-terminal zinc-binding peptide from the HIV-1 nucleocapsid protein (10(-8)M

Magnetic Properties of Group 8 Metal-Metal-Bonded Porphyrin and Tetraazaporphyrin Dimers.

Solid state magnetic susceptibility data (2-350 K) are presented for the metal-metal doubly bonded dimers [Ru(OEP)](2) (OEP = octaethylporphyrin), [Ru(OETAP)](2) (OETAP = octaethyltetraazaporphyrin), and [Os(OEP)](2). The data are consistent with strong zero-field splitting of the triplet ground state (D approximately 240-630 cm(-)(1)). Variable temperature (200-300 K) (1)H NMR data are presented for [Os(OEP)](2) and [Ru(OETAP)](2) and for two heterodimers, [(OEP)RuRu(OETAP)] and [(OEP)OsRu(OETAP)].