On the Competition for Available Zinc

Abstract

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C 2 H 2 ) and a tetrathiolate (C 4 ) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C 2 H 2 showed the expected Cys 2 His 2 -ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptidepeptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C 2 H 2 appeared as a simple second order process, the suggested mechanism for C 4 comprises a zinc-bridged Zn-(C 4 ) 2 species as well as a Zn-C 4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.Within the last decade the generally accepted roles of protein-bound zinc in catalysis and structure stabilization were complemented by regulatory functions. Protein binding sites have been classified as "catalytic," "co-catalytic," and "structural" zinc sites (1). A fourth type, namely "interface" was added recently (2). The possible role of a variable zinc occupancy of protein sites as a modification that provides a pathway for intracellular information transfer has been discussed (3), and the steadily growing number of known zinc proteins involved in gene regulation led to the suggestion that some classes of proteins might transduce changes in available zinc levels into changes in patterns of gene expression (4). An update of recent findings in zinc biology (5) now supports the view of zinc as a key element in cellular regulation.An important question with respect to a regulatory function, however, concerns the controversially discussed concentration of free zinc ions in different physiological environments. Cells react on a changed zinc supply by e.g. an activated transcription of metallothionein (Ref. 6 and references therein) or changes in the activity of zinc-sensing transcription activators like Zap1 (7) and MTF-1 (8). In the latter cases zinc binding to individual zinc finger motifs is supposed to induce structural changes, which in turn modify the functionality of the proteins. If the transduction ...

Zinc Ion-induced Domain Organization in Metallo-β-lactamases

“…Such a mechanism has been suggested for zinc transfer between MBLs and EDTA (14). It has been shown earlier for zinc finger peptides that substoichiometric availability of metal ions might lead to the formation of metal ion bridged complexes (51). Thus, we propose the ability of MBLs to form such complexes if the metal ion binding site concentration exceeds the concentration of available metal ions.…”

Metallothioneins with unusual residues: Histidines as modulators of zinc affinity and reactivity

“…It has been estimated that the interior of cells contain an over-abundance of zinc-binding units, essentially precluding the existence of ''free" zinc [34,35]. Accordingly, recent studies on zinc finger peptides have suggested that in biological systems, zinc exchange is likely to occur not through a dissociation/association mechanism, but rather via mixed ternary complexes, e.g.…”

“…Accordingly, recent studies on zinc finger peptides have suggested that in biological systems, zinc exchange is likely to occur not through a dissociation/association mechanism, but rather via mixed ternary complexes, e.g. with kinetically labile structural sites [35]. In the light of the idea that the regulation of zinc fluxes, in particular, reversible zinc binding to zinc fingers, is an important mechanism in cellular development and differentiation, we need to better understand the thermodynamics and kinetics of zinc binding to proteins in general.…”

Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals

“…A major issue regarding the question of how zinc is re-distributed in cells is whether zinc is chaperoned and transferred between proteins by zincmediated protein/protein interactions, i.e. without ever being free, 1 or whether zinc ions are generated intermittently from zinc proteins (Heinz et al 2005). It has been argued that re-distribution of zinc cannot be based on specific chaperone proteins because too many would be required to supply all of the many zinc proteins (Bozym et al 2006).…”