On the competition for available zinc.

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) 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 C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 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.