title: 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 (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.
authors.affiliation: Department of Natural Sciences, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark.
authors.forename: Uwe
authors.initials: U
authors.lastname: Heinz
authors.forename: Martin
authors.initials: M
authors.lastname: Kiefer
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.forename: Hans-Werner
authors.initials: HW
authors.lastname: Adolph
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D013052:Spectrometry, X-Ray Emission; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D002614:Chelating Agents; D008024:Ligands; D010455:Peptides; D002104:Cadmium; D004492:Edetic Acid; D015032:Zinc