title: Oxidation of Zn(Cys)4 zinc finger peptides by O2 and H2O2: products, mechanism and kinetics.
abstract: The reactivity of a series of Zn(Cys)(4) zinc finger model peptides towards H(2)O(2) and O(2) has been investigated. The oxidation products were identified by HPLC and ESI-MS analysis. At pH<7.5, the zinc complexes and the free peptides are oxidised to bis-disulfide-containing peptides. Above pH 7.5, the oxidation of the zinc complexes by H(2)O(2) also yields sulfinate- and sulfonate-containing overoxidised peptides. At pH 7.0, monitoring of the reactions between the zinc complexes and H(2)O(2) by HPLC revealed the sequential formation of two disulfides. Several techniques for the determination of the rate constant for the first oxidation step corresponding to the attack of H(2)O(2) by the Zn(Cys)(4) site have been compared. This rate constant can be reliably determined by monitoring the oxidation by HPLC, fluorescence, circular dichroism or absorption spectroscopy in the presence of excess ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid. In contrast, monitoring of the release of zinc with 4-(2-pyridylazo)resorcinol or of the thiol content with 5,5'-dithiobis(2-nitrobenzoate) did not yield reliable values of this rate constant for the case in which the formation of the second disulfide is slower than the formation of the first. The kinetic measurements clearly evidence a protective effect of zinc on the oxidation of the cysteines by both H(2)O(2) and O(2), which points to the fact that zinc binding diminishes the nucleophilicity of the thiolates. In addition, the reaction between the zinc finger and H(2)O(2) is too slow to consider zinc fingers as potential sensors for H(2)O(2) in cells.
authors: Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS/CEA/Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
authors: Emilie
authors: E
authors: Bourlès
authors: Manon
authors: M
authors: Isaac
authors: Colette
authors: C
authors: Lebrun
authors: Jean-Marc
authors: JM
authors: Latour
authors: Olivier
authors: O
authors: Sénèque
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D012118:Resorcinols / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D010455:Peptides; D012118:Resorcinols; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol; D010100:Oxygen