Oxidation of Zn(Cys)4 Zinc Finger Peptides by O2 and H2O2: Products, Mechanism and Kinetics

The reactivity of a series of Zn(Cys)4 zinc finger model peptides towards H2O2 and O2 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 H2O2 also yields sulfinate- and sulfonate-containing overoxidised peptides. At pH 7.0, monitoring of the reactions between the zinc complexes and H2O2 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 H2O2 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 H2O2 and O2, which points to the fact that zinc binding diminishes the nucleophilicity of the thiolates. In addition, the reaction between the zinc finger and H2O2 is too slow to consider zinc fingers as potential sensors for H2O2 in cells.