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.

On the Design of Zinc‐Finger Models with Cyclic Peptides Bearing a Linear Tail

“…[79,80] For instance, we were able to demonstrate that the kinetics of their oxidation by H 2 O 2 is linked to their dynamic behavior in solution. Their small size allows characterization at the molecular level that is difficult with an entire protein.…”

“…Small peptidic models of ZFs have allowed us to gain new insights about the reactivity of ZFs. [79,80] For instance, we were able to demonstrate that the kinetics of their oxidation by H 2 O 2 is linked to their dynamic behavior in solution. [80] However, the reactivity of ZFs is still not well understood and many questions remain.…”

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines

“…Possible reaction pathway for the photooxidation of thiolates, either coordinated to zinc or free. Zn·CPF(17 AE 2) 10 6 (0.7 AE 0.07) 10 6 Zn·CP-1(CCHH) 0.0083 [50] Zn·L TC (14 AE 1) 10 6 (4.3 AE 0.4) 10 6 0.23 [71] [a] Ratec onstantsi nLmol À1 s À1 .…”

Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins

“…Moreover, metalloproteins, particularly those that bind metal ions through sulfur donors of cysteine residues, might be oxidized or chemically modified depending on the number of biological reactive species that typically decrease metal ion-to-protein affinity [3,4]. The best examples of such cysteine-containing proteins are the metallothioneins and zinc finger domains [5][6][7][8][9][10][11].…”