The Reactions of H2O2 and GSNO with the Zinc Finger Motif of XPA. Not A Regulatory Mechanism, But No Synergy with Cadmium Toxicity.

Aleksandra Witkiewicz-Kucharczyk,Jacek Olędzki,Wojciech Goch,Wojciech Bal,Andrea Hartwig

doi:10.3390/molecules25184177

Abstract

Tetrathiolate zinc fingers are potential targets of oxidative assault under cellular stress conditions. We used the synthetic 37-residue peptide representing the tetrathiolate zinc finger domain of the DNA repair protein XPA, acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) as a working model to study the reaction of its Zn(II) complex (ZnXPAzf) with hydrogen peroxide and S-nitrosoglutathione (GSNO), as oxidative and nitrosative stress agents, respectively. We also used the Cd(II) substituted XPAzf (CdXPAzf) to assess the situation of cadmium assault, which is accompanied by oxidative stress. Using electrospray mass spectrometry (ESI-MS), HPLC, and UV-vis and circular dichroism spectroscopies we demonstrated that even very low levels of H2O2 and GSNO invariably cause irreversible thiol oxidation and concomitant Zn(II) release from ZnXPAzf. In contrast, CdXPAzf was more resistant to oxidation, demonstrating the absence of synergy between cadmium and oxidative stresses. Our results indicate that GSNO cannot act as a reversible modifier of XPA, and rather has a deleterious effect on DNA repair.

Highlights

IntroductionThe zinc fingers (ZF) comprise one of the most abundant and diverse motifs in protein biochemistry, well beyond the classical structures in which the Zn(II) ion bound to two Cys and two His residues serves as a tetrahedral pin to create a DNA-recognizing loop between a β-sheet and an α-helix [1]
The zinc fingers (ZF) comprise one of the most abundant and diverse motifs in protein biochemistry, well beyond the classical structures in which the Zn(II) ion bound to two Cys and two His residues serves as a tetrahedral pin to create a DNA-recognizing loop between a β-sheet and an α-helix [1].The object of our interest, XPAzf, is a nonclassical four-Cys ZF present in the 273aa XPA nuclear protein belonging to the Nucleotide Excision Repair (NER) DNA repair pathway [2]
In our previous studies we demonstrated that a direct substitution of Zn(II) in ZnXPAzf by any of these metal ions is mechanistically possible and leads to structural alterations which are likely responsible for this inhibition [8,9]

Summary

Introduction

The zinc fingers (ZF) comprise one of the most abundant and diverse motifs in protein biochemistry, well beyond the classical structures in which the Zn(II) ion bound to two Cys and two His residues serves as a tetrahedral pin to create a DNA-recognizing loop between a β-sheet and an α-helix [1]. In our previous studies we demonstrated that a direct substitution of Zn(II) in ZnXPAzf by any of these metal ions is mechanistically possible and leads to structural alterations which are likely responsible for this inhibition [8,9]. Being uncharged and non-radical, it can diffuse to long distances and penetrates membranes It has a number of physiological functions as mildly reactive redox messenger, but elevated during oxidative stress it is disruptive to cellular metabolism. We demonstrated that it can destroy ZnXPAzf at a 10-fold molar excess by gradual formation of disulfide bonds and Zn(II) expulsion [12]. We used synthetic acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide as XPAzf model, as in our previous work [6,7,8,9,12,16]

Experimental Procedures

Results and Discussion

The exemplary of HPLC chromatograms obtained reaction of μM

The kinetics ofof reactions of 10