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

Abstract

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ββα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ββα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.