Nucleophilic reactivity of Zinc-bound thiolates: subtle interplay between coordination set and conformational flexibility

Abstract

During the past decades it has been established that zinc-bound cysteines in proteins can react with various electrophiles to perform physiological functions such as alkyl transfer reactions or oxidative stress sensing. Electrophiles targeting especially vulnerable structural zinc fingers have also been proposed as therapeutic agents against cancers or HIV. However, the nucleophilic reactivity of zinc fingers remains poorly understood. In this article, we investigate the nucleophilic reaction of zinc finger model peptides with H₂O₂ in order to get deeper insight into the factors governing the reactivity of zinc-bound cysteines of zinc finger sites. We use a set of nine peptides belonging to two different peptide families with (Cys)₄, (Cys)₃(His) and (Cys)₂(His)₂ coordination sets. One family is derived from the consensus peptide of classical ββα zinc fingers and the other one derived from the zinc finger site of the oxidative stress sensor Hsp33 that adopts a loosened zinc ribbon fold. The coordination properties and the structural behaviors of the new members of the latter family were carefully characterized. The rate constants of the reaction of the nine zinc finger models with H₂O₂ were measured at various temperatures to determine the activation parameters. In all cases, the reaction is characterized by a small enthalpy of activation, which shows that the nucleophilic reaction of zinc-bound cysteines is easy, and large unfavorable negative entropy of activation. Neutral Zn(Cys)₂(His)₂ cores are intrinsically less reactive than negatively charged Zn(Cys)₄ and Zn(Cys)₃(His). Interestingly, we observe that the more flexible zinc finger sites are the more reactive. Indeed the entropies of activation are strongly linked to the conformational behavior of the peptide in solution. This work reveals important factors that govern the reactivity of zinc-bound cysteines in these two structural classes of zinc fingers and can be used to identify reactive zinc fingers in proteins.