Role of protons in the thermodynamic contribution of a Zn(II)-Cys4 site toward metalloprotein stability

The current limited understanding of the free energy contributions of metal-protein interactions toward metalloprotein stability is largely due to an inability to separate the energetics of the metal-ligand and protein-protein interactions. In order to elucidate the thermodynamic contribution of a Zn(II)-(S center dot Cys)(4) site toward metalloprotein stability relevant to classic structural Zn(II) sites, the reaction of {Zn(II)(H2O)(6)}(2+) with a minimal, unstructured, tetracysteine 16-mer peptide, GGG, is described. Isothermal titration fluorimetry over the pH range of 4.5 to 9.0 is used to measure the free energy of Zn(II) binding to the model peptide GGG. The data show that, in the absence of proton competition, Zn(II) binds to the Cys(4) coordination sphere with a K-d of 60 aM, indicating that the Zn(II)-(S center dot Cys)(4) interaction can provide up to 22.1 kcal mol(-1) in driving force for protein stabilization, folding, and/or assembly. Isothermal titration calorimetry shows that Zn(II)-GGG formation is entropy driven because of water release from both the metal and the peptide scaffold. At pH 7.0, where the Zn(II)-GG K-d value is 8.0 pM, the reaction releases 3.8 protons, is endothermic with Delta H-rxn of +6.4 kcal mol(-1), and entropy driven with Delta S-rxn of +72 cal K-1 mol(-1). At pH 8.0, where the peptide is partially deprotonated prior to Zn(II) binding, the 1.0 fM Zn(II)-GGG K-d value reflects a Zn(II) complexation reaction involving the release of 2.5 protons, which is slightly exothermic, with Delta H-rxn of -2.0 kcal mol(-1), and largely entropy driven, with Delta S-rxn of +61 cal K-1 mol(-1). At pH 5.5, where proton competition weakens the K-d to 4.0 mu M, only 3.2 protons are released upon Zn(II) binding, the reaction is endothermic, with Delta H-rxn of +7.7 kcal mol(-1), and entropy driven, with Delta S-rxn of +51 cal K-1 mol(-1). Likely an intrinsic property of Zn(II)-(S center dot Cys)(4) sites, the entropy driven binding of Zn(II) reflects the proton dependent chemical speciation of the Zn(II)-(S center dot Cys)(4) peptide complex and its effects on modulating the dehydration of both the peptide and metal. Furthermore, the Zn(II) binding thermodynamics of a variety of Zn(II) proteins at pH 7.0 reveals the presence of enthalpy-entropy compensation (EEC) phenomena in nature.