Thermodynamics of Ni²⁺, Cu²⁺, and Zn²⁺ Binding to the Urease Metallochaperone UreE^†

The two Ni²⁺ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni²⁺, Cu²⁺, and Zn²⁺ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni²⁺ binding to the wild-type K. aerogenes UreE protein, and Ni²⁺ and Zn²⁺ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 μM, three Ni²⁺ or Cu²⁺ ions bind to 25 μM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)₄ and then (Ni/Cu)₂(H144*UreE)₄, or Ni/Cu(H144*UreE)₂, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni²⁺ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni²⁺ coordination to the two proteins. A relatively small negative value of ΔC_p is associated with Ni²⁺ and Cu²⁺ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.