The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (wt) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by Differential Scanning Calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry-Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, G, is 31 kJ mol-1 higher. Both enthalpic and entropic factors contribute to the observed G increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin wt as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ mol-1, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterised by the highest value of the activation energy, Ea, as derived from the kinetic stability analysis.