The effect of redox state on the folding free energy of azurin

The unfolding of oxidized and reduced azurin by guanidine hydrochloride has been monitored by circular dichroism. Dilution experiments showed the unfolding to be reversible, and the equilibrium data have been interpreted in terms of a two-state model, The protein is stabilized by the strong metal binding in the native state, so that the folding free energy is as high as -52.2 kJ mol(-1) for the oxidized protein. The reduced protein is less stable, with a folding free energy of -40.0 kJ mol(-1). A thermodynamic cycle shows, as a consequence, that unfolded azurin has a reduction potential 0.13 V above that of the folded protein. This is explained by the bipyramidal site in the native fold stabilizing Cu(II) by a rack mechanism. with the same geometry being maintained in the Cu(I) form. In the unfolded protein, on the other hand, the coordination geometries are expected to differ for the two oxidation states, Cu(I) being stabilized by the cysteine thiol group in a linear or trigonal symmetry, whereas Cu(II) prefers oxygen ligands in a tetragonal geometry.