Structural heterogeneity of blue copper proteins: an EPR study of amicyanin and of wild-type and Cys3Ala/Cys26Ala mutant azurin

A comparative investigation of the effects of cooling rate and solvent physicochemical properties on the structural heterogeneity of wild-type and disulfide bond depleted azurin (Cys3Ala/Cys26Ala) and of amicyanin has been performed by EPR spectroscopy and computer simulation. By describing the spectral features of the EPR spectra in terms of Gaussian distributions of the components of the $$\mathop{\bf g}\limits^{\leftrightarrow}$$ and $$\mathop{\bf A}\limits^{\leftrightarrow}$$ tensors of the spin Hamiltonian, we have shown that either the cooling rate or the solvent composition affect the structural heterogeneity of the proteins. Such a heterogeneity has been quantified by the standard deviations σg || and σA || of the parallel components of the axially symmetric tensors. In particular, both parameters become smaller after the slow cooling cycle; such a reduction is more significant when glycerol is added as co-solvent to the protein solutions. The comparison of the σg || and σA || values found, for the copper proteins investigated, highlights that the reduction is more marked in the azurins compared to amicyanin and that the Cys3Ala/Cys26Ala azurin mutant has a structural heterogeneity lower than that shown by the wild-type protein. The remarkable similarity of the copper coordination sphere of the proteins suggests a more rigid structure of the azurin protein matrix in the absence of the disulfide bridge compared to wild-type azurin and of amicyanin with respect to both forms of azurin. The former result establishes an important role for the -SS- bond in modulating the flexibility of wild-type azurin.