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Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, $${\text{N}}_{3} \overset k \longleftrightarrow {\text{U}}_{3} \xrightarrow{k}{\text{F}} $$ (N—native, U—unfolded and F—final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T max ≈ 85°C, i.e., 15°C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model $${\text{N}}_{3} \xrightarrow{k}{\text{F}}_{3} , $$ where the protein irreversibly passes from the native to the final state. No evidence of subunits’ dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

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.