The electronic spectra of three rhombic type 1 blue copper proteins, nitrite reductase, pseudoazurin, and cucumber basic protein, have been studied by ab initio multiconfigurational second-order perturbation theory (the CASPT2 method). The six lowest excitations have been calculated and assigned with an error of less than 1800 cm-1. The singly occupied orbital in the ground-state forms a strongly covalent antibond between the copper ion and the thiolate group of the cysteine ligand with a mixture of σ and π character. This is in contrast to the axial type 1 copper protein plastocyanin which has an almost pure Cu−SCys π interaction. The two brightest lines in the absorption spectrum originate from transitions to the corresponding σ (∼460 nm) and π (∼600 nm) bonding orbitals. The relative intensity of these two lines is determined by the character of the ground-state orbital. It is possible to obtain a structure closely similar to the one found in nitrite reductase by geometry optimizations with the hybrid density functional B3LYP method in vacuum.It is a tetragonal structure with bonds of mainly σ character to the four ligands like normal square-planar Cu(II) complexes, but the cysteine thiolate group donates much charge to the copper ion and thereby makes the structure strongly distorted toward a tetrahedron. Both this structure and a trigonal π-bonded structure, which also can be obtained for all complexes and is an excellent model of plastocyanin, are equilibrium structures (although usually not with the same ligand models). They have virtually the same energy (within ∼7 kJ/mol), and the barrier between them is low. Therefore, small differences in the structure and electrostatics of different proteins may lead to stabilization of one or the other of the structures. The results indicate that axial type 1 proteins have a trigonal structure with an almost pure Cu−SCys π bond, whereas rhombic type 1 proteins have tetragonal structures with a significant σ character in this bond. Type 1.5 and 2 copper−cysteinate proteins arise when the tetragonal structure becomes more flattened than in nitrite reductase, probably by the inclusion of stronger (type 1.5) and more (type 2) ligands.

The electronic spectrum of the blue copper protein plastocyanin has been studied by ab initio multiconfigurational second-order perturbation theory (the CASPT2 method). The six lowest electronic transitions have been calculated and assigned with an error of less than 2000 cm-1. The singly occupied orbital in the ground state is Cu 3d−SCys 3pπ antibonding with some NHis 2pσ character. The bright blue color originates from an electron transfer to this orbital from the corresponding Cu 3d−SCys 3pπ bonding orbital. The influence of different ligand models on the spectrum has been thoroughly studied; Cu(imidazole)2(SCH3)(S(CH3)2)+ as a model of CuHis2CysMet is the smallest system that gives converged results. The spectrum is surprisingly sensitive to changes in the geometry, especially in the Cu−S bond distances; a 5 pm change in the Cu−SCys bond length may change the excitation energies by as much as 2000 cm-1. The effect of the surrounding protein and solvent on the transition energies has been modeled by point charges and is found to be significant for some of the transitions (up to 2000 cm-1).

Theoretical investigations of the structure and function of the blue copper proteins are described. We have studied the optimum vacuum geometry of oxidised and reduced copper sites, the relative stability of trigonal and tetragonal Cu(II) structures, the relation between the structure and electronic spectra, the reorganisation energy, and reduction potentials. Our calculations give no support to the suggestion that strain plays a significant role in the function of these proteins; on the contrary, our results show that the structures encountered in the proteins are close to their optimal vacuum geometries (within 7 kJ/mol). We stress the importance of defining what is meant by strain and of quantifying strain energies or forces in order to make strain hypotheses testable.

The electronic spectrum of the azurin Met121Gln mutant, a model of the blue copper protein stellacyanin, has been studied by ab initio multiconfigurational second-order perturbation theory (the CASPT2 method), including the effect of the protein and solvent by point charges. The six lowest electronic transitions have been calculated and assigned with an error of less than 2400 cm -1 . The ground-state singly occupied orbital is found to be a predominantly π antibonding orbital involving Cu3d and S cys 3p π . However, it also contains a significant amount (18%) of Cu-S cys σ antibonding character. This σ interaction is responsible for the appearance in the absorption spectrum of a band at 460 nm, with a significantly higher intensity than observed for other, axial, type 1 copper proteins (i.e., plastocyanin or azurin). The π-σ mixing is caused by the axial glutamine ligand binding at a much shorter distance to copper than the corresponding methionine ligand in the normal blue copper proteins. This explains why, based on its spectral properties, stellacyanin is classified among the rhombic type 1 copper proteins, although its structure is clearly trigonal, as it is for the axial proteins. Calculations have also been performed on structures where the glutamine model coordinates to the copper ion via the deprotonated N atom instead of the O atom. However, the resulting transition energies do not resemble the experimental spectrum obtained at normal or elevated pH. Thus, the results do not confirm the suggestion that the coordinating atom of glutamine changes at high pH.