Low-temperature optical absorption, circular dichroism, magnetic circular dichroism, and sulfur K-edge X-ray absorption spectra have been measured for the green "blue" copper center (type 1) in Achromobacter cycloclastes nitrite reductase. Combined with density functional calculations, the results of these spectroscopies have been used to define the extremely "perturbed" electronic structure of this site relative to that of the prototypical "classic" site found in plastocyanin. Experimentally calibrated density functional calculations have been further used to determine the specific geometric distortions which generate the perturbed electronic structure. These studies indicate that the principal electronic structure changes in nitrite reductase, relative to plastocyanin, are a rotation of the Cu d 2 . These changes in Cu-ligand interactions result in the redistribution of absorption intensity in the charge transfer and ligand field transitions. Additionally, the new S(Met)-Cu interaction accounts for the unexpectedly high sulfur covalency in the HOMO. The increase in ligand field strength shifts all the d f d transitions in nitrite reductase to ∼1000 cm -1 higher energy than their counterparts in plastocyanin, which accounts for the EPR spectral differences between the type 1 sites in these complexes. The geometric distortion primarily responsible for the electronic structure changes in nitrite reductase, relative to plastocyanin, is determined to involve a coupled angular movement of the Cys and Met residues toward a more flattened tetrahedral (toward square planar) structure. This movement is consistent with a tetragonal Jahn-Teller distortion resulting from the shorter Cu-S(Met) bond in nitrite reductase relative to plastocyanin. This increased Jahn-Teller distortion implies that the type 1 site is "less entatic" than that in plastocyanin.

The electronic structures of the perturbed blue copper proteins stellacyanin (STC) and cucumber basic protein (CBP, also called plantacyanin, PNC) are defined relative to that of the well-understood “classic” site found in plastocyanin (PLC) by combining the results of low-temperature optical absorption, circular dichroism, and magnetic circular dichroism spectra with density functional calculations. Additionally, absorption and magnetic circular dichroism spectra of Alcaligenes denitrificans wild-type and M121Q azurin are presented and compared to PLC and STC, respectively. These studies show that the principal electronic structure changes in CBP/PNC, with respect to PLC, are a small shift of the ligand field transitions to higher energy and a rotation of the Cu d x 2 - y 2 half-filled HOMO which increases the pseudo-σ and decreases the π interactions of the cysteine (Cys) sulfur with Cu d x 2 - y 2 and, in addition, mixes some methionine (Met) sulfur character into the HOMO. The geometrical distortion responsible for the perturbed electronic structure, relative to PLC, involves a coupled angular movement of the Cys and Met residues toward a more flattened tetragonal structure. In contrast to CBP/PNC, STC (which has the axial Met substituted by Gln) has its ligand field transitions shifted to lower energy and undergoes much smaller degrees of HOMO rotation and Cys pseudo-σ/π mixing; no axial glutamine character is displayed in the HOMO. These changes indicate a tetrahedral distortion in STC. Therefore, perturbed spectral features are consistent with both tetragonal and tetrahedral geometric distortions relative to PLC. These perturbations are discussed in terms of the increased axial ligand strength in these proteins (i.e., short Cu−S(Met) in CBP/PNC and Oε(Gln) in STC). This induces an ∼ε(u)-like distorting force which either results in a tetragonal distortion of the site (CBP/PNC) or is structurally restrained by the protein (STC and M121Q).