The orientation and the axial, Deltachiax, and rhombic, Deltachirh, components of the magnetic susceptibility tensor anisotropy for the cobalt(II) and nickel(II) derivatives of azurin from Pseudomonas aeruginosa have been determined from 1H NMR data. For both derivatives, the axial geometry of the system determines the orientation of the chi-tensor, whose z-axis forms an angle of 18.6 and 20.1 degrees with the Cu-OGly45 axial bond in the cobalt(II) and nickel(II) derivatives, respectively. For protons close to this axis, large negative pseudocontact shifts are observed, while those close to the NNS plane of the equatorial ligands experience lower and positive pseudocontact shifts for the same distance. Dipolar shifts are larger in the cobalt derivative, not only because of the larger spin number but also due to its intrinsically higher anisotropy. The contact contribution to the hyperfine shifts for the coordinated residues has been evaluated and analyzed in terms of unpaired spin delocalization mechanisms and geometry considerations. The results are extended to other blue copper proteins whose cobalt derivatives have been studied by 1H NMR. The electronic structure and its implications in the redox properties of the native copper proteins are also commented.

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)-azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N2S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.

Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gln in stellacyanin.

The paramagnetic 1H NMR spectra of the Co(II) and Ni(II) substituted forms of the type 1 blue copper protein (cupredoxin) amicyanin have been assigned. This is the first such analysis of a cupredoxin, which has a distorted tetrahedral active site with the ligands provided by two histidines, a cysteine and a methionine. The isotropic shifts of the resonances in these spectra are compared with those of Co(II) and Ni(II) azurin. A number of interesting similarities and differences are found. The coordination of the metal by the two equatorial histidine ligands is very similar in both proteins. The interaction between the introduced metal and the thiolate sulfur of the equatorial cysteine ligand is enhanced in the amicyanin derivatives. Resonances belonging to the weak axial methionine ligand exhibit much larger shifts in the amicyanin derivatives, indicative of shorter M(II)-S(Met) distances. The presence of shorter axial M(II)-S(Met) and equatorial M(II)-S(Cys) distances in both Co(II) and Ni(II) amicyanin is ascribed to the absence of a second axially interacting amino acid at the active site of this cupredoxin.