| Abstract
| - Using cobalt or nickel to replace copper in native azurin allowsone to fingerprint the metalcoordination site of the protein. The metal sites of wild typeAlcaligenes denitrificans azurin and itsM121Q mutant are clearly distinguishable through the paramagnetic1H NMR spectra of the Ni(II) andCo(II) derivatives. In the wild type azurin, Gly45coordinates to nickel or cobalt, while Met121 appearsas a weak metal ligand. On the contrary, in the M121Q azurinmutant, the metal exhibits a clear preferencefor the Gln121, which coordinates through the side chain carbonyloxygen, and Gly45 is not a ligand.Changes in the isotropic shifts and relaxation properties ofsignals from the Cys112, His46, and His117metal ligands suggest a movement of the metal ion out of the equatorialplane, indicating that the metalsite is tetrahedral. These effects are less pronounced in theNi(II) M121Q azurin than in the Co(II)metalloderivative. The similarity between the NMR spectra of theCo(II) derivatives of stellacyanin andthe M121Q azurin is in agreement with a very similar metal site in bothproteins and supports the existenceof a coordinated Gln in stellacyanin.
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