CREATION OF TYPE-1 AND TYPE-2 COPPER SITES BY ADDITION OF EXOGENOUS LIGANDS TO THE PSEUDOMONAS-AERUGINOSA AZURIN HIS117GLY MUTANT

The imidazole side chain of the copper-coordinating histidine117 of the type-1 copper protein Pseudomonas aeruginosa azurin protrudes through the surface of the protein where it has contact with the solvent. Replacement of this histidine by a glycine, using site-directed mutagenesis, makes the copper center amenable to direct manipulation through the addition of external ligands. Depending on the kind of the externally added ligand, different type-1 and type-2 copper centers are obtained with UV/vis and EPR spectroscopic characteristics that encompass the known range of proteins with one copper in their active sites. In a Vanngard-Peisach-Blumberg plot the A(parallel-to) and g(parallel-to) EPR parameters of the His117Gly type-1 sites appear to cluster in three distinct regions, possibly indicating a preference of the mutant protein for a few distinct configurations of the metal site. After addition of various substituted imidazoles, the His117Gly mutant obtains UV/vis and EPR spectroscopic characteristics that are virtually identical to those of wild-type (WT) azurin. This implies that the structure of the mutant protein and the geometry of the metal site are maintained despite the replacement of the histidine. The importance of His117 in WT azurin for electron transfer is illustrated by the observation that the reconstituted mutant is not functional in reversible electron transfer.