SPECTROSCOPIC AND MECHANISTIC STUDIES OF TYPE-1 AND TYPE-2 COPPER SITES IN PSEUDOMONAS-AERUGINOSA AZURIN AS OBTAINED BY ADDITION OF EXTERNAL LIGANDS TO MUTANT HIS46GLY

The spectroscopic and mechanistic properties of the Cu-containing acti ve site of azurin from Pseudomonas aeruginosa were investigated by the construction of a mutant in which one of the ligands of the metal, Hi s46, was replaced by a glycine. Although the mutation creates a hole i n the interior of the protein, the 3D structure of the protein does no t change to any appreciable extent. However, the spectroscopic (optica l, resonance Raman, EPR) properties of the mutant protein are strongly affected by the mutation. In the presence of external ligands, the pr operties of the original wild-type protein are restored to a smaller o r larger extent, depending on the ligand. It is concluded that the hol e created by the mutation, even though it is completely buried inside the protein, can be filled by external ligands, often resulting in the creation of a mixture of so-called type-1 and type-2 copper sites. Al so, the redox properties (midpoint potential, kinetics of reduction/ox idation) appeared to be strongly affected by the mutation and the pres ence of external ligands. The results are compared with previous resul ts obtained on the mutant His117Gly.