title: 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.
abstract: The spectroscopic and mechanistic properties of the Cu-containing active site of azurin from Pseudomonas aeruginosa were investigated by the construction of a mutant in which one of the ligands of the metal, His46, was replaced by a glycine. Although the mutation creates a hole in the interior of the protein, the 3D structure of the protein does not change to any appreciable extent. However, the spectroscopic (optical, resonance Raman, EPR) properties of the mutant protein are strongly affected by the mutation. In the presence of external ligands, the properties of the original wild-type protein are restored to a smaller or larger extent, depending on the ligand. It is concluded that the hole 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. Also, the redox properties (midpoint potential, kinetics of reduction/oxidation) appeared to be strongly affected by the mutation and the presence of external ligands. The results are compared with previous results obtained on the mutant His117Gly.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: van Pouderoyen
authors.forename: C R
authors.initials: CR
authors.lastname: Andrew
authors.forename: T M
authors.initials: TM
authors.lastname: Loehr
authors.forename: J
authors.initials: J
authors.lastname: Sanders-Loehr
authors.forename: S
authors.initials: S
authors.lastname: Mazumdar
authors.forename: H A
authors.initials: HA
authors.lastname: Hill
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D003300:Copper; D004563:Electrochemistry; D008024:Ligands; D009154:Mutation; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D001400:Azurin; D003300:Copper