Crystal structures of modified apo-His117Gly and apo-His46Gly mutants of Pseudomonas aeruginosa azurin.

The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion. The crystal structure of His117Gly azurin at 2.4 A resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112. There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets. In the structure of the wild-type holo-azurin from P. aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers. The completely different packing of the apo-His117Gly mutant can be explained by the disruption of the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115. The structure of apo-His46Gly azurin at 2.5 A resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation. In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion. The imidazole ring of His117 is shifted by about 1 A towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure. This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis. This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2.