title: Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa.
abstract: A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B.G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.
authors.affiliation: Molecular Biophysics Group, CCLRC Daresbury Laboratory, Warrington, Cheshire, U.K.
authors.forename: R W
authors.initials: RW
authors.lastname: Strange
authors.forename: L M
authors.initials: LM
authors.lastname: Murphy
authors.forename: B G
authors.initials: BG
authors.lastname: Karlsson
authors.forename: B
authors.initials: B
authors.lastname: Reinhammar
authors.forename: S S
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D015502:Absorptiometry, Photon; D001400:Azurin / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D001400:Azurin; D018698:Glutamic Acid; D003300:Copper; D008715:Methionine