title: Electron-transfer properties of Pseudomonas aeruginosa [Lys44, Glu64]azurin.
abstract: In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Van Pouderoyen
authors.forename: G
authors.initials: G
authors.lastname: Cigna
authors.forename: G
authors.initials: G
authors.lastname: Rolli
authors.forename: F
authors.initials: F
authors.lastname: Cutruzzolà
authors.forename: F
authors.initials: F
authors.lastname: Malatesta
authors.forename: M C
authors.initials: MC
authors.lastname: Silvestrini
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009572:Nitrite Reductases / Q000378:metabolism; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D001400:Azurin; C017167:cytochrome C(551); D009572:Nitrite Reductases