Modification of the electron-transfer sites of Pseudomonas aeruginosa azurin by site-directed mutagenesis.
Site-directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron-transfer sites have been changed: His-35-Lys and Glu-91-Gln at one site and Phe-114-Ala at the other. The charge-transfer band and the EPR spectrum are the same as in the wild-type protein in the first two mutants, whereas in the Phe-114-Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4.10(-4)/cm. This protein also shows an increase of 20-40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c551 is unchanged compared to the wild-type protein in the Phe-114-Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu-91 is not important for the interaction with cytochrome c551 and that His-35 plays no critical role in the electron transfer to the copper site.
Pascher
T
T
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Bergström
J
J
Malmström
B G
BG
Vänngård
T
T
Lundberg
L G
LG
D001400:Azurin / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D007700:Kinetics; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D013053:Spectrophotometry
D001426:Bacterial Proteins; D001400:Azurin