Cassette mutagenesis of Met121 in azurin from Pseudomonas aeruginosa.
Cassette mutagenesis was used to exchange the suggested copper ligand Met121 in azurin to all other amino acids, and a stop codon. The mutant proteins were characterized by optical absorption spectroscopy and EPR. At low pH, all mutants exhibit the characteristics of a blue type 1 copper protein, indicating that methionine is not needed to create a blue copper site. At high pH, the Glu121 and the Lys121 mutants constitute a new form of protein-bound copper that is outside the range of type 1 copper.
Karlsson
B G
BG
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Nordling
M
M
Pascher
T
T
Tsai
L C
LC
Sjölin
L
L
Lundberg
L G
LG
D001400:Azurin / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D013053:Spectrophotometry
D015345:Oligonucleotide Probes; D001400:Azurin; D003300:Copper; D008715:Methionine