Gene synthesis, expression, and mutagenesis of the blue copper proteins azurin and plastocyanin.
Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Site-saturation mutagenesis has been used to alter two copper-binding residues of azurin (Met-121 and His-46) and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.
Chang
T K
TK
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Iverson
S A
SA
Rodrigues
C G
CG
Kiser
C N
CN
Lew
A Y
AY
Germanas
J P
JP
Richards
J H
JH
D000595:Amino Acid Sequence; D001400:Azurin / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D017343:Genes, Plant; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D015345:Oligonucleotide Probes; D010944:Plants / Q000235:genetics; D010970:Plastocyanin / Q000235:genetics*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D001400:Azurin; D010970:Plastocyanin