X-ray structure determination and characterization of the Pseudomonas aeruginosa azurin mutant Met121Glu.
The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.
Karlsson
B G
BG
Department of Biochemistry and Biophysics, Goteborg University, Sweden. Goran.Karlsson@bcbp.gu.se
Tsai
L C
LC
Nar
H
H
Sanders-Loehr
J
J
Bonander
N
N
Langer
V
V
Sjölin
L
L
D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D001400:Azurin; D018698:Glutamic Acid; D003300:Copper; D008715:Methionine