A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa.
The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.
Frank
P
P
Licht
A
A
Tullius
T D
TD
Hodgson
K O
KO
Pecht
I
I
D001400:Azurin / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008433:Mathematics; D011550:Pseudomonas aeruginosa / Q000032:analysis* / Q000254:growth & development; D012643:Selenium / Q000032:analysis*; D012645:Selenomethionine / Q000032:analysis*; D013053:Spectrophotometry
D001426:Bacterial Proteins; D001400:Azurin; D003300:Copper; D012645:Selenomethionine; D012643:Selenium