Spectroscopic characterization of recombinant Cu,Zn superoxide dismutase from Photobacterium leiognathi expressed in Escherichia coli: evidence for a novel catalytic copper binding site.
Cu,Zn superoxide dismutase from Photobacterium leiognathi has been cloned and expressed in Escherichia coli. The circular dichroism spectrum in the UV region of the recombinant protein indicates an higher content of random coil structure with respect to the eukaryotic enzymes. Investigation of the active site by optical, CD, and EPR spectroscopy indicates a different coordination geometry around the catalytic copper site with respect to the eukaryotic enzymes. In particular a different orientation of the metal bridging histidine is suggested. The pH dependence of the copper EPR spectrum shows the presence of a single equilibrium which is at least one unit lower than the pK value observed for the bovine enzyme. Despite such structural differences the catalytic rate of this enzyme is identical to that observed for the eukaryotic Cu,Zn superoxide dismutase, suggesting that the overall electric field distribution is similar to that observed in the eukaryotic enzymes.
Foti
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D
Department of Biological and Organic Chemistry, University of Messina, Italy.
Lo Curto
B
B
Cuzzocrea
G
G
Stroppolo
M E
ME
Polizio
F
F
Venanzi
M
M
Desideri
A
A
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism*; D004274:DNA, Recombinant / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D010776:Photobacterium; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D003300:Copper; D013482:Superoxide Dismutase