Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.
Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.
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Max-Planck-Institut für Biochemie, Abteilung für Strukturforschung, Martinsried, Federal Republic of Germany.
Huber
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R
Messerschmidt
A
A
Filippou
A C
AC
Barth
M
M
Jaquinod
M
M
van de Kamp
M
M
Canters
G W
GW
D001400:Azurin / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D014961:X-Ray Diffraction; D015032:Zinc / Q000378:metabolism*
D007477:Ions; D001400:Azurin; D003300:Copper; D015032:Zinc