Site saturation of the histidine-46 position in Pseudomonas aeruginosa azurin: characterization of the His46Asp copper and cobalt proteins.
Cassette mutagenesis has been used to replace the copper ligand His46 of Pseudomonas aeruginosa azurin with 19 other amino acids and a stop codon. Several mutant proteins were expressed in Escherichia coli and isolated; however, only the variant in which His was replaced by Asp exhibited the spectral characteristics of a blue (type 1) center. The spectroscopic and electrochemical properties of this mutant protein show that the copper site is perturbed relative to wild-type azurin. The absorption spectrum of Cu(II)(His46Asp) azurin exhibits a S(Cys)-->Cu(II) band at 612 nm, as well as weaker features at approximately 300, 454, and approximately 850 nm; its EPR spectrum is rhombic (g parallel = 2.327(1), gx approximately 2.03, and gy approximately 2.07; A parallel = 22(2) x 10(-4), Ax approximately 46 x 10(-4), and Ay approximately 22 x 10(-4) cm-1). The reduction potential of the mutant (260 mV vs NHE at pH 8.5; 297 mV at pH 5.0) is lower than that of wild-type azurin (288 mV at pH 8.5; 349 mV at pH 5.0). The S(Cys)-->Co(II) absorption bands (approximately 300 and 362 nm) in Co(II)(His46Asp) azurin are strongly blue-shifted relative to those (330 and 375 nm) in the spectrum of the Co(II) (His46) protein, whereas the intensities of the ligand-field bands in the 500-650-nm region (epsilon approximately 100 M-1 cm-1) indicate a five-coordinate Co(II) environment.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
J P
JP
Germanas
A J
AJ
Di Bilio
H B
HB
Gray
J H
JH
Richards
D001400:Azurin / Q000737:chemistry*; D001483:Base Sequence; D003035:Cobalt / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D004277:DNA, Single-Stranded; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006639:Histidine / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D004277:DNA, Single-Stranded; D001400:Azurin; D003035:Cobalt; D006639:Histidine; D003300:Copper