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.