Site saturation of the histidine-46 position in Pseudomonas aeruginosa azurin: Characterization of the His46Asp copper and cobalt proteins

Abstract

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 ~300, 454, and ~850 nm; its EPR spectrum is rhombic (g|| = 2.327(1), g_x ≈ 2.03, and g_y, ≈ 2.07; A|| ≈ 22(2) X 10^(-4), A_x, ≈ 46 X 10^(-4), and A_y, ≈ 22 X 10^(-4) cm^(-l)). The reduction potential of the mutant (260 mV us 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 (~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 (∈ ≈ 100 M^(-1) cm^(-l)) indicate a five-coordinate Co(I1) environment.

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