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 e xhibited the spectral characteristics of a blue (type 1) center. The s pectroscopic and electrochemical properties of this mutant protein sho w 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, 4 54, and approximately 850 nm; its EPR spectrum is rhombic (g(parallel- to) = 2.327(1), g(x) almost-equal-to 2.03, and g(y) almost-equal-to 2. 07; A(parallel-to) = 22(2) x 10(-4), A(x) almost-equal-to 46 x 10(-4), and A(y) almost-equal-to 22 x 10(-4) cm-1). The reduction potential o f 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 an d 375 nm) in the spectrum of the Co(II)(His46) protein, whereas the in tensities of the ligand-field bands in the 500-650-nm region (epsilon almost-equal-to 100 M-1 cm-1) indicate a five-coordinate Co(II) enviro nment.