Probing copper ligands in denatured Pseudomonas aeruginosa azurin: unfolding His117Gly and His46Gly mutants

Azurin is a single-domain beta -barrel protein with a redox-active copper c ofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one hi stidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single- site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are inv estigated here. Equilibrium denaturation experiments of His46Gly azurin loa ded with copper demonstrate that copper remains bound to this mutant in hig h urea concentrations where the protein's secondary structure is lost. In c ontrast, for copper-loaded His117Gly azurin, copper does not stay coordinat ed upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corr esponding to residues 111-123 in azurin, suggesting similar metal coordinat ion. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximi ty of histidine-117 to cysteine-112 in the primary sequence.