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 cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine 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 investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.
Pozdnyakova
I
I
Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Guidry
J
J
Wittung-Stafshede
P
P
D001400:Azurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D008024:Ligands; D001400:Azurin; D003300:Copper; D014508:Urea; D019791:Guanidine