Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the Cu^(II(Cys112Asp) protein

Abstract

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^(II)(Cys112Asp) azurin can be reduced by excess [Ru^(II)(NH_3)_6]^(2+), resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c_(551) (μ = 0.1 M sodium phosphate (pH 7.0);E°(Cu^(II/I)) = 180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^(II)(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^(II)–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.