Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(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. CuII(Cys112Asp) azurin can be reduced by excess [RuII(NH3)6]2+, resulting in a CuI protein with an electronic absorption spectrum very similar to that of wild-type CuI azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c 551 (μ = 0.1 M sodium phosphate (pH 7.0);(CuII/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 CuII(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 Nδ 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 CuII–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.

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Received: 30 December 1996 / Accepted: 8 May 1997

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Faham, S., Mizoguchi, T., Adman, E. et al. Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein. JBIC 2, 464–469 (1997). https://doi.org/10.1007/s007750050157

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  • Key words Azurin
  • Cytochrome c551
  • Electron transfer
  • Site-directed mutagenesis
  • Protein crystallography