Environment of Copper in Pseudomonas aeruginosa Azurin Probed by Binding of Exogenous Ligands to Met121X (X = Gly, Ala, Val, Leu, or Asp) Mutants

• Published in: Biochemistry (Easton) 1996-02, Vol.35 (7), p.2429-2436
• Main Authors: Bonander, Nicklas, Karlsson, B. Göran, Vänngård, Tore
• Format: Article
• Language: English
• Subjects: Amino Acids - genetics; Amino Acids - metabolism; Azurin - chemistry; Azurin - genetics; Azurin - metabolism; Copper - chemistry; Electron Spin Resonance Spectroscopy; Ligands; Molecular Probes; Mutagenesis, Site-Directed; Pseudomonas aeruginosa; Pseudomonas aeruginosa - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9522110

The binding of small exogenous ligands to mutants of the blue copper protein azurin from Pseudomonas aeruginosa, altered in the axial position, Met121X (X = Gly, Ala, Val, Leu, or Asp), has been studied with optical and electron paramagnetic resonance (EPR) spectroscopy. The results show that small molecules can enter the pocket left by the side chain of Met121. For azide, the dissociation constants are Leu > Val > Ala, reflecting the increasing space available. The Gly and Asp mutants bind azide less strongly than the Ala mutant, due to competition with water (Gly) and the polar side chain (Asp). Similar trends are found for thiocyanate. Cyanide binds equally well to the Ala and Val mutants. A number of other small potential ligands were tried. Alcohols do not affect room-temperature optical spectra, but at low temperatures, the EPR spectrum is stellacyanin-like, indicative of a weak axial interaction. Ligands binding with a carboxyl group or nitrogen (e.g. acetate or azide) convert the metal center to a form intermediate between regular types 1 and 2, presumably by pulling the copper ion out of the trigonal plane formed by Cys(S) and two His(N). Cyanide interacts strongly as shown by the hyperfine coupling to the 13C nucleus. With increasing strength of the axial interaction, the two major bands in the visible region (600 and 400−500 nm) shift in parallel to higher energy, and at the same time, the strength of the latter transition increases at the expense of the former. This demonstrates that these transitions have a common origin, namely S-to-Cu charge transfer transition.