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

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• 作者：Nicklas Bonander ; B. Gö ; ran Karlsson ; and Tore Vä ; nngå ; rd
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：February 20, 1996
• 年：1996
• 卷：35
• 期：7
• 页码：2429 - 2436
• 全文大小：551K
• 年卷期：v.35,no.7(February 20, 1996)
• ISSN：1520-4995

文摘

The binding of small exogenous ligands to mutants ofthe blue copper protein azurin fromPseudomonas aeruginosa, altered in the axial position,Met121X (X = Gly, Ala, Val, Leu, or Asp), hasbeen studied with optical and electron paramagnetic resonance (EPR)spectroscopy. The results showthat small molecules can enter the pocket left by the side chain ofMet121. For azide, the dissociationconstants are Leu > Val > Ala, reflecting the increasing spaceavailable. The Gly and Asp mutants bindazide less strongly than the Ala mutant, due to competition with water(Gly) and the polar side chain(Asp). Similar trends are found for thiocyanate. Cyanidebinds equally well to the Ala and Val mutants.A number of other small potential ligands were tried.Alcohols do not affect room-temperature opticalspectra, but at low temperatures, the EPR spectrum isstellacyanin-like, indicative of a weak axial interaction.Ligands binding with a carboxyl group or nitrogen (e.g. acetate orazide) convert the metal center to aform intermediate between regular types 1 and 2, presumably by pullingthe copper ion out of the trigonalplane formed by Cys(S) and two His(N). Cyanide interactsstrongly as shown by the hyperfine couplingto the ¹³C nucleus. With increasing strength of theaxial interaction, the two major bands in the visibleregion (600 and 400-500 nm) shift in parallel to higher energy, andat the same time, the strength of thelatter transition increases at the expense of the former. Thisdemonstrates that these transitions have acommon origin, namely S-to-Cu charge transfer transition.