Probing the Role of Axial Methionine in the Blue Copper Center of Azurin with Unnatural Amino Acids

Citations Over TimeTop 13% of 2003 papers

Abstract

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.

Related Papers

• → Gene synthesis, expression, and mutagenesis of the blue copper proteins azurin and plastocyanin.(1991)60 cited
• → Structural, dynamical and functional aspects of the inner motions in the blue copper protein azurin(2006)22 cited
• → Incorporation of the red copper nitrosocyanin binding loop into blue copper azurin(2010)20 cited
• → Structural heterogeneity of blue copper proteins: an EPR study of amicyanin and of wild-type and Cys3Ala/Cys26Ala mutant azurin(2001)8 cited
• → Structure and Electron Transfer Reactions of Blue Copper Proteins(1977)4 cited