Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids.
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.
Berry
Steven M
SM
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Ralle
Martina
M
Low
Donald W
DW
Blackburn
Ninian J
NJ
Lu
Yi
Y
D015502:Absorptiometry, Photon; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008024:Ligands; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009646:Norleucine / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D012645:Selenomethionine / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D008024:Ligands; D001400:Azurin; D009646:Norleucine; D012645:Selenomethionine; D008715:Methionine