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

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.

Probing the role of the backbone carbonyl interaction with the CuA center in azurin by replacing the peptide bond with an ester linkage

“…38,39 After successful synthesis and purification of the 19-mer depsipeptide, it was used in EPL reactions according to previously published procedures. 12,[14][15][16] During the EPL reaction, we noticed poor protein yields with depsipeptides lacking a Cys 116 -StBu protection, which was attributed to disulfide bond formation with the Cys 116 thiol. Furthermore, both the 19-mer depsipeptide and the ester Cu A azurin protein product demonstrated high sensitivity of the ester linkage to hydrolysis at any pH above 7.5.…”

Recent Applications of Hybrid Ab Initio Quantum Mechanics – Molecular Mechanics Simulations to Biological Macromolecules

“…In plastocyanin and azurin, this Met residue is important to control their reduction potentials (Berry et al, 2003;Garner et al, 2006). By analogy to the blue Cu proteins, Met207 of the bovine CcO may also be involved in regulating the electron transfer rate.…”

Design of Biomimetic Models Related to the Active Sites of Fe-Only Hydrogenase

“…In addition, energies of LUMOs in the NH and NHFeH species are decreased, which in turn makes reductions easier. Effects of interactions in the secondary coordination sphere on reduction potentials have been studied www.intechopen.com in several classes of metalloproteins such as iron-sulfur proteins (ferredoxins and highpotential iron proteins) (Low & Hill, 2000) and cupredoxins (Berry et al, 2003). A shift of the working potential greater than 1 V is observed.…”