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

Abstract

The role of a backbone carbonyl interaction with an engineered Cu A center in azurin was investigated by developing a method of synthesis and incorporation of a depsipeptide where one of the amide bonds in azurin is replaced by an ester bond using expressed protein ligation. Studies by electronic absorption and electron paramagnetic resonance spectroscopic techniques indicate that, while the substitution does not significantly alter the geometry of the site, it weakens the axial interaction to the Cu A center and strengthens the Cu-Cu bond, as evidenced by the blue shift of the near-IR absorption that has been assigned to the Cu-Cu ψ→ψ* transition. Interestingly, the changes in the electronic structure from the replacement did not result in a change in the reduction potential of the Cu A center, suggesting that the diamond core structure of Cu 2 S Cys2 is resistant to variations in axial interactions.Exploring the structure and function of metalloproteins requires intimate knowledge about the roles of residues around the metal-binding sites. Such knowledge forms a strong basis for successful design and engineering of novel metalloproteins with tunable functional properties. [1][2][3][4][5][6] The most common way to acquire such knowledge is site directed mutagenesis (SDM). Because SDM is limited to only ∼20 proteinogenic amino acids, determining the precise role of the residues is difficult, as SDM often simultaneously changes more than one factor, such as electronic and steric effects, at the same time. Therefore, it is nearly impossible to de-convolute the contributions of each individual factor in such instances. To overcome this limitation, non-proteinogenic analogs of natural amino acids have been incorporated into metalloproteins. [7][8][9][10][11] For example, we have previously employed Expressed Protein Ligation (EPL) to replace conserved metal-binding amino acids with their isostructural nonproteinogenic analogs. 12,13 Such replacements allowed us to de-convolute steric factors from other factors such as electronic effects and hydrophobicity, thereby firmly establishing the roles of these residues in metalloprotein function. 12 HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptWhile most residues use side chains to interact with metal centers, increasing numbers of examples have appeared in the literature showing backbone carbonyl oxygens as metalcoordinating ligands. One such example is the Cu A center found in cytochrome c oxidase (CcO) and nitrous oxide reductase (N 2 OR). [17][18][19][20] Cu A contains a dinuclear copper center bridged by two cysteine thiolates wherein each copper is also coordinated by a histidine (Figure 1). More interestingly, perpendicular to this "diamond core" structural plane, are three carbonyl oxygens of the backbone peptide linkage that are within 2.17 to 4.07 Å of the two copper centers, suggesting that these backbone carbonyl oxygens may play a key role in the formation and fine-tuning of the Cu A center. 21 However, their roles in CcO, N 2 OR...

Probing Backbone Hydrogen Bonds in Proteins by Amide‐to‐Ester Mutations

“…Recently, another metalloprotein has been studied by using a similar strategy. Here, the active site of the Cu A center of azurin was probed, and the authors found that the results were opposite to those obtained with the HiPIP upon modifying a backbone carbonyl interaction. Interestingly, the A‐to‐E variant of this metalloprotein, prepared by EPL, did not lower the reduction potential of the Cu A center in azurin.…”

Bioinspired Synthesis of Cu2+‐Modified Covalent Triazine Framework: A New Highly Efficient and Promising Peroxidase Mimic

“…Notably, copper ions are commonly found as the active sites in natural enzymes, such as laccase and amine oxidases. In this context, excellent works by Lu and co‐workers showed that copper is the element of choice for the design and synthesis of enzymes; however, the skeleton of these modified Cu‐containing enzymes still consists of proteins or amino acids. Thus, the development of new artificial protein‐like ligands containing anchored copper ions to mimic the activity of native enzymes is of great interest.…”

“…In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids. In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids.…”

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

“…26–31 Cupredoxins have been the target of many metalloprotein design efforts due to their uniquely constrained metal binding centers and spectral properties. 4,6,7,17,32 Approaches employing protein redesign using azurin as a scaffold, 20,28,33–35 de novo design within three or four helical bundles, 1 , 36–38 and artificial metalloproteins 39 have been explored. DeGrado et al designed a single polypeptide that formed an antiparallel 3-helix bundle called α 3 D, 40,41 and this scaffold has been modified to serve as a heavy metal binding protein, 42 capable of binding Cd(II) or iron in a tetrathiolate center.…”