Copper-containing metalloenzymes constitute a major class of proteins that catalyze a myriad of reactions in nature. Inspired by the structural and functional characteristics of this unique class of metalloenzymes, we report the conception, design, characterization, and functional studies of a de novo artificial copper peptide (ArCuP) within a trimeric self-assembled polypeptide scaffold that activates and reduces peroxide. Using a first-principles approach, the ArCuP was designed to coordinate one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallography, UV–vis, and electron paramagnetic resonance data demonstrate that Cu binds via the Nε atoms of His forming a T2Cu environment. When reacted with hydrogen peroxide, the putative copper-hydroperoxo species is formed where a reductive priming step accelerates the rate of its formation and reduction. Mass spectrometry was used to identify specific residues undergoing oxidative modification, which showed His oxidation only in the reduced state. The redox behavior of the ArCuP was elucidated by protein film voltammetry. Detailed characterization of the electrocatalytic behavior of the ArCuP led us to determine the catalytic parameters (K M and k cat), which established the peroxidase activity of the ArCuP. Combined spectroscopic and electrochemical data showed a pH dependence on the reactivity, which was optimum at pH 7.5.

Transition-metal-catalyzed oxidative stress is a widespread concern in the pathogenesis of Alzheimer's disease. However, the exact role of amyloid beta oligomers towards oxidative stress is widely debated. Assessing the oxidative nature of the oligomers in vitro is complicated by the different experimental conditions under which they are prepared. We have investigated Cu -catalyzed reactive oxygen species (ROS) generation by using oligomers prepared in phosphate-buffered saline (Aβ ) and in cell culture medium (Aβ ), and compared their activities with respect to the monomers and fibrils prepared at neutral and acidic pH. Although both are deca- to dodecamers, the Aβ oligomers have a spherical morphology and are smaller than the Aβ . The Aβ behaved as pro-oxidants; in contrast, Aβ quench OH generation attributed to CCM itself. Although the pro-oxidant oligomers showed oxidation, they also partially protect themselves from radical damage and maintain their overall spherical arrangement. The monomers and fibrils manifested antioxidant properties: radical scavenging as opposed to redox silencing. A dual role of Aβ species depending on the stage of the disease is proposed. In the earlier stages, the monomers can act as antioxidants, whereas at the later stages, the oligomers take on a pro-oxidant role. Kaempferol, a natural flavonoid, bound Cu in 2:1 ratio and abolished ROS production in all Aβ species. It also distinctly modified the folding landscape of Aβ species into new or altered morphologies.

We report a series of de novo designed Artificial Cu Peptides (ArCuPs) that oxidize and peroxygenate C-H bonds of model abiotic substrates via electrochemically generated Cu-oxygen species using H2O2 as the terminal oxidant, akin to native Cu enzymes. Detailed assessment of kinetic parameters established the catalytic nature of the ArCuPs. Selective alteration of outer sphere steric at the d layers above and below the Cu site allows facilitated access of substrates, where a more pronounced effect on catalysis is observed when space is created at the d layer below the Cu site via Ile to Ala mutation producing a kcat of 6.2 s-1, TONmax of 14800 and catalytic proficiency (kcat/KM/kuncat) of 340 M-1 for the oxidation of benzyl alcohol. Independent spectroscopic studied revealed that the rate of formation of the Cu-oxygen species and the spectroscopic feature of the most active variant is distinct compared to the other ArCuPs. Systematic alteration of outer sphere hydrophobicity led to a correlated tuning of the T2 Cu site redox potentials by ~80 mV. The enhanced activity of the ArCuP variant is attributed to a combination of steric effect that allows easy access of substrates, the nature of Cu-oxygen species, and stability of this construct compared to others, where Ile to Ala mutation unexpectedly leads to a higher thermostability which is further augmented by Cu binding.

The cover feature picture shows that Ab oligomers perform a dual role in Cu‐catalyzed reactive oxygen species (ROS) generation. The pro‐oxidant nature of the oligomers severely damages cells, but these species can partially shield themselves from ROS‐induced self‐damage. Ab monomers and fibrils prepared under similar conditions behave as antioxidants by scavenging OH.. With the progress of disease, a dual antioxidant versus pro‐oxidant role has been proposed, as the monomers morph into oligomers. A natural flavonoid, Kampferol, which inhibits ROS toxicity and remodels the folding landscape of the Ab species has been studied. More information can be found in the full paper by S. Chakraborty et al. on page 2360 in Issue 22, 2018 (DOI: 10.1002/cbic.201800446).

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