Stability and Pseudocatecholase Activity of Artificial Bis-Histidyl Copper Peptides

In this paper, 4-methylcatechol (4-MC) and L/D-Dopa are selected as target substrates for de novo protein design, serving as a proof-of-concept system for evaluating the catalytic activity of metal coordination sites. We report two novel water-soluble bis-histidyl peptides that exhibit catalytic activity, where structural constraints around the active site would play a pivotal role in metalloenzyme development. The particular tandem His-His motif in the peptides and favorable E₀' enable them to catalyze catechol oxidation reactions efficiently. The coordination behavior of the peptides with Cu(II) and Cu(I) ions is thoroughly investigated using a combination of analytical techniques, including potentiometric titration and fluorescence, ultraviolet-visible (UV-vis), and circular dichroism (CD) spectroscopies. The insights gained into the catalytic binding site and associated pseudocatecholase activity of these peptides contribute to the development of copper-based bioinspired artificial metalloenzymes.