Tuning the function of de novo designed artificial Cu proteins by modulating reorganization energies

Artificial metalloenzyme (ArM) design is an attractive approach for deciphering the functional determinants of native enzymes or imparting new functions. Metalloproteins with redox cofactors catalyze critical reactions enabled by optimized primary, secondary, and outer-sphere interactions that facilitate efficient electron transfer. Controlling outer-sphere interactions to tune reactivity remains a challenge. Inspired by the common coordination motifs of copper (Cu) proteins, we have designed artificial Cu proteins (ArCuPs), extensively characterized them, and demonstrated their H₂O₂, O₂, and C-H oxidation reactivity to abiotic substrates. By selectively modifying outer-sphere solvent reorganization energy, we have shown that we can control C-H peroxidation activity. This chapter describes methods to design ArCuPs and their detailed characterization, including electrochemical C-H oxidation, and the procedures for determining reorganization energies using electrochemistry as a readily available laboratory tool.