Mechanism of Oxidative C─H Bond Activation by De Novo Designed Artificial Cu Metalloenzymes Using H₂O₂ and O₂

De novo-designed artificial Cu protein (ArCuP), 3SCC, featuring a trigonal Cu(His)₃ binding environment, activates H₂O₂, O₂, and benzylic C─H bonds of abiotic substrates. We outline the mechanism of ArCuP-catalyzed C─H peroxidation of one such abiotic substrate, the peroxidation of benzyl alcohol (BA) to benzaldehyde. The Cu^I(H₂O₂) complex undergoes homolytic cleavage, producing Cu^II-OH and ^•OH, akin to the lytic polysaccharide monooxygenases (LPMOs). The rate-limiting step is found in a PCET process, where the Cu^II-OH species accepts the benzylic C─H proton, accompanied by electron transfer from the O-centered substrate radical, to produce benzaldehyde. The C─H peroxidation is modulated by outer-sphere modifications, with the I12A-3SCC variant having the highest catalytic proficiency. Combined ^•OH monitoring and proteomics analysis reveal that the I12A variant produces the least amount of ^•OH, indicating no oxidation of the active site His residues, unlike other ArCuPs. The chemical and thermal stability of the I12A variant contributes to its superior reactivity. The presence of substrate significantly lowers protein-level oxidation, similar to LPMOs and other Cu and heme-based enzymes.