Molecular Insights into the Activation of a Fungal Copper Radical Oxidase by Peroxidases

Copper radical oxidases (CROs) are monocopper enzymes that catalyze primary alcohol oxidation, making them valuable for industrial applications. Their activation requires an initial oxidation step, typically mediated by peroxidases, chemical oxidants, or electrochemical methods. The horseradish peroxidase (HRP) is empirically used for CRO activation. We recently identified the natural peroxidase partner of CROs in the fungal plant pathogen <i>Colletotrichum orbiculare</i>. However, the molecular interactions and electron transfer mechanisms between CROs and their peroxidase partners have not yet been elucidated yet. We investigated the molecular interaction between the model CRO (<i>Cor</i>AlcOx) and its natural peroxidase partner (<i>Cor</i>PerOx) or HRP, hypothesizing that specific surface residues affect interaction strength and activation efficiency. Monte Carlo simulations allowed the identification of key interaction sites, including three residues in <i>Cor</i>PerOx and a helix-loop-helix (HLH) motif in HRP. Mutagenesis studies confirmed that disrupting these sites impairs protein complex formation and CRO activation. Notably, while computational analysis indicated that the Δ _<i>HLH</i> HRP variant displayed stronger binding to <i>Cor</i>AlcOx, experimental data revealed a reduced activation capacity, highlighting the critical importance of the precise orientation of residues involved in electron transfer. Unexpectedly, we found that peroxidase activity alone is not crucial for CRO activation as the <i>Cor</i>PerOx or HRP variants retained peroxidase activity despite minimal activation capacity. Using Monte Carlo simulations, we propose an electron transfer pathway between CROs and their natural peroxidase partner. This study elucidates the structural interactions and key residue orientations on the peroxidase side (<i>Cor</i>PerOx), essential for CRO activation, with residues from both sides involved in protein complex formation and electron transfer. These findings deepen our understanding of CRO activation mechanisms and open avenues for designing alternative activators and targeted inhibitors.