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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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 Colletotrichum orbiculare . 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 ( Cor AlcOx) and its natural peroxidase partner ( Cor 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 Cor 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 HLH HRP variant displayed stronger binding to Cor 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 Cor 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 ( Cor 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.

Source: Chem & Bio Engineering

DOI: 10.1021/cbe.5c00060