A self-assembled nanozyme featuring precise active centers and topography exhibits controlled catalytic interplay with mitochondrial protein while regulating electron flow during bioinspired oxygen reduction

Abstract

Enzymes orchestrate a profound regulation over several reactions, transforming small molecules and protein-based substrates and tightly controlling electron interplays through their sophisticated well-defined active sites and microenvironment. Achieving this level of precision in the active site design, crucial for specific reactions and tight control over electron transfer dynamics, remains a significant challenge in current-generation nanozymes. Inefficient enzyme mimetics with undefined active sites can lead to undesired effects and unsystematic electron transfer, resulting in electron leakage and reactive oxygen species (ROS) generation, which can adversely affect cellular energy pathways and lead to oxidative stress. Therefore, the evergrowing biotechnological prospects of nanozymes demand crucial attention on developing precision-engineered next-generation nanozymes having well-defined active sites that can electronically communicate with protein-based electron donor substrates. Given their pivotal role in cellular energy pathways, nanozymes are required to demonstrate great control over electron flow. In our ongoing efforts to broaden the field of nanozymes, we unveil an unprecedented electron interplay between a self-assembled nanozyme (Cu-Phen) and cytochrome c (cyt c), a crucial electron donor protein involved in the electron-transport chain. Cu-Phen, with its precision-engineered structure featuring a well-defined active site, exhibits a receptor-like hydrophobic surface that facilitates electronic communication with cyt c preferentially through hydrophobic interactions, efficiently mimicking the function of the cyt c oxidase (CcO) enzyme, under physiologically relevant conditions. Through kinetic isotopic effects, we also show for the first time that the electron transfer at the Cu-Phen self-assembled nanozyme and cyt c interface occurs through a proton-coupled electron transfer (PCET) mechanism in a controlled manner, with the phenylalanine ligand that may cooperatively participate in proton relays during the catalytic cycle. We also systematically delineate why Cu²⁺ ions alone exhibit uncontrolled electron transfer, setting Cu-Phen nanozymes apart with unique catalytic characteristics. Remarkably, the controlled electron transfer majorly results in the complete reduction of oxygen to water, with negligible production of superoxide and H₂O₂. The topography-modulated binding and electron transfer mechanism distinguishes Cu-Phen from general nanozymes, highlighting its unique potential for advanced applications. Our investigation also offers a comprehensive analysis of nanozyme engineering, intending to shape the future development of robust artificial enzyme systems for bio-energy applications.