Oxygen-sensitive mechanisms and protective strategies of metalloenzymes

The highly reactive metal cofactors enable metalloenzymes to catalyze a wide range of biologically important reactions, including carbon dioxide fixation, nitrogen fixation, and hydrogen evolution, but also render many of them vulnerable to oxygen (O₂)-induced damage. O₂ sensitivity has long been a major bottleneck limiting the mechanistic study and industrial applications of metalloenzymes. In this review, we systematically analyze the molecular basis of O₂ lability and the naturally evolved O₂ resistance across five representative O₂-sensitive metalloenzyme systems. Our analysis reveals that O₂ penetrates their buried active sites primarily through hydrophobic gas tunnels, resulting in a direct oxidative attack or reactive oxygen species (ROS)-mediated secondary oxidation that disrupts or decomposes metal cofactors. To protect these enzymes from oxidative damage, natural evolution has mainly developed three strategies, including steric hindrance of O₂ diffusion, stabilization of protective states in metal cofactors, and utilization of unique redox relays to mitigate ROS. In addition, we comprehensively summarize current artificial O₂-tolerance-engineering approaches, including rational engineering of O₂-access tunnels and active-site microenvironments, directed evolution and de novo protein binder design, as well as material-assisted protections using redox polymers and carboxysome shells. This review finally highlights future research priorities in resolving dynamic structural mechanisms, distinguishing diffusion kinetics of O₂ and gaseous substrates, and optimizing synergistic strategies of precision enzyme engineering and multifunctional material encapsulation for developing highly efficient O₂-tolerant, metalloenzyme-based biocatalysts.