J Inorg Biochem. 2026 Oct 12;286:113473. doi: 10.1016/j.jinorgbio.2026.113473. Online ahead of print.
ABSTRACT
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.
PMID:42887174 | DOI:10.1016/j.jinorgbio.2026.113473