Classification, catalytic mechanisms, and functional characterization of non-heme diiron oxygenases: A review

Non-heme diiron oxygenases (NDOs) are a widely distributed family of metalloenzymes that use a dinuclear iron center to activate molecular O₂ and perform a broad range of oxidative reactions. These enzymes play central roles in carbon-nitrogen cycling and in the biosynthesis of many natural products. Unlike systems that rely on a single conserved active-site architecture, NDOs feature diverse metal coordination environments. These include the classic histidine/carboxylate-coordinated diiron center, systems in which the diiron cofactor assembles only upon substrate binding, and other noncanonical arrangements. In all cases, O₂ binds at the diiron core and is converted into reactive oxygen species that drive regio-, chemo-, and stereoselective oxidation of CH, NH, and CC bonds under physiologically mild conditions. Owing to favorable biocompatibility, broad substrate scope and mechanistic versatility, NDOs represent attractive catalysts for applications in synthetic biology and sustainable biomanufacturing. In the present review, current knowledge on family classification and representative catalytic functions were synthesized, active-site architectures were analyzed, catalytic cycles and substrate-binding strategies were described, and recent advances from structural biology, spectroscopy, computational modeling, and kinetic analysis were integrated. Through the evaluation of translational prospects in engineered biosynthetic pathways and green transformation platforms, the aim was to provide a rigorous conceptual framework to guide future mechanistic studies and protein-engineering efforts.