Miniature Photoenzyme Enables Organelle‐Specific Cellular Control via Deboronative Hydroxylation

Artificial photoenzymes hold transformative potential for in vitro biocatalysis, but their translation to live-cell environments demands minimal cellular perturbation and aerobic compatibility. Here, we present miniSOG, a 12 kDa miniature photoenzyme that enables bioorthogonal deboronative hydroxylation via superoxide radical anion (O₂ ^•-) generation under blue light irradiation. Leveraging the inherent photochemistry of flavins, miniSOG facilitates the photoactivation of 27 structurally diverse organoboronates-including aryl/alkyl boronates, fluorophores, anticancer agents, and epigenetic modulators-through a unified O₂ ^•--mediated mechanism. This system achieves spatiotemporally precise photocatalysis in live cells, where miniSOG's compact size and subcellular targeting enable organelle-specific localization and confined reactivity due to short-range O₂ ^•- diffusion (∼0.2 µm). We demonstrate its utility in light-gated cellular modulation: i) mitochondrial depolarization via localized release of 2,4-dinitrophenol (DNP) to disrupt energy metabolism, and ii) nuclear m⁶A methylation enhancement to epigenetically upregulate autophagy. By repurposing miniSOG's photochemistry for bioorthogonal deboronative hydroxylation, this work establishes a versatile, genetically encoded platform for manipulating fundamental cellular pathways with minimal off-target effects.