Miniature Photoenzyme Enables Organelle‐Specific Cellular Control via Deboronative Hydroxylation

Abstract 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 (O 2 •− ) 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 O 2 •− ‐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 O 2 •− 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 m 6 A 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.