An expanded dinuclear‑zinc centre enables H₃O₂⁻‑mediated enzymatic aromatic substitution

Abstract Nucleophilic aromatic substitution (SNAr) is central to synthetic chemistry, biocatalysis and biodegradation. However, SNAr enzymes have rarely been reported. They often show comparatively constrained substrate spectra or rely on cofactors. Here we establish the molecular mechanism of a 2,4-dinitroanisole (DNAN) hydrolase (DnhAB) from Nocardioides sp. JS1661. It converts DNAN, an alternative explosive to 2,4,6-trinitrotoluene (TNT), to 2,4-dinitrophenol (DNP). Isotope-labelling and biochemical analyses verify the SNAr activity of DnhAB with broad substrate scope. It targets activated nitroarenes and heteroaromatics, and enables efficient dehalogenation including fluoronitroarenes. Crystal structures disclose an atypical dinuclear zinc centre with an expanded Zn–Zn distance (~4.6 Å) that is inconsistent with a typical μ-hydroxide bridge, but accommodates a hydrogen-bonded H₃O₂⁻-like species. Mutagenesis and calculations delineate its catalytic mechanism with H₃O₂⁻-like species acting as the nucleophile. Our findings expand the repertoire of enzymatic SNAr, define a non-typical zinc-dependent paradigm, and provide evidence for proton-shared Zn–O₂H₃–Zn chemistry in biological systems.