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Abstract Cupin dioxygenases such as salicylate 1,2‐dioxygense (SDO) perform aromatic C−C bond scission via a 3‐His motif tethered iron cofactor. Here, transient kinetics measurements are used to monitor the catalytic cycle of SDO by using a nitro‐substituted substrate analog, 3‐nitrogentisate. Compared to the natural substrate, the nitro group reduces the enzymatick_catby 500‐fold, thereby facilitating the detection and kinetic characterization of reaction intermediates. Sums and products of reciprocal relaxation times derived from kinetic measurements were found to be linearly dependent on O₂concentration, suggesting reversible formation of two distinct intermediates. Dioxygen binding to the metal cofactor takes place with a forward rate of 5.9×10³ M⁻¹ s⁻¹: two orders of magnitude slower than other comparable ring‐cleaving dioxygenses. Optical chromophore of the first intermediate is distinct from thein situgenerated SDO Fe(III)−O₂⋅⁻complex but closer to the enzyme‐substrate precursor.