Oxygen Activation in Aromatic Ring Cleaving Salicylate Dioxygenase: Detection of Reaction Intermediates with a Nitro-substituted Substrate Analog

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 enzymatic k_cat by 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 O₂ concentration, 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 the in situ generated SDO Fe(III)-O₂⋅^- complex but closer to the enzyme-substrate precursor.