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

Abstract

AbstractCupin 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 kcat 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.