Figure (a) Catalytic cycle of wt 2NTDO and enzyme variants. Upon substrate binding to the 6-coordinate non-heme Fe II resting state 1, O 2 is bound concomitant with electron transfer from the reduced Rieske cluster (Fe II -Fe III ). O 2 uncoupling and release of unreacted substrate and ROS (H 2 O 2 ) (dashed arrows) occur from the Fe III -oxo-peroxo species 2. Substrate oxygenation can be catalyzed by species 2 and high-valent Fe-oxo(hydroxo) species 3. formation of Fe III -(hydro)peroxo species (2 in Figure ). This interpretation is corroborated by the detection of H 2 O 2 from O 2 uncoupling in assays of variants (Table ). Almost identical observations in terms of O-KIE were made with wt 2NTDO (1.016 ± 0.002) and another nitroarene dioxygenase with an even larger set of substrates implying that the mechanism of O 2 activation remained identical in 2NTDO variants. We do, however, also find three O-KIE values between 1.022 and 1.026 for oxygenation of nitrobenzene and nitrotoluene isomers by I204 variants which exceed the values of the other O-KIE in rest of our data. These values approach theoretical O equilibrium isotope effects ( 18 O-EIEs) that are considered representative of the rate-limiting formation of Fe IV =O species ( 18 O-EIE of 1.0287). This mechanism of O 2 activation is also consistent with reactions of other Rieske oxygenases. Whether this finding hints at changes in O 2 activation mechanisms in I204 variants is currently unclear.