The higher O-KIE values make up 30% of the data for I204 variants but less than 6% of the approx. 50 O-KIE values that we have determined so far for nitroarene dioxygenases. C-Kinetic isotope effects ( 13 C-KIEs) of nitroaromatic substrate hydroxylation were used as probes for changes at later stages of the catalytic cycle in the variants. Specifically, we characterized the timing of substrate hydroxylation vs. O 2 uncoupling (green arrows in Figure ). The 13 C-KIE values for substrate hydroxylation by the different 2NTDO variants were, again, similar to what was observed previously for wt 2NTDO (0.998 -1.011, Figure , Tables 1 and S8). These data indicate a lack of reversibility of species 3 formation, from which hydroxylation occurs with an intrinsic C-KIE of up to 1.039. As a consequence, the unreacted substrate released upon O 2 uncoupling was not subject to any isotope fractionation from reaction 3 → 4 (Figure ). The two exceptions to this interpretation with large 13 C-KIE values (I204T and I204A of 1.013 ± 0.002 and 1.022 ± 0.008, respectively, Figure ) also come from reactions catalyzed by I204 variants. In analogy to the interpretation of O-KIE data above, these data could be seen as first and thus preliminary evidence for an alteration of the hydroxylation mechanism in enzyme variants. Larger 13 C-KIE, by contrast, are not uncommon.