Previous laboratory evolution experiments with nitroarene dioxygenases offer a promising avenue to examine the above questions. These works showed that Acidovorax sp. strain JS42 expressing the Rieske oxygenase 2-nitrotoluene dioxygenase (2NTDO) altered its sub-strate specificity from 2-nitrotoluene 3-and 4-nitrotoluene as growth-supporting substrates, if exposed to these alternative substrates over weeks and month, respectively. This adaptation process was accompanied by one and two amino acid substitutions in the enzyme variants compared to the wild-type (wt) 2NTDO. We recently quantified in vitro oxygenation efficiencies of Rieske oxygenases as the fraction of consumed O 2 used for substrate hydroxylation. Our studies of oxygenation efficiencies of wt 2NTDO revealed that the share of productively activated O 2 by 2NTDO with 3-and 4-nitrotoluene amounts to less than 16% and 6%, respectively. Conversely, 98% of O 2 was recovered in hydroxylation products when 2-nitrotoluene was used as the substrate. Here, we revisit the work of Parales et al. to examine two hypotheses regarding the adaptation of 2NTDO towards oxygenation of 3-and 4-nitrotoluene. (i) Enzyme evolution in such biodegradation processes correlate with increasingly efficient substrate hydroxylation and thus reduction of ROS generation. (ii) This process occurs as mutations at residues that are located in structural "hotspots" for catalytic performance. To that end, we carried out the following three tasks. (1) We applied previously developed methodology to quantify the substrate oxygenation efficiencies from the O 2 uncoupling for four nitroaromatic substrates with wt 2NTDO as well as five adapted variants exhibiting single and double amino acid substitutions. Based on this data set for multiple (variant) enzyme-substrate combinations, we related the changes of oxygenation efficiency to amino acid substitutions and studied their relevance for enabling growth of Acidovorax sp. strain JS42 with each of the four substrates. (2) We examined whether the kinetic mechanism associated with the catalytic cycle of Rieske oxygenases was conserved in enzyme variants by evaluating the kinetics of O 2 activation and substrate hydroxylation. (3) We studied how the known amino acid substitutions and the newly determined oxygenation efficiency of wild-type and variant 2NTDO concur with current hypothesis for relationships between structure and reactivity of Rieske oxygenases. Specifically, we analyzed whether the amino acid substitutions are localized at or near the active site or in substrate transport tunnels and whether these structural modifications modulate substrate binding and/or intra-molecular substrate transport in ways that could affect the extent of O 2 uncoupling. Finally, we assessed our results in an environmental context by examining the relevance of the variants identified by Parales et al. through bioinformatic analysis of naturally-occurring Rieske oxygenase sequences.