Here, we deliberately focus on poses with the shortest Ar-NO 2 -Asn258 binding distance. The comparison of all substrate binding affinities with the extent of O 2 uncoupling, f O 2 -uc , is shown in Figure . Figure shows the interaction network of docked 4-nitrotoluene into the homology model of variant L238V-M248I and the correlation of binding affinities with f O 2 -uc -values of this substrate. We found that binding affinities of 4-nitrotoluene and 3-nitrotoluene in wt 2NTDO increase (i.e., become more negative) as a consequence of mutations M248, L238-M248, and I204 (Figure and). Moreover, the increase of binding affinities correlates approximately linearly with f O 2 -uc and supports the conclusion that tighter substrate binding might be advantageous to hold the substrate in place for efficient hydroxylation as invoked for variations of O 2 uncoupling observed for α-ketoglutarate dependent non-heme ferrous iron oxygenase. However, such correlations were not observed for each enzyme/variant-substrate combination (Figure ). While selected enzyme variants stemming from the 4NT + and 3NT + experiments show tighter substrate binding compared to wt 2NTDO for some of the tested substrates, systematic trends were only observed for substrates 3-and 4-nitrotoluene (Figure ). According to common views of enzyme catalysis, one expects tuning of transition state stabilization to occur with a concomitant optimization of the enzymes's specific activity (i.e., k cat /K m ) 127 according to metabolic needs. A preferred optimization of substrate binding alone, that is lowering of K m , under the conditions of the adaptation experiments where substrate concentrations exceed K m might not necessarily improve catalysis. Modulation of enzymatic activity through tighter binding of the substrate as means to avoid O 2 uncoupling and concomitant production of ROS, by contrast, would appear as a quite meaningful adaptation strategy.