We carried out the identical type of experiments for the quantification of oxygenation efficiency and O 2 uncoupling for the 3-nitrotoluene adapted variants I204A, I204T, and I204V denoted henceforth as 3-NT + experiments (Figure ). These enzymes originate from laboratory evolution experiments with Acidovorax sp. strain JS42 adapted on 3-nitrotoluene over one month. In analogy to 4-NT + variants M248I and L238V-M248I, we found that each of these substitutions improved the efficiency of O , entry 3 vs. Table , entries 15, 18 and 21), again reflecting an adaptation to the new nitrotoluene substrate isomer.