One example is the documented formation of 2-methyl-5-nitrophenol in reactions of 2NTDO with 4-nitrotoluene in whole cell assays. Previously, we did not observe any formation of the monooxygenated product in purified enzyme assays. However, due to the high extent of O 2 uncoupling and the lower activity of purified enzyme compared to the whole cell assay, turnover documented in Bopp et al. was only 20 µM compared to roughly 500 µM in Pati et al. and 2-methyl-5-nitrophenol was likely below detection limit. In contrast, the two 4NT + variants, M248I and L238V-M248I, catalyzed turnover of 120 µM and 160 µM 4-nitrotoluene, respectively. We did not monitor for 2-methyl-5-nitrophenol during the HPLC measurement of these experiments. However, peaks that are consistent with 2-methyl-5-nitrophenol UV/Vis spectrum eluted at reasonable retention times (data not shown). Unfortunately, a quantitative evaluation was not possible but rough estimates based on previous calibrations suggest that 2methyl-5-nitrophenol concentrations would not significantly alter the discrepancy to substrate consumption. Due to the lack of other unknown peaks and information on additional potential reaction products, we assume that either 3-and 4-nitrotoluene form other products and/or the monooxygenated products not evident in NO - 2 formation, i.e. nitrobenzylalcohol and 2-methyl-5-nitrophenol, react further. At low concentrations, detection of these unknown products in the chromatogram is unlikely. The unknown nature of these reactions results in an uncertainty in the quantification of f O 2 -uc . Customarily, efficient oxygenations are quantified by the sum of NO - 2 and, if applicable, nitrobenzylalcohol (equation 2 in the main manuscript). Due to the uncertainty in determining the initial concentration, [S] 0 , quantification based on substrate consumption as in equation S4 is usually less accurate.