We examined the catalytic cycle of 2NTDO variants shown in Figure and probed for changes in rate-limiting steps of O 2 activation and substrate hydroxylation upon amino acid substitution by evaluating nitroaromatic substrate and O 2 kinetic isotope effects (KIEs). Competitive 18 O and C kinetic isotope effects of dissolved O 2 and the nitroaromatic substrate, respectively, were quantified following the methodology of our previous works and the data are compiled in Tables and. Figure