Biodegradation of aromatic contaminants can typically be assessed with compound-specific isotope analysis (CSIA) based on changes in the stable C and H isotopic composition of the contaminant. However, isotope fractionation associated with aromatic oxygenations, one of the most important microbial strategies to initiate biodegradation, is only poorly understood. To facilitate the successful application of CSIA for quantifying biodegradation in the environment, we have investigated the dioxygenation of five nitroaromatic compounds to the corresponding catechols by nitrobenzene dioxygenase (NBDO) to elucidate the substrate-specificity of C and H isotope fractionation. Apparent 13C- and 2H-kinetic isotope effects (KIEs) varied significantly among the studied substrates despite a common reaction mechanism, which was corroborated by quantum mechanical calculations. We further investigated the substrate-specificity of this reaction by determining the Michaelis-Menten kinetics of product formation and O2 consumption as well as the 18O-KIE of O2 activation by NBDO. Experimental 18O-KIEs were independent of the nitroaromatic substrate and in agreement with the calculated 18O-KIE for an initial and irreversible formation of an enzyme-bound peroxide species. We further found that, instead of a stoichiometric consumption of O2 and substrate, O2 activation is partially uncoupled from substrate dioxygenation resulting in the release of H2O2 from the enzyme’s active site. The relative amount of H2O2 formation compared to total O2 consumption varied with respect to the nitroaromatic substrate bound to the active site of NBDO. This correlation was in qualitative agreement with the magnitude of apparent KIEs measured in the substrate, which indicates that the more efficient O2 is incorporated into the substrate, the smaller the measurable isotope fractionation becomes. Our findings consequently imply that for dioxygenase enzymes, which are further evolved than NBDO, such as naphthalene dioxygenase, quantifying biodegradation in the field by CSIA becomes increasingly challenging. This conclusion is indeed supported by generally small isotope effects measured for other aromatic contaminants including naphthalene and alkylated benzenes. Thus, the key to predict whether CSIA is applicable for certain biodegradation reactions is knowledge about the kinetic mechanism of the enzymes involved, particularly the sequence of substrate binding and O2 activation.

Substrate specificity of kinetic isotope effects associated with the dioxygenation of (nitro)aromatic contaminants is due to uncoupling of O2 activation

253rd National Meeting (2017)

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