Isotope Effects of Enzymatic Dioxygenation of Nitrobenzene and 2-Nitrotoluene by Nitrobenzene Dioxygenase

Abstract

Oxygenation of aromatic rings is a frequent initial step in the biodegradation of persistent contaminants, and the accompanying isotope fractionation is increasingly used to assess the extent of transformation in the environment. Here, we systematically investigated the dioxygenation of two nitroaromatic compounds (nitrobenzene and 2-nitrotoluene) by nitrobenzene dioxygenase (NBDO) to obtain insights into the factors governing its C, H, and N isotope fractionation. Experiments were carried out at different levels of biological complexity from whole bacterial cells to pure enzyme. C, H, and N isotope enrichment factors and kinetic isotope effects (KIEs) were derived from the compound-specific isotope analysis of nitroarenes, whereas C isotope fractionation was also quantified in the oxygenated reaction products. Dioxygenation of nitrobenzene to catechol and 2-nitrotoluene to 3-methylcatechol showed large C isotope enrichment factors, ϵC, of -4.1 ± 0.2‰ and -2.5 ± 0.2‰, respectively, and was observed consistently in the substrates and dioxygenation products. ϵH- and ϵN-values were smaller, that is -5.7 ± 1.3‰ and -1.0 ± 0.3‰, respectively. C isotope fractionation was also identical in experiments with whole bacterial cells and pure enzymes. The corresponding (13)C-KIEs for the dioxygenation of nitrobenzene and 2-nitrotoluene were 1.025 ± 0.001 and 1.018 ± 0.001 and suggest a moderate substrate specificity. Our study illustrates that dioxygenation of nitroaromatic contaminants exhibits a large C isotope fractionation, which is not masked by substrate transport and uptake processes and larger than dioxygenation of other aromatic hydrocarbons.

Isotope Effects as New Proxies for Organic Pollutant Transformation

“…[48] We are currently exploring the magnitude and origin of isotope fractionation of such reactions with an investigation of NAC dioxygenation in different biological model systems ranging from pure cultures of wild-type bacteria capable of biodegrading NACs to pure enzymes of Rieske non-heme Fe dioxygenases. [33] Our most recent results show that 13 C-KIEs for dioxygenations of nitroarenes can be as high as 1.024 (i.e. for reaction (iii) in Fig.…”

“…(b) Three possible pathways of enzymatic nitrobenzene dioxygenation to catechol through concerted and stepwise addition of dioxygen. [33] tration changes systematically. An application of CSIA in the environment is shown in the inset of Fig.…”

Characterization of Substrate, Cosubstrate, and Product Isotope Effects Associated With Enzymatic Oxygenations of Organic Compounds Based on Compound-Specific Isotope Analysis

“…In the following sections, we illustrate the mathematical procedures for evaluating isotope fractionation data and deriving apparent kinetic isotope effects for enzymatic oxygenations based on two examples, in which the pollutants nitrobenzene and 2-nitrotoluene function as substrates for nitrobenzene dioxygenase. The two data sets from Pati et al (2014) show how different evaluation procedures for substrate and product isotope fractionation data can be applied to obtain insights into the mechanisms of pollutant oxygenation and catalytic cycle of a Rieske non-heme ferrous iron oxygenase.…”

“…This reaction leads to a cis-dihydrodiol intermediate 2, which undergoes spontaneous elimination of nitrite (NO -2 ) to catechol (3). In laboratory experiments, we determined the dynamics of substrate and product concentrations by conventional methods, that is gas chromatography / mass spectrometry (GC/MS) for 1, high-performance liquid chromatography (HPLC) for 3, and a colorimetric assay for nitrite (Pati et al, 2014). Figure 2 (a) Time-course of nitrobenzene concentration and change in C isotope signature during the dioxygenation by NBDO.…”

“…The isotope fractionation observed during enzyme-catalyzed oxygenations of organic pollutants can vary substantially even for identical reactions because of multiple rate-limiting steps. We recently observed highly variable kinetic isotope effects of dioxygenations of different nitroarenes by nitrobenzene and nitrotoluene dioxygenases (Pati, Kohler, Bolotin, Parales, & Hofstetter, 2014;. In these works, we hypothesized that different steps of the pathway for activation of molecular O 2 contribute in a substrate-specific manner to the catalytic cycle of Rieske non-heme ferrous iron oxygenases.…”