Department Environmental Chemistry

Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O₂, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O₂ uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O₂ activation to substrate hydroxylation, depending on the extent of O-O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O₂ uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.

Enzymatic oxygenations are among the most important biodegradation and detoxification reactions of organic pollutants. In the environment, however, such natural attenuation processes are extremely difficult to monitor. Changes of stable isotope ratios of aromatic pollutants at natural isotopic abundances serve as proxies for isotope effects associated with oxygenation reactions. Such isotope fractionations offer new avenues for revealing the pathway and extent of pollutant transformation and provide new insights into the mechanisms of catalysis by Rieske non-heme ferrous iron oxygenases. Based on compound-specific C, H, N, and O isotope analysis, we present a comprehensive methodology with which isotope effects can be derived from the isotope fractionation measured in substrates, the cosubstrate O₂, and organic oxygenation products. We use dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase as illustrative examples to introduce different mathematical procedures for deriving apparent substrate and product isotope effects. We present two experimental approaches to control reactant and product turnover for isotope fractionation analysis in experimental systems containing purified enzymes, E. coli clones, and pure strains of environmental microorganisms. Finally, we present instrumental procedures and sample treatment instructions for analysis of C, H, and N isotope analysis in organic compounds and O isotope analysis in aqueous O₂ by gas and liquid chromatography coupled to isotope ratio mass spectrometry.