Elucidating the Role of O2 Uncoupling in the Oxidative Biodegradation of Organic Contaminants by Rieske Non-heme Iron Dioxygenases.

Oxygenations of aromatic soil and water contaminants with molecular O2 catalyzed by Rieske dioxygenases are frequent initial steps of biodegradation in natural and engineered environments. Many of these non-heme ferrous iron enzymes are known to be involved in contaminant metabolism, but the understanding of enzyme-substrate interactions that lead to successful biodegradation is still elusive. Here, we studied the mechanisms of O2 activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation. Experiments in enzyme assays of 2-nitrotoluene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fluoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that typically 20-100% of the enzyme's activity involves unproductive paths of O2 activation with generation of reactive oxygen species through so-called O2 uncoupling. The 18O and 13C kinetic isotope effects of O2 activation and nitroaromatic substrate hydroxylation, respectively, suggest that O2 uncoupling occurs after generation of FeIII-(hydro)peroxo species in the catalytic cycle. While 2NTDO hydroxylates ortho-substituted nitroaromatic substrates more efficiently, NBDO favors meta-substituted, presumably due to distinct active site residues of the two enzymes. Our data implies, however, that the O2 uncoupling and hydroxylation activity cannot be assessed from simple structure-reactivity relationships. By quantifying O2 uncoupling by Rieske dioxygenases, our work provides a mechanistic link between contaminant biodegradation, the generation of reactive oxygen species, and possible adaptation strategies of microorganisms to the exposure of new contaminants.

Managing argon interference during measurements of 18O/16O ratios in O2 by continuous-flow isotope ratio mass spectrometry.

Monitoring changes in stable oxygen isotope ratios in molecular oxygen allows for studying many fundamental processes in bio(geo)chemistry and environmental sciences. While the measurement of [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] in gaseous samples can be carried out conveniently and from extracting moderately small aqueous samples for analyses by continuous-flow isotope ratio mass spectrometry (CF-IRMS), oxygen isotope signatures, [Formula: see text]O, could be overestimated by more than 6[Formula: see text] because of interferences from argon in air. Here, we systematically evaluated the extent of such Ar interferences on [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] for measurements by gas chromatography/IRMS and GasBench/IRMS and propose simple instrumental modifications for improved Ar and [Formula: see text] separation as well as post-measurement correction procedures for obtaining accurate [Formula: see text]O. We subsequently evaluated the consequences of Ar interferences for the quantification of O isotope fractionation in terms of isotope enrichment factors, [Formula: see text], and [Formula: see text]O kinetic isotope effects ([Formula: see text]O KIEs) in samples where [Formula: see text] is consumed and Ar:[Formula: see text] ratios increase steadily and substantially over the course of a reaction. We show that the extent of O isotope fractionation is overestimated only slightly and that this effect is typically smaller than uncertainties originating from the precision of [Formula: see text]O measurements and experimental variability. Ar interferences can become more relevant and bias [Formula: see text] values by more than [Formula: see text] in aqueous samples where fractional [Formula: see text] conversion exceeds 90%. Practically, however, such samples would typically contain less than 25 [Formula: see text]M of [Formula: see text] at ambient temperature, an amount that is close to the method detection limit of [Formula: see text]O/[Formula: see text]O ratio measurement by CF-IRMS.

Substrate-Specific Coupling of O2 Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases.

Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme FeII center, elementary steps leading to O2 activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O2 uncoupling for a series of substituted nitroaromatic compounds with 18O and 13C kinetic isotope effects of dissolved O2 and aromatic substrates, respectively, we show that O2 uncoupling occurs after the rate-limiting formation of FeIII-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O2 uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.

Enzyme Kinetics of Organic Contaminant Oxygenations.

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.

Assessing Aerobic Biotransformation of Hexachlorocyclohexane Isomers by Compound-Specific Isotope Analysis.

Contamination of soils and sediments with the highly persistent hexachlorocyclohexanes (HCHs) continues to be a threat for humans and the environment. Despite the existence of bacteria capable of biodegradation and cometabolic transformation of HCH isomers, such processes occur over time scales of decades and are thus challenging to assess. Here, we explored the use of compound-specific isotope analysis (CSIA) to track the aerobic biodegradation and biotransformation pathways of the most prominent isomers, namely, (-)-α-, (+)-α-, ß-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes. Dehydrochlorination of (+)-α-, γ-, and δ-HCH catalyzed by LinA2 was subject to substantial C and H isotope fraction with apparent 13C- and 2H-kinetic isotope effects (AKIEs) of up to 1.029 ± 0.001 and 6.7 ± 2.9, respectively, which are indicative of bimolecular eliminations. Hydrolytic dechlorination of δ-HCH by LinB exhibited even larger C but substantially smaller H isotope fractionation with 13C- and 2H-AKIEs of 1.073 ± 0.006 and 1.41 ± 0.04, respectively, which are typical for nucleophilic substitutions. The systematic evaluation of isomer-specific phenomena showed that, in addition to contaminant uptake limitations, diffusion-limited turnover ((-)-α-HCH), substrate dissolution (ß-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.

Biochar as possible long-term soil amendment for phytostabilisation of TE-contaminated soils.

Soils contaminated by trace elements (TEs) pose a high risk to their surrounding areas as TEs can spread by wind and water erosion or leaching. A possible option to reduce TE transfer from these sites is phytostabilisation. It is a long-term and cost-effective rehabilitation strategy which aims at immobilising TEs within the soil by vegetation cover and amendment application. One possible amendment is biochar. It is charred organic matter which has been shown to immobilise metals due to its high surface area and alkaline pH. Doubts have been expressed about the longevity of this immobilising effect as it could dissipate once the carbonates in the biochar have dissolved. Therefore, in a pot experiment, we determined plant metal uptake by ryegrass (Lolium perenne) from three TE-contaminated soils treated with two biochars, which differed only in their pH (acidic, 2.80; alkaline, 9.33) and carbonate (0.17 and 7.3 %) content. Root biomass was increased by the application of the alkaline biochar due to the decrease in TE toxicity. Zinc and Cu bioavailability and plant uptake were equally reduced by both biochars, showing that surface area plays an important role in metal immobilisation. Biochar could serve as a long-term amendment for TE immobilisation even after its alkalinity effect has dissipated.