Characterization of O uncoupling in biodegradation reactions of nitroaromatic contaminants catalyzed by rieske oxygenases.

Rieske oxygenases are known as catalysts that enable the cleavage of aromatic and aliphatic C-H bonds in structurally diverse biomolecules and recalcitrant organic environmental pollutants through substrate oxygenations and oxidative heteroatom dealkylations. Yet, the unproductive O activation, which is concomitant with the release of reactive oxygen species (ROS), is typically not taken into account when characterizing Rieske oxygenase function. Even if considered an undesired side reaction, this O uncoupling allows for studying active site perturbations, enzyme mechanisms, and how enzymes evolve as environmental microorganisms adapt their substrates to alternative carbon and energy sources.

Advances in Oxygen Isotope Analysis of Phosphate by Electrospray Orbitrap Mass Spectrometry for Studying the Microbial Metabolism of Microorganisms.

Understanding the impact of human activities on the metabolic state of soil and aquatic environments is of paramount importance to implement measures for maintaining ecosystem services. Variations of natural abundance 18O/16O ratios in phosphate have been proposed as proxies for the holistic assessment of metabolic activity given the crucial importance of phosphoryl transfer reactions in fundamental biological processes. However, instrumental and procedural limitations inherent to oxygen isotope analysis in phosphate and organophosphorus compounds have so far limited the stable isotope-based evaluation of metabolic processes.

Hydrogen Peroxide Formation during Ozonation of Olefins and Phenol: Mechanistic Insights from Oxygen Isotope Signatures.

Mitigation of undesired byproducts from ozonation of dissolved organic matter (DOM) such as aldehydes and ketones is currently hampered by limited knowledge of their precursors and formation pathways. Here, the stable oxygen isotope composition of HO formed simultaneously with these byproducts was studied to determine if it can reveal this missing information. A newly developed procedure, which quantitatively transforms HO to O for subsequent O/O ratio analysis, was used to determine the δO of HO generated from ozonated model compounds (olefins and phenol, pH 3-8).

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

Oxygenations of aromatic soil and water contaminants with molecular O 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 O activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation.

Managing argon interference during measurements of O/O ratios in O 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.

Substrate-Specific Coupling of O 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 Fe center, elementary steps leading to O 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 O activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle.

The Apparently Unreactive Substrate Facilitates the Electron Transfer for Dioxygen Activation in Rieske Dioxygenases.

Rieske dioxygenases belong to the non-heme iron family of oxygenases and catalyze important cis-dihydroxylation as well as O-/N-dealkylation and oxidative cyclization reactions for a wide range of substrates. The lack of substrate coordination at the non-heme ferrous iron center, however, makes it particularly challenging to delineate the role of the substrate for productive activation. Here, we studied the role of the substrate in the key elementary reaction leading to activation from a theoretical perspective by systematically considering (i) the 6-coordinate to 5-coordinate conversion of the non-heme Fe upon abstraction of a water ligand, (ii) binding of , and (iii) transfer of an electron from the Rieske cluster.

Thermodynamic controls on rates of iron oxide reduction by extracellular electron shuttles.

Anaerobic microbial respiration in suboxic and anoxic environments often involves particulate ferric iron (oxyhydr-)oxides as terminal electron acceptors. To ensure efficient respiration, a widespread strategy among iron-reducing microorganisms is the use of extracellular electron shuttles (EES) that transfer two electrons from the microbial cell to the iron oxide surface. Yet, a fundamental understanding of how EES-oxide redox thermodynamics affect rates of iron oxide reduction remains elusive.

Triple-Element Compound-Specific Stable Isotope Analysis (3D-CSIA): Added Value of Cl Isotope Ratios to Assess Herbicide Degradation.

Multielement isotope fractionation studies to assess pollutant transformation are well-established for point-source pollution but are only emerging for diffuse pollution by micropollutants like pesticides. Specifically, chlorine isotope fractionation is hardly explored but promising, because many pesticides contain only few chlorine atoms so that "undiluted" position-specific Cl isotope effects can be expected in compound-average data. This study explored combined Cl, N, and C isotope fractionation to sensitively detect biotic and abiotic transformation of the widespread herbicides and groundwater contaminants acetochlor, metolachlor, and atrazine.

Exploring the Utility of Compound-Specific Isotope Analysis for Assessing Ferrous Iron-Mediated Reduction of RDX in the Subsurface.

Subsurface contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at ordnance production and testing sites is a problem because of the persistence, mobility, and toxicity of RDX and the formation of toxic products under anoxic conditions. While the utility of compound-specific isotope analysis for inferring natural attenuation pathways from stable isotope ratios has been demonstrated, the stable isotope fractionation for RDX reduction by iron-bearing minerals remains unknown. Here, we evaluated N and C isotope fractionation of RDX during reduction by Fe(II) associated with Fe minerals and natural sediments and applied N isotope ratios to the assessment of mineral-catalyzed RDX reduction in a contaminant plume and in sediment columns treated by chemical reduction.

Role of Carbonate in Thermodynamic Relationships Describing Pollutant Reduction Kinetics by Iron Oxide-Bound Fe.

The reduction of environmental pollutants by Fe bound to iron oxides is an important process that determines pollutant toxicities and mobilities. Recently, we showed that pollutant reduction rates depend on the thermodynamic driving force of the reaction in a linear free energy relationship that was a function of the solution pH value and the reduction potential, , of the interfacial Fe/Fe redox couple. In this work, we studied how carbonate affected the free energy relationship by examining the effect that carbonate has on nitrobenzene reduction rates by Fe bound to goethite (α-FeOOH).

Carbon Isotope Fractionation of Substituted Benzene Analogs during Oxidation with Ozone and Hydroxyl Radicals: How Should Experimental Data Be Interpreted?

Oxidative processes frequently contribute to organic pollutant degradation in natural and engineered systems, such as during the remediation of contaminated sites and in water treatment processes. Because a systematic characterization of abiotic reactions of organic pollutants with oxidants such as ozone or hydroxyl radicals by compound-specific stable isotope analysis (CSIA) is lacking, stable isotope-based approaches have rarely been applied for the elucidation of mechanisms of such transformations. Here, we investigated the carbon isotope fractionation associated with the oxidation of benzene and several methylated and methoxylated analogs, namely, toluene, three xylene isomers, mesitylene, and anisole, and determined their carbon isotope enrichments factors () for reactions with ozone ( = -3.

Assessment of 2,4-Dinitroanisole Transformation Using Compound-Specific Isotope Analysis after Chemical Reduction of Iron Oxides.

Ferrous iron-bearing minerals are important reductants in the contaminated subsurface, but their availability for the reduction of anthropogenic pollutants is often limited by competition with other electron acceptors including microorganisms and poor accessibility to Fe(II) in complex hydrogeologic settings. The supply of external electron donors through chemical reduction (ISCR) has been proposed as one remediation approach, but the quantification of pollutant transformation is complicated by the perturbations introduced to the subsurface by ISCR. Here, we evaluate the application of compound specific isotope analysis (CSIA) for monitoring the reduction of 2,4-dinitroanisole (DNAN), a component of insensitive munitions formulations, by mineral-bound Fe(II) generated through ISCR of subsurface material from two field sites.

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.

Dual-Element Isotope Analysis of Desphenylchloridazon to Investigate Its Environmental Fate in a Systematic Field Study: A Long-Term Lysimeter Experiment.

Desphenylchloridazon (DPC), the main metabolite of the herbicide chloridazon (CLZ), is more water soluble and persistent than CLZ and frequently detected in water bodies. When assessing DPC transformation in the environment, results can be nonconclusive if based on concentration analysis alone because estimates may be confounded by simultaneous DPC formation from CLZ. This study investigated the fate of DPC by combining concentration-based methods with compound-specific C and N stable isotope analysis (CSIA).

Decreases in Iron Oxide Reducibility during Microbial Reductive Dissolution and Transformation of Ferrihydrite.

Ferrous iron formed during microbial ferric iron reduction induces phase transformations of poorly crystalline into more crystalline and thermodynamically more stable iron (oxyhydr)oxides. Yet, characterizing the resulting decreases in the reactivity of the remaining oxide ferric iron toward reduction (i.e.

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-, β-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes.

Solid-phase extraction method for stable isotope analysis of pesticides from large volume environmental water samples.

Compound-specific isotope analysis (CSIA) is a valuable tool for assessing the fate of organic pollutants in the environment. However, the requirement of sufficient analyte mass for precise isotope ratio mass spectrometry combined with prevailing low environmental concentrations currently limits comprehensive applications to many micropollutants. Here, we evaluate the upscaling of solid-phase extraction (SPE) approaches for routine CSIA of herbicides.

Electrochemical Analysis of Changes in Iron Oxide Reducibility during Abiotic Ferrihydrite Transformation into Goethite and Magnetite.

Electron transfer to ferric iron in (oxyhydr-)oxides (hereafter iron oxides) is a critical step in many processes that are central to the biogeochemical cycling of elements and to pollutant dynamics. Understanding these processes requires analytical approaches that allow for characterizing the reactivity of iron oxides toward reduction under controlled thermodynamic boundary conditions. Here, we used mediated electrochemical reduction (MER) to follow changes in iron oxide reduction extents and rates during abiotic ferrous iron-induced transformation of six-line ferrihydrite.

Kinetic Isotope Effects of the Enzymatic Transformation of γ-Hexachlorocyclohexane by the Lindane Dehydrochlorinase Variants LinA1 and LinA2.

Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers.

C- and N-Isotope Analysis of Desphenylchloridazon by Liquid Chromatography-Isotope-Ratio Mass Spectrometry and Derivatization Gas Chromatography-Isotope-Ratio Mass Spectrometry.

The widespread application of herbicides impacts surface water and groundwater. Metabolites (e.g.

Mineral identity, natural organic matter, and repeated contaminant exposures do not affect the carbon and nitrogen isotope fractionation of 2,4-dinitroanisole during abiotic reduction.

The recent development of insensitive munitions, such as 2,4-dinitroanisole (DNAN), as components of military explosives has generated concern for potential subsurface contamination and created a need to fully characterize their transformation processes. Compound specific isotope analysis (CSIA) has proven to be a useful means of assessing transformation pathways according to characteristic stable isotope fractionation patterns. The C and N isotope fractionation of DNAN associated with abiotic and enzymatic hydrolysis was recently assessed.

Molecularly Imprinted Polymers for Compound-Specific Isotope Analysis of Polar Organic Micropollutants in Aquatic Environments.

Compound-specific isotope analysis (CSIA) of polar organic micropollutants in environmental waters requires a processing of large sample volumes to obtain the required analyte masses for analysis by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). However, the accumulation of organic matter of unknown isotopic composition in standard enrichment procedures currently compromises the accurate determination of isotope ratios. We explored the use of molecularly imprinted polymers (MIPs) for selective analyte enrichment for C/C and N/N ratio measurements by GC/IRMS using 1 H-benzotriazole, a typical corrosion inhibitor in dishwashing detergents, as example of a widely detected polar organic micropollutant.

Linking Thermodynamics to Pollutant Reduction Kinetics by Fe Bound to Iron Oxides.

Numerous studies have reported that pollutant reduction rates by ferrous iron (Fe) are substantially enhanced in the presence of an iron (oxyhydr)oxide mineral. Developing a thermodynamic framework to explain this phenomenon has been historically difficult due to challenges in quantifying reduction potential ( E) values for oxide-bound Fe species. Recently, our group demonstrated that E values for hematite- and goethite-bound Fe can be accurately calculated using Gibbs free energy of formation values.