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.

Elucidating the Role of O Uncoupling for the Adaptation of Bacterial Biodegradation Reactions Catalyzed by Rieske Oxygenases.

Oxygenation of aromatic and aliphatic hydrocarbons by Rieske oxygenases is the initial step of various biodegradation pathways for environmental organic contaminants. Microorganisms carrying Rieske oxygenases are able to quickly adapt their substrate spectra to alternative carbon and energy sources that are structurally related to the original target substrate, yet the molecular events responsible for this rapid adaptation are not well understood. Here, we evaluated the hypothesis that reactive oxygen species (ROS) generated by unproductive activation of O, the so-called O uncoupling, in the presence of the alternative substrate exert a selective pressure on the bacterium for increasing the oxygenation efficiency of Rieske oxygenases.

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.

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.

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.