Substrate-Specific Coupling of O2 Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases
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SUBMITTER: Pati S
PROVIDER: S-EPMC9171724 | biostudies-literature | 2022 May
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC9502038 | biostudies-literature
Project description:BackgroundRieske non-heme iron aromatic ring-hydroxylating oxygenases (RHOs) are multi-component enzyme systems that are remarkably diverse in bacteria isolated from diverse habitats. Since the first classification in 1990, there has been a need to devise a new classification scheme for these enzymes because many RHOs have been discovered, which do not belong to any group in the previous classification. Here, we present a scheme for classification of RHOs reflecting new sequence information and interactions between RHO enzyme components.ResultWe have analyzed a total of 130 RHO enzymes in which 25 well-characterized RHO enzymes were used as standards to test our hypothesis for the proposed classification system. From the sequence analysis of electron transport chain (ETC) components of the standard RHOs, we extracted classification keys that reflect not only the phylogenetic affiliation within each component but also relationship among components. Oxygenase components of standard RHOs were phylogenetically classified into 10 groups with the classification keys derived from ETC components. This phylogenetic classification scheme was converted to a new systematic classification consisting of 5 distinct types. The new classification system was statistically examined to justify its stability. Type I represents two-component RHO systems that consist of an oxygenase and an FNRC-type reductase. Type II contains other two-component RHO systems that consist of an oxygenase and an FNRN-type reductase. Type III represents a group of three-component RHO systems that consist of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNRN-type reductase. Type IV represents another three-component systems that consist of oxygenase, [2Fe-2S]-type ferredoxin and GR-type reductase. Type V represents another different three-component systems that consist of an oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase.ConclusionThe new classification system provides the following features. First, the new classification system analyzes RHO enzymes as a whole. RwithSecond, the new classification system is not static but responds dynamically to the growing pool of RHO enzymes. Third, our classification can be applied reliably to the classification of incomplete RHOs. Fourth, the classification has direct applicability to experimental work. Fifth, the system provides new insights into the evolution of RHO systems based on enzyme interaction.
| S-EPMC2358900 | biostudies-literature
Project description: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 O2 activation. Here, we studied the role of the substrate in the key elementary reaction leading to O2 activation from a theoretical perspective by systematically considering (i) the 6-coordinate to 5-coordinate conversion of the non-heme FeII upon abstraction of a water ligand, (ii) binding of O2 , and (iii) transfer of an electron from the Rieske cluster. We systematically evaluated the spin-state-dependent reaction energies and structural effects at the active site for all combinations of the three elementary processes in the presence and absence of substrate using naphthalene dioxygenase as a prototypical Rieske dioxygenase. We find that reaction energies for the generation of a coordination vacancy at the non-heme Fe II center through thermoneutral H2 O reorientation and exothermic O2 binding prior to Rieske cluster oxidation are largely insensitive to the presence of naphthalene and do not lead to formation of any of the known reactive Fe-oxygen species. By contrast, the role of the substrate becomes evident after Rieske cluster oxidation and exclusively for the 6-coordinate non-heme Fe II sites in that the additional electron is found at the substrate instead of at the iron and oxygen atoms. Our results imply an allosteric control of the substrate on Rieske dioxygenase reactivity to happen prior to changes at the non-heme Fe II in agreement with a strategy that avoids unproductive O2 activation.
| S-EPMC9306888 | biostudies-literature
Project description:Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of cis-dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology, e.g. chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of Pseudomonas fluorescens IP01 and its variant CDO M232A expressed in E. coli JM109 (DE3) cells, employing R-limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale.•Preservation of biocatalysts in recombinant whole-cells.•Ready-to-use enzymatic reaction.•Semi-preparative biotransformation with lyophilized whole-cells.
| S-EPMC8374376 | biostudies-literature
Project description:Dioxygenase (dOx) utilizes stereospecific oxidation on aromatic molecules; consequently, dOx has potential applications in bioremediation and stereospecific oxidation synthesis. The reactive components of dOx comprise a Rieske structure Cys2[2Fe-2S]His2 and a non-heme reactive oxygen center (ROC). Between the Rieske structure and the ROC, a universally conserved Asp residue appears to bridge the two structures forming a Rieske-Asp-ROC triad, where the Asp is known to be essential for electron transfer processes. The Rieske and ROC share hydrogen bonds with Asp through their His ligands; suggesting an ideal network for electron transfer via the carboxyl side chain of Asp. Associated with the dOx is an itinerant charge carrying protein Ferredoxin (Fdx). Depending on the specific cognate, Fdx may also possess either the Rieske structure or a related structure known as 4-Cys-[2Fe-2S] (4-Cys). In this study, we extensively explore, at different levels of theory, the behavior of the individual components (Rieske and ROC) and their interaction together via the Asp using a variety of density function methods, basis sets, and a method known as Generalized Ionic Fragment Approach (GIFA) that permits setting up spin configurations manually. We also report results on the 4-Cys structure for comparison. The individual optimized structures are compared with observed spectroscopic data from the Rieske, 4-Cys and ROC structures (where information is available). The separate pieces are then combined together into a large Rieske-Asp-ROC (donor/bridge/acceptor) complex to estimate the overall coupling between individual components, based on changes to the partial charges. The results suggest that the partial charges are significantly altered when Asp bridges the Rieske and the ROC; hence, long range coupling through hydrogen bonding effects via the intercalated Asp bridge can drastically affect the partial charge distributions compared to the individual isolated structures. The results are consistent with a proton coupled electron transfer mechanism.
| S-EPMC5033481 | biostudies-literature
Project description:An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
| S-EPMC7219546 | biostudies-literature
Project description:Various mechanisms have been proposed for the initial O(2) attack in intradiol dioxygenases based on different electronic descriptions of the enzyme-substrate (ES) complex. We have examined the geometric and electronic structure of the high-spin ferric ES complex of protocatechuate 3,4-dioxygenase (3,4-PCD) with UV/visible absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopies. The experimental data were coupled with DFT and INDO/S-CI calculations, and an experimentally calibrated bonding description was obtained. The broad absorption spectrum for the ES complex in the 6000-31000 cm(-1) region was resolved into at least five individual transitions, assigned as ligand-to-metal charge transfer (LMCT) from the protocatechuate (PCA) substrate and Tyr408. From our DFT calculations, all five LMCT transitions originate from the PCA and Tyr piop orbitals to the ferric dpi orbitals. The strong pi covalent donor interactions dominate the bonding in the ES complex. Using hypothetical Ga(3+)-catecholate/semiquinone complexes as references, 3,4-PCD-PCA was found to be best described as a highly covalent Fe(3+)-catecholate complex. The covalency is distributed unevenly among the four PCA valence orbitals, with the strongest interaction between the piop-sym and Fe dxz orbitals. This strong pi interaction, as reflected in the lowest energy PCA-to-Fe(3+) LMCT transition, is responsible for substrate activation for the O(2) reaction of intradiol dioxygenases. This involves a multi-electron-transfer (one beta and two alpha) mechanism, with Fe3+ acting as a buffer for the spin-forbidden two-electron redox process between PCA and O(2) in the formation of the peroxy-bridged ESO2 intermediate. The Fe ligand field overcomes the spin-forbidden nature of the triplet O(2) reaction, which potentially results in an intermediate spin state (S = 3/2) on the Fe(3+) center which is stabilized by a change in coordination along the reaction coordinate.
| S-EPMC2536531 | biostudies-literature
Project description:Rieske non-heme iron oxygenases, which have a Rieske-type [2Fe-2S] cluster and a non-heme catalytic iron center, are an important family of oxidoreductases involved mainly in regio- and stereoselective transformation of a wide array of aromatic hydrocarbons. Though present in all domains of life, the most widely studied Rieske non-heme iron oxygenases are found in mesophilic bacteria. The present study explores the potential for isolating novel Rieske non-heme iron oxygenases from thermophilic sources. Browsing the entire bacterial genome database led to the identification of 45 homologs from thermophilic bacteria distributed mainly among Chloroflexi, Deinococcus-Thermus and Firmicutes. Thermostability, measured according to the aliphatic index, showed higher values for certain homologs compared with their mesophilic relatives. Prediction of substrate preferences indicated that a wide array of aromatic hydrocarbons could be transformed by most of the identified oxygenase homologs. Further identification of putative genes encoding components of a functional oxygenase system opens up the possibility of reconstituting functional thermophilic Rieske non-heme iron oxygenase systems with novel properties.
| S-EPMC5209329 | biostudies-literature
Project description: Not available
| S-EPMC2720596 | biostudies-literature
Project description:Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
| S-EPMC2628974 | biostudies-literature