Functional analysis of an α-ketoglutarate-dependent non-heme iron oxygenase in fungal meroterpenoid biosynthesis.

α-Ketoglutarate-dependent non-heme iron (α-KG NHI) oxygenases compose one of the largest superfamilies of tailoring enzymes that play key roles in structural and functional diversifications. During the biosynthesis of meroterpenoids, α-KG NHI oxygenases catalyze diverse types of chemical reactions, including hydroxylation, desaturation, epoxidation, endoperoxidation, ring-cleavage, and skeletal rearrangements. Due to their catalytic versatility, keen attention has been focused on functional analyses of α-KG NHI oxygenases. This chapter provides detailed methodologies for the functional analysis of the fungal α-KG NHI oxygenase SptF, which plays an important role in the structural diversification of andiconin-derived meroterpenoids. The procedures included describe how to prepare the meroterpenoid substrate using a heterologous fungal host, measure the in vitro enzymatic activity of SptF, and how to perform structural and mutagenesis studies on SptF. These protocols are also applicable to functional analyses of other α-KG NHI oxygenases.

Spectroscopic definition of ferrous active sites in non-heme iron enzymes.

Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O2 activation is controlled to occur only in the presence of all cosubstrates.

Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme.

Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp3)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.

Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

Cyclopropanation and aziridination catalyzed by non-heme iron and 2-oxoglutarate dependent enzymes.

Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs) involved in unusual peptide modifications.

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs), formerly known as domain of unknown function 692 (DUF692), are involved in the post-translational modification of peptides during the biosynthesis of peptide-based natural products. These enzymes catalyze highly unusual and diverse chemical modifications. Several class-defining features of this large family (>14 000 members) are beginning to emerge. Structurally, the enzymes are characterized by a TIM-barrel fold and a set of conserved residues for a di- or tri-iron binding site. They use molecular oxygen to modify peptide substrates, often in a four-electron oxidation taking place at a cysteine residue. This review summarizes the current understanding of MNIOs. Four modifications are discussed in detail: oxazolone-thioamide formation, ß-carbon excision, hydantoin-macrocycle formation, and 5-thiooxazole formation. Briefly discussed are two other reactions that do not take place on Cys residues.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis.

Talaromyolides (1-6) are a group of unusual 6/6/6/6/6/6 hexacyclic meroterpenoids with (3R)-6-hydroxymellein and 4,5-seco-drimane substructures, isolated from the marine fungus Talaromyces purpureogenus. We have identified the biosynthetic gene cluster tlxA-J by heterologous expression in Aspergillus, in vitro enzyme assays, and CRISPR-Cas9-based gene inactivation. Remarkably, the heterodimer of non-heme iron (NHI) enzymes, TlxJ-TlxI, catalyzes three steps of oxidation including a key reaction, hydroxylation at C-5 and C-9 of 12, the intermediate with 3-ketohydroxydrimane scaffold, to facilitate a retro-aldol reaction, leading to the construction of the 4,5-secodrimane skeleton and characteristic ketal scaffold of 1-6. The products of TlxJ-TlxI, 1 and 4, were further hydroxylated at C-4'ß by another NHI heterodimer, TlxA-TlxC, and acetylated by TlxB to yield the final products, 3 and 6. The X-ray structural analysis coupled with site-directed mutagenesis provided insights into the heterodimer TlxJ-TlxI formation and its catalysis. This is the first report to show that two NHI proteins form a heterodimer for catalysis and utilizes a novel methodology to create functional oxygenase structures in secondary metabolite biosynthesis.

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement.

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in streptozotocin. The intramolecular oxidative rearrangement process is known to proceed at the FeII-containing active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent FeIV═O species. In the catalytic reaction pathway, the O2 binds to the iron center and attacks on the substrate to form the peroxo bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the Cε-Nω bond of the substrate is homolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O-O bond cleavage, the following reaction steps proceed effortlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

Supplementation with ribonucleotide-based ingredient (Ribodiet®) lessens oxidative stress, brain inflammation, and amyloid pathology in a murine model of Alzheimer.

Alzheimer's disease (AD) is the most common type of dementia worldwide, characterized by the deposition of neurofibrillary tangles and amyloid-ß (Aß) peptides in the brain. Additionally, increasing evidence demonstrates that a neuroinflammatory state and oxidative stress, iron-dependent, play a crucial role in the onset and disease progression. Besides conventional therapies, the use of natural-based products represents a future medical option for AD treatment and/or prevention. We, therefore, evaluated the effects of a ribonucleotides-based ingredient (Ribodiet®) in a non-genetic mouse model of AD. To this aim, mice were injected intracerebroventricularly (i.c.v.) with Aß1-42 peptide (3 µg/3 µl) and after with Ribodiet® (0.1-10 mg/mouse) orally (p.o.) 3 times weekly for 21 days following the induction of experimental AD. The mnemonic and cognitive decline was then evaluated, and, successively, we have assessed ex vivo the modulation of different cyto-chemokines on mice brain homogenates. Finally, the level of GFAP, S100ß, and iron-related metabolic proteins were monitored as markers of reactive gliosis, neuro-inflammation, and oxidative stress. Results indicate that Ribodiet® lessens oxidative stress, brain inflammation, and amyloid pathology via modulation of iron-related metabolic proteins paving the way for its rationale use for the treatment of AD and other age-related diseases.

Maternal Iron Deficiency Modulates Placental Transcriptome and Proteome in Mid-Gestation of Mouse Pregnancy.

BACKGROUND: Maternal iron deficiency (ID) is associated with poor pregnancy and fetal outcomes. The effect is thought to be mediated by the placenta but there is no comprehensive assessment of placental responses to maternal ID. Additionally, whether the influence of maternal ID on the placenta differs by fetal sex is unknown. OBJECTIVES: To identify gene and protein signatures of ID mouse placentas at mid-gestation. A secondary objective was to profile the expression of iron genes in mouse placentas across gestation. METHODS: We used a real-time PCR-based array to determine the mRNA expression of all known iron genes in mouse placentas at embryonic day (E) 12.5, E14.5, E16.5, and E19.5 (n = 3 placentas/time point). To determine the effect of maternal ID, we performed RNA sequencing and proteomics in male and female placentas from ID and iron-adequate mice at E12.5 (n = 8 dams/diet). RESULTS: In female placentas, 6 genes, including transferrin receptor (Tfrc) and solute carrier family 11 member 2, were significantly changed by maternal ID. An additional 154 genes were altered in male ID placentas. A proteomic analysis quantified 7662 proteins in the placenta. Proteins translated from iron-responsive element (IRE)-containing mRNA were altered in abundance; ferritin and ferroportin 1 decreased, while TFRC increased in ID placentas. Less than 4% of the significantly altered genes in ID placentas occurred both at the transcriptional and translational levels. CONCLUSIONS: Our data demonstrate that the impact of maternal ID on placental gene expression in mice is limited in scope and magnitude at mid-gestation. We provide strong evidence for IRE-based transcriptional and translational coordination of iron gene expression in the mouse placenta. Finally, we discover sexually dimorphic effects of maternal ID on placental gene expression, with more genes and pathways altered in male compared with female mouse placentas.

How Do Electrostatic Perturbations of the Protein Affect the Bifurcation Pathways of Substrate Hydroxylation versus Desaturation in the Nonheme Iron-Dependent Viomycin Biosynthesis Enzyme?

The viomycin biosynthesis enzyme VioC is a nonheme iron and α-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of l-arginine at the C3-position for antibiotics biosynthesis. Interestingly, experimental studies showed that using the substrate analogue, namely, l-homo-arginine, a mixture of products was obtained originating from C3-hydroxylation, C4-hydroxylation, and C3-C4-desaturation. To understand how the addition of one CH2 group to a substrate can lead to such a dramatic change in selectivity and activity, we decided to perform a computational study using quantum mechanical (QM) cluster models. We set up a large active-site cluster model of 245 atoms that includes the oxidant with its first- and second-coordination sphere influences as well as the substrate binding pocket. The model was validated against experimental work from the literature on related enzymes and previous computational studies. Thereafter, possible pathways leading to products and byproducts were investigated for a model containing l-Arg and one for l-homo-Arg as substrate. The calculated free energies of activation predict product distributions that match the experimental observation and give a low-energy C3-hydroxylation pathway for l-Arg, while for l-homo-Arg, several barriers are found to be close in energy leading to a mixture of products. We then analyzed the origins of the differences in product distributions using thermochemical, valence bond, and electrostatic models. Our studies show that the C3-H and C4-H bond strengths of l-Arg and l-homo-Arg are similar; however, external perturbations from an induced electric field of the protein affect the relative C-H bond strengths of l-Arg dramatically and make the C3-H bond the weakest and guide the reaction to a selective C3-hydroxylation channel. Therefore, the charge distribution in the protein and the induced electric dipole field of the active site of VioC guides the l-Arg substrate activation to C3-hydroxylation and disfavors the C4-hydroxylation pathway, while this does not occur for l-homo-Arg. Tight substrate positioning and electrostatic perturbations from the second-coordination sphere residues in VioC also result in a slower overall reaction for l-Arg; however, they enable a high substrate selectivity. Our studies highlight the importance of the second-coordination sphere in proteins that position the substrate and oxidant, perturb charge distributions, and enable substrate selectivity.

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

The nonheme iron enzyme OrfP reacts with l-Arg selectively to form the 3R,4R-dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l-Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C3 -H hydrogen atom abstraction. This happens despite the fact that the C3 -H and C4 -H bond strengths of l-Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C3 -H activation to a low-energy 5 σ-pathway, while substrate positioning destabilizes the C4 -H abstraction and sends it over the higher-lying 5 π-pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.

Nonheme iron-thiolate complexes as structural models of sulfoxide synthase active sites.

Two mononuclear iron(ii)-thiolate complexes have been prepared that represent structural models of the nonheme iron enzymes EgtB and OvoA, which catalyze the O2-dependent formation of carbon-sulfur bonds in the biosynthesis of thiohistidine compounds. The series of Fe(ii) complexes reported here feature tripodal N4 chelates (LA and LB) that contain both pyridyl and imidazolyl donors (LA = (1H-imidazol-4-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine; LB = N,N-bis((1-methylimidazol-2-yl)methyl)-2-pyridylmethylamine). Further coordination with monodentate aromatic or aliphatic thiolate ligands yielded the five-coordinate, high-spin Fe(ii) complexes [FeII(LA)(SMes)]BPh4 (1) and [FeII(LB)(SCy)]BPh4 (2), where SMes = 2,4,6-trimethylthiophenolate and SCy = cyclohexanethiolate. X-ray crystal structures revealed that 1 and 2 possess trigonal bipyramidal geometries formed by the N4S ligand set. In each case, the thiolate ligand is positioned cis to an imidazole donor, replicating the arrangement of Cys- and His-based substrates in the active site of EgtB. The geometric and electronic structures of 1 and 2 were analyzed with UV-vis absorption and Mössbauer spectroscopies in tandem with density functional theory (DFT) calculations. Exposure of 1 and 2 to nitric oxide (NO) yielded six-coordinate FeNO adducts that were characterized with infrared and electron paramagnetic resonance (EPR) spectroscopies, confirming that these complexes are capable of binding diatomic molecules. Reaction of 1 and 2 with O2 causes oxidation of the thiolate ligands to disulfide products. The implications of these results for the development of functional models of EgtB and OvoA are discussed.

Nonheme Iron- and 2-Oxoglutarate-Dependent Dioxygenases in Fungal Meroterpenoid Biosynthesis.

This review summarizes the recent progress in research on the non-heme Fe(II)- and 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of pharmaceutically important fungal meroterpenoids. This enzyme class activates a selective C-H bond of the substrate and catalyzes a wide range of chemical reactions, from simple hydroxylation to dynamic carbon skeletal rearrangements, thereby significantly contributing to the structural diversification and complexification of the molecules. Structure-function studies of these enzymes provide an excellent platform for the development of useful biocatalysts for synthetic biology to create novel molecules for future drug discovery.

Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy.

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO2, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (FAFB), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s-1). These data unambiguously show that reduced ferredoxin, or possibly reduced FAFB, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (FAFB) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.

Chlorination versus hydroxylation selectivity mediated by the non-heme iron halogenase WelO5.

The selectivity of halogenation versus hydroxylation in α-KG de-pendent halogenases is vital to their function and has been widely studied, particularly using the halogenase SyrB2 as a model. WelO5, a new member of α-KG dependent halogenases, catalyzes the chlorination of 12-epi-fischerindole U in the welwitindolinone biosynthetic pathway. Herein, we give a detailed insight into the selectivity of WelO5 through combined quantum mechanical/molecular mechanical (QM/MM) calculations for the whole catalytic cycle. O2 activation leads to a Fe(iv)[double bond, length as m-dash]O moiety which adopts an equatorial conformation (in the plane consisting of His164, chloride and Fe atom), in contrast to axial conformation (perpendicular to the plane). Key to the conformational selectivity is a serine residue (Ser189) in the equatorial plane, that brings the precursor of the Fe(iv)[double bond, length as m-dash]O intermediate (a Fe(ii)-peracid complex) to the equatorial conformation through hydrogen bonding. Hydrogen abstraction of the substrate by the equatorial Fe(iv)[double bond, length as m-dash]O leads to a five-coordinated HO-Fe(iii)-Cl complex, where the hydroxyl ligand is still equatorial and thus relatively far from the substrate radical in the axial direction compared to the chloride ligand. This smoothly explains the extremely high selectivity of chlorination in WelO5 and provides a microscopic explanation for the experimental finding that S189A WelO5 ceases to display any chlorination selectivity versus hydroxylation. Notably, although Ser189 is vital for the selectivity of the enzyme, it is not part of the substrate binding pocket. Therefore, WelO5 serves as an excellent example how chemoselectivity can be achieved in directed evolution without the tedious redesign of the substrate binding pocket.

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

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.

Computational studies of DNA base repair mechanisms by nonheme iron dioxygenases: selective epoxidation and hydroxylation pathways.

DNA base repair mechanisms of alkylated DNA bases is an important reaction in chemical biology and particularly in the human body. It is typically catalyzed by an α-ketoglutarate-dependent nonheme iron dioxygenase named the AlkB repair enzyme. In this work we report a detailed computational study into the structure and reactivity of AlkB repair enzymes with alkylated DNA bases. In particular, we investigate the aliphatic hydroxylation and C[double bond, length as m-dash]C epoxidation mechanisms of alkylated DNA bases by a high-valent iron(iv)-oxo intermediate. Our computational studies use quantum mechanics/molecular mechanics methods on full enzymatic structures as well as cluster models on active site systems. The work shows that the iron(iv)-oxo species is rapidly formed after dioxygen binding to an iron(ii) center and passes a bicyclic ring structure as intermediate. Subsequent cluster models explore the mechanism of substrate hydroxylation and epoxidation of alkylated DNA bases. The work shows low energy barriers for substrate activation and consequently energetically feasible pathways are predicted. Overall, the work shows that a high-valent iron(iv)-oxo species can efficiently dealkylate alkylated DNA bases and return them into their original form.