Mononuclear Iron Center Research Articles

New Frontiers in Nonheme Enzymatic Oxyferryl Species.

Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S=2 oxyferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S=1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S=2 Fe(IV)-oxo intermediates, the discovery of the first S=1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S=2 and the S=1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S=1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.

Construction and Function of Thiolate-Bridged Diiron NxHy Nitrogenase Model Complexes.

ConspectusBiological nitrogen fixation mediated by nitrogenases has garnered significant research interest due to its critical importance to the development of efficient catalysts for mild ammonia synthesis. Although the active center of the most studied FeMo-nitrogenases has been determined to be a complicated [Fe7S9MoC] hetero-multinuclear metal-sulfur cluster known as the FeMo-cofactor, the exact binding site and reduction pathway of N2 remain a subject of debate. Over the past decades, the majority of studies have focused on mononuclear molybdenum or iron centers as potential reaction sites. In stark contrast, cooperative activation of N2 through bi- or multimetallic centers has been largely overlooked and underexplored, despite the renewed interest sparked by recent biochemical and computational studies. Consequently, constructing bioinspired bi- or multinuclear metallic model complexes presents an intriguing yet challenging prospect. In this Account, we detail our long-standing research on the design and synthesis of novel thiolate-bridged diiron complexes as nitrogenase models and their application to chemical simulations of potential biological N2 reduction pathways.Inspired by the structural and electronic features of the potential diiron active center in the belt region of the FeMo-cofactor, we have designed and synthesized a series of new thiolate-bridged diiron nitrogenase model complexes, wherein iron centers with +2 or +3 oxidation states are coordinated by Cp* as carbon-based donors and thiolate ligands as sulfur donors. Through the synergistic interaction between the two iron centers, unstable diazene (NH═NH) species can be trapped to generate the first example of a [Fe2S2]-type complex bearing a cis-μ-η1:η1-NH═NH subunit. Significantly, this species can not only catalyze the reductive N-N bond cleavage of hydrazine to ammonia but also trigger a stepwise reduction sequence NH═NH → [NH2-NH]- → [NH]2-(+NH3) → [NH2]- → NH3. Furthermore, an unprecedented thiolate-bridged diiron μ-nitride featuring a bent Fe-N-Fe moiety was successfully isolated and structurally characterized. Importantly, this diiron μ-nitride can undergo successive proton-coupled electron transfer processes to efficiently release ammonia in the presence of separate protons and electrons and can even be directly hydrogenated using H2 as a combination of protons and electrons for high-yield ammonia formation. Based on combined experimental and computational studies, we proposed two distinct reductive transformation sequences on the diiron centers, which involve a series of crucial NxHy intermediates. Moreover, we also achieved catalytic N2 reduction to silylamines with [Fe2S2]-type complexes by ligand modulation.Our bioinspired diiron cooperative scaffold may provide a suitable model for probing the potential N2 stepwise reduction pathways from the molecular level. Different from the traditional alternating and distal pathways dominated by mononuclear iron or molybdenum complexes, our proposed alternating transformation route based on the diiron centers may not involve the N2H4 intermediate, and the convergence point of the alternating and terminal pathways is imide, not amide. Our research strategy could inform the design and development of new types of bioinspired catalysts for mild and efficient nitrogen reduction in the future.

Functional and spectroscopic approaches to determining thermal limitations of Rieske oxygenases.

The biotechnological potential of Rieske Oxygenases (ROs) and their cognate reductases remains unmet, in part because these systems can be functionally short-lived. Here, we describe a set of experiments aimed at identifying both the functional and structural stability limitations of ROs, using terephthalate (TPA) dioxygenase (from Comamonas strain E6) as a model system. Successful expression and purification of a cofactor-complete, histidine-tagged TPA dioxygenase and reductase protein system requires induction with the Escherichia coli host at stationary phase as well as a chaperone inducing cold-shock and supplementation with additional iron, sulfur, and flavin. The relative stability of the Rieske cluster and mononuclear iron center can then be assessed using spectroscopic and functional measurements following dialysis in an iron chelating buffer. These experiments involve measurements of the overall lifetime of the system via total turnover number using both UV-Visible absorbance and HPLC analyses, as well specific activity as a function of temperature. Important methods for assessing the stability of these multi-cofactor, multi-protein dependent systems at multiple levels of structure (secondary to quaternary) include differential scanning calorimetry, circular dichroism, and metallospectroscopy. Results can be rationalized in terms of three-dimensional structures and bioinformatics. The experiments described here provide a roadmap to a detailed characterization of the limitations of ROs. With a few notable exceptions, these issues are not widely addressed in current literature.

Custom tuning of Rieske oxygenase reactivity

Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding how this catalytic scaffold can be predictively tuned to catalyze divergent reactions. Therefore, in this work, using a combination of structural analyses, as well as substrate and rational protein-based engineering campaigns, we elucidate the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM. We identify structural features that permit a substrate to be functionalized by TsaM and pinpoint active-site residues that can be targeted to manipulate reactivity. Exploiting these findings allowed for custom tuning of TsaM reactivity: substrates are identified that support divergent TsaM-catalyzed reactions and variants are created that exclusively catalyze dioxygenation or sequential monooxygenation chemistry. Importantly, we further leverage these trends to tune the reactivity of additional monooxygenase and dioxygenase enzymes, and thereby provide strategies to custom tune Rieske oxygenase reaction outcomes.

Repurposing Iron‐ and 2‐Oxoglutarate‐Dependent Oxygenases to Catalyze Olefin Hydration

AbstractMononuclear nonheme iron(II) and 2‐oxoglutarate (Fe/2OG)‐dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C−H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration—a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron‐oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG‐independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.

Repurposing Iron- and 2-Oxoglutarate-Dependent Oxygenases to Catalyze Olefin Hydration.

Mononuclear nonheme iron(II) and 2-oxoglutarate (Fe/2OG)-dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C-H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration-a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron-oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG-independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.

Mechanism of Nitrogen Reduction to Ammonia in a Diiron Model of Nitrogenase.

Nitrogenase is a fascinating enzyme in biology that reduces dinitrogen from air to ammonia through stepwise reduction and protonation. Despite it being studied in detail by experimental and computational groups, there are still many unknown factors in the catalytic cycle of nitrogenase, especially related to the addition of protons and electrons and their order. A recent biomimetic study characterized a potential dinitrogen-bridged diiron cluster as a synthetic model of nitrogenase. Using strong acid and reductants, the dinitrogen was converted into ammonia molecules, but details of the mechanism remains unknown. In particular, it was unclear from the experimental studies whether the proton and electron transfer steps are sequential or alternating. Moreover, the work failed to establish what the function of the diiron core is and whether it split into mononuclear iron fragments during the reaction. To understand the structure and reactivity of the biomimetic dinitrogen-bridged diiron complex [(P2P'PhFeH)2(μ-N2)] with triphenylphosphine ligands, we performed a density functional theory study. Our computational methods were validated against experimental crystal structure coordinates, Mössbauer parameters, and vibrational frequencies and show excellent agreement. Subsequently, we investigated the alternating and consecutive addition of electrons and protons to the system. The calculations identify a number of possible reaction channels, namely, same-site protonation, alternating protonation, and complex dissociation into mononuclear iron centers. The calculations show that the overall mechanism is not a pure sequential set of electron and proton transfers but a mixture of alternating and consecutive steps. In particular, the first reaction steps will start with double proton transfer followed by an electron transfer, while thereafter, there is another proton transfer and a second electron transfer to give a complex whereby ammonia can split off with a low energetic barrier. The second channel starts with alternating protonation of the two nitrogen atoms, whereafter the initial double proton transfer, electrons and protons are added sequentially to form a hydrazine-bound complex. The latter split off ammonia spontaneously after further protonation. The various reaction channels are analyzed with valence bond and orbital diagrams. We anticipate the nitrogenase enzyme to operate with mixed alternating and consecutive protonation and electron transfer steps.

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.

Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure-function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes. Therefore, in this work, by leveraging a combination of available structural information and state-of-the-art protein modeling tools, we show that three "hotspot" regions can be targeted to alter the site selectivity, substrate preference, and substrate scope of the Rieske oxygenase p-toluenesulfonate methyl monooxygenase (TsaM). Through mutation of six to 10 residues distributed between three protein regions, TsaM was engineered to behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase (DdmC). This engineering feat means that TsaM was rationally engineered to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its favored native para position, and that TsaM was redesigned to perform chemistry on dicamba, a substrate that is not natively accepted by the enzyme. This work thus contributes to unlocking our understanding of structure-function relationships in the Rieske oxygenase enzyme class and expands foundational principles for future engineering of these metalloenzymes.

Structure‐Based Strategies for Rieske Oxygenase Catalysis

Rieske Oxygenases are enzymes that showcase the remarkable ability to functionalize an inert C–H bond. These enzymes use a Rieske [2Fe‐2S] cluster and a mononuclear iron center to bind and activate molecular oxygen. Using these metallocenters, Rieske Oxygenases have been shown to catalyze a variety of reactions, including one, two, and sequential hydroxylation reactions. This chemistry is important to the biosynthetic pathways of multiple organisms and is thus attractive for industrially building and tailoring natural product scaffolds. Similarly, the chemistry of the Rieske Oxygenases is abundant in degradative pathways and therefore offers compelling strategies for degrading environmental pollutants. However, a traditional lack of structure–function information for this expansive class of enzymes has restricted our ability to capitalize on these important and diverse catalytic strategies. Therefore, our laboratory is working to understand the architectural features of these enzymes that allow them to control and facilitate catalysis. Here, we present our current understanding of how these enzymes selectively catalyze reactions and use structure to guide chemical outcome.

Side-on coordination of diphosphorus to a mononuclear iron center.

The diagonal relationship in the periodic table between phosphorus and carbon has set an expectation that the triple-bonded diatomic diphosphorus molecule (P2) should more closely mimic the attributes of acetylene (HC≡CH) rather than its group 15 congener dinitrogen (N2). Although acetylene has well-documented coordination chemistry with mononuclear transition metals, coordination complexes that feature P2 bound to a single metal center have remained elusive. We report the isolation and x-ray crystallographic characterization of a mononuclear iron complex featuring P2 coordination in a side-on, η2-binding mode. An analogous η2-bound bis-timethylsilylacetylene iron complex is reported for comparison. Nuclear magnetic resonance, infrared, and Mössbauer spectroscopic analysis-in conjunction with density functional theory calculations-demonstrate that η2-P2 and η2-acetylene ligands exert a similar electronic demand on mononuclear iron centers but exhibit different reactivity profiles.

Retraction Note: Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme.

The X-ray crystal structures of FtmOx1, the first known α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction, are presented, along with further biochemical analyses which reveal the catalytic versatility of mononuclear non-haem iron enzymes, and help to unravel the mechanisms of endoperoxide biosyntheses. Verruculogen, a non-ribosomal peptide produced by various Aspergillus and Penicillium strains, contains an unusual and highly reactive endoperoxide (R-O-O-R) functional group that is generated from fumitremorgin B by the mononuclear non-haem iron enzyme FtmOx1. Pinghua Liu and colleagues report the X-ray crystal structures of FtmOx1 alone and in the presence of fumitremorgen B or α-ketoglutarate. Together with further biochemical analyses, these structures reveal the catalytic versatility of mononuclear non-haem iron enzymes, and help to unravel the mechanisms of endoperoxide biosyntheses. Verruculogen, a non-ribosomal peptide produced by various Aspergillus and Penicillium strains, contains an unusual and highly reactive endoperoxide (R-O-O-R) functional group that is generated from fumitremorgin B by the mononuclear non-haem iron enzyme FtmOx1. Pinghua Liu and colleagues report the X-ray crystal structures of FtmOx1 alone and in the presence of fumitremorgen B or α-ketoglutarate. Together with further biochemical analyses, these structures reveal the catalytic versatility of mononuclear non-haem iron enzymes, and help to unravel the mechanisms of endoperoxide biosyntheses. Many peroxy-containing secondary metabolites1,2 have been isolated and shown to provide beneficial effects to human health3,4,5. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases6,7,8, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme9. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction10,11,12. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Computational Study on O–O Bond Formation on a Mononuclear Non‐Heme Iron Center

Cleavage and formation of the O–O bond in dioxygen is among the most important metal‐dependent processes in Nature. The former process has been mimicked structurally and functionaly with a number of biomimetic iron complexes. The reverse process, namely the dioxygen bond formation, however, appears unfavorable with iron. Over the years many attempts have been made to oxidize water on a mononuclear iron center through reactions with peroxide. In this work we present a computational study focused on the mechanism of dioxygen formation on two mononuclear non‐heme iron(IV)‐oxo species upon addition of peracid (m‐chloroperbenzoic acid, mCPBA). A range of test reactions and sequences are analyzed with thermochemical cycles with respect to the thermodynamic and kinetic feasibility of product formation. The work shows that iron‐borne O–O bond formation through peracid attack at FeIVO indeed is feasible through two competing pathways. These are initiated by homolytic inner‐sphere splitting of the RC(O)OO‐H and the RC(O)O‐OH bond in the peracid, respectively.

A 2‐Tyr‐1‐carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase

AbstractN,N‐dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2β2 or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

A 2‐Tyr‐1‐carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase

N,N‐dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2β2 or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

Effect of Ligand Fields on the Reactivity of O2‐Activating Iron(II)‐Benzilate Complexes of Neutral N5 Donor Ligands

Three new iron(II)-benzilate complexes [(N4Py)Fe II (benzilate)]ClO 4 ( 1 ), [(N4Py Me2 )Fe II (benzilate)]ClO 4 ( 2 ) and [(N4Py Me4 )Fe II (benzilate)]ClO 4 ( 3 ) of neutral pentadentate nitrogen donor ligands have been isolated and characterized to study their dioxygen reactivity. Single crystal X-ray structures reveal a mononuclear six-coordinate iron(II) centre in each case, where benzilate binds to the iron center in monodentate mode via one carboxylate oxygen. Introduction of methyl groups in the 6-positions of the pyridine rings makes the N4Py Me2 and N4Py Me4 ligands weaker field compared to the parent N4Py ligand. All the complexes ( 1-3 ) react with dioxygen to decarboxylate the coordinated benzilate to benzophenone quantitatively. The decarboxylation takes lesser time for the complex of more sterically hindered ligand and follows the order 3 > 2 > 1 . The complexes display oxygen atom transfer reactivity to thioanisole and also exhibit hydrogen atom transfer reactions with substrates containing weak C-H bonds. Based on interception studies with external substrates, labelling experiment and Hammett analysis, a nucleophilic iron(II)-hydroperoxo species is proposed to form upon two-electron reductive activation of dioxygen by each iron(II)-benzilate complex. The nucleophilic oxidants are converted to the corresponding electrophilic iron(IV)-oxo oxidant upon treatment with a protic acid. The high-spin iron(II)-benzilate complex with the weakest ligand field results in the formation of a more reactive iron-oxygen oxidant.

Nitrogen Reduction to Ammonia on a Biomimetic Mononuclear Iron Centre: Insights into the Nitrogenase Enzyme.

Nitrogenases catalyse nitrogen fixation to ammonia on a multinuclear Fe‐Mo centre, but their mechanism and particularly the order of proton and electron transfer processes that happen during the catalytic cycle is still unresolved. Recently, a unique biomimetic mononuclear iron model was developed using tris(phosphine)borate (TPB) ligands that was shown to convert N2 into NH3. Herein, we present a computational study on the [(TPB)FeN2]− complex and describe its conversion into ammonia through the addition of electrons and protons. In particular, we tested the consecutive proton transfer on only the distal nitrogen atom or alternated protonation of the distal/proximal nitrogen. It is found that the lowest energy pathway is consecutive addition of three protons to the same site, which forms ammonia and an iron‐nitrido complex. In addition, the proton transfer step of complexes with the metal in various oxidation and spin states were tested and show that the pK a values of biomimetic mononuclear nitrogenase intermediates vary little with iron oxidation states. As such, the model gives several possible NH3 formation pathways depending on the order of electron/proton transfer, and all should be physically accessible in the natural system. These results may have implications for enzymatic nitrogenases and give insight into the catalytic properties of mononuclear iron centres.

Reductive Activation of O2 by Non-Heme Iron(II) Benzilate Complexes of N4 Ligands: Effect of Ligand Topology on the Reactivity of O2-Derived Oxidant.

A series of iron(II) benzilate complexes (1-7) with general formula [(L)FeII(benzilate)]+ have been isolated and characterized to study the effect of supporting ligand (L) on the reactivity of metal-based oxidant generated in the reaction with dioxygen. Five tripodal N4 ligands (tris(2-pyridylmethyl)amine (TPA in 1), tris(6-methyl-2-pyridylmethyl)amine (6-Me3-TPA in 2), N1,N1-dimethyl-N2,N2-bis(2-pyridylmethyl)ethane-1,2-diamine (iso-BPMEN in 3), N1,N1-dimethyl-N2,N2-bis(6-methyl-2-pyridylmethyl)ethane-1,2-diamine (6-Me2-iso-BPMEN in 4), and tris(2-benzimidazolylmethyl)amine (TBimA in 7)) along with two linear tetradentate amine ligands (N1,N2-dimethyl-N1,N2-bis(2-pyridylmethyl)ethane-1,2-diamine (BPMEN in 5) and N1,N2-dimethyl-N1,N2-bis(6-methyl-2-pyridylmethyl)ethane-1,2-diamine (6-Me2-BPMEN in 6)) were employed in the study. Single-crystal X-ray structural studies reveal that each of the complex cations of 1-3 and 5 contains a mononuclear six-coordinate iron(II) center coordinated by a monoanionic benzilate, whereas complex 7 contains a mononuclear five-coordinate iron(II) center. Benzilate binds to the iron center in a monodentate fashion via one of the carboxylate oxygens in 1 and 7, but it coordinates in a bidentate chelating mode through carboxylate oxygen and neutral hydroxy oxygen in 2, 3, and 5. All of the iron(II) complexes react with dioxygen to exhibit quantitative decarboxylation of benzilic acid to benzophenone. In the decarboxylation pathway, dioxygen becomes reduced on the iron center and the resulting iron-oxygen oxidant shows versatile reactivity. The oxidants are nucleophilic in nature and oxidize sulfide to sulfoxide and sulfone. Furthermore, complexes 2 and 4-6 react with alkenes to produce cis-diols in moderate yields with the incorporation of both the oxygen atoms of dioxygen. The oxygen atoms of the nucleophilic oxidants do not exchange with water. On the basis of interception studies, nucleophilic iron(II) hydroperoxides are proposed to generate in situ in the reaction pathways. The difference in reactivity of the complexes toward external substrates could be attributed to the geometry of the O2-derived iron-oxygen oxidant. DFT calculations suggest that, among all possible geometries and spin states, high-spin side-on iron(II) hydroperoxides are energetically favorable for the complexes of 6-Me3-TPA, 6-Me2-iso-BPMEN, BPMEN, and 6-Me2-BPMEN ligands, while high spin end-on iron(II) hydroperoxides are favorable for the complexes of TPA, iso-BPMEN, and TBimA ligands.

A Rieske-Type Oxygenase of Pseudomonas sp. BIOMIG1 Converts Benzalkonium Chlorides to Benzyldimethyl Amine.

Recently, an array of eight genes involved in the biotransformation of benzalkonium chlorides (BACs)-an active ingredient of many disinfectants-to benzyldimethyl amine (BDMA) was identified in the genome of Pseudomonas sp. BIOMIG1, which is a bacterium present in various environments and mineralizes BACs. In this study, we showed that heterologous expression of an oxygenase gene (oxyBAC) present in this gene array in E. coli resulted in formation of BDMA from BACs at a rate of 14 μM h-1. oxyBAC is phylogenetically classified as a Rieske-type oxygenase (RO) and belongs to a group which catalyzes the cleavage of C-N+ bond between either methyl or alkyl ester and a quaternary nitrogen (N) of natural quaternary ammonium compounds such as stachydrine, carnitine, and trimethylglycine. Insertion of two glycines into the Rieske domain and substitution of tyrosine with leucine in the mononuclear iron center differentiate oxyBAC from other ROs that cleave C-N+, and presumably facilitate the cleavage of saturated alkyl chain from quaternary N via N-dealkylation reaction. In addition, unlike other ROs, oxyBAC did not require a specific reductase to function. Our results demonstrate that oxyBAC represents a new member of RO associated with BAC degradation, and have applications for controlling the fate of BACs in the environment.

Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme.

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Theoretical Model of the Structure and the Reaction Mechanisms of Sulfur Oxygenase Reductase in <i>Acidithiobacillus thiooxidans</i>

Sulfur oxygenase reductase (SOR), which is thought to be an important enzyme involved in sulfur oxidation in many microorganisms, may play a key role in sulfur oxidation in Acidithiobacillusthiooxidans. Draft genome sequence of A. thiooxidans A01 indicated the presence of sulfur oxygenase reductase gene (sor). The complementary DNA fragment was speculated to encode a putative 311-aa full-length protein SOR. Structural analysis of SOR revealed that three cysteines located in the two conserved domains, C32 at V-G-P-K-V-C32 as well as C102 and C105 at C102-X-X-C105, might form the substrate activation and binding site. It was proposed that conserved motif H87-X3-H91-X23-E115 acted as ligands might combine with iron atom to constitute a mononuclear non-heme iron center, catalyzing the oxidation reaction of substrate.