Diiron Cluster Research Articles

Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our invitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with invivo experiments in bacteria in the future.

Aqueous pH influence on the electrocatalytic hydrogen evolution reaction with carbon nanotube-supported diiron dithiolato compound

Biomimetic chemistry on structure, function and external environment of [FeFe]-hydrogenases for hydrogen evolution reaction (HER) has shown a promising way to develop non-precious metal electrocatalysts for H2 production in the current Pt-dominated HER system. Herein, three new diiron dithiolato compounds [{(μ-SCH2)2N(C6H4CH2CO2R)}Fe2(CO)6] (R = H (1), C6H4CHO-p (2), and C6H4Me-m (3)) were first prepared and identified as a library of [FeFe]-hydrogenase models in this work. Subsequently, the as-prepared diiron molecule 2 can be covalently attached onto carbon nanotubes (CNTs), resulting in the obtainment of target CNT-supported [FeFe]-hydrogenase model labeling as covalent hybrid 2Fe2S-f-CNT. Notably, the electrocatalytic HER performance and stability of the resulted hybrid 2Fe2S-f-CNT immobilized respectively on a gassy carbon (GC) electrode are systematically studied and compared in 0.1 M KOH (pH = 13), 0.1 M phosphate buffer (pH = 7), and 0.05 M H2SO4 (pH = 1) aqueous solutions by various spectroscopic and electrochemical techniques. The result has shown that the higher electrocatalytic HER activity is observed in alkaline solution (pH = 13) relative to neutral and acidic solutions (pH = 7, 1), being attributed to the different electrochemical HER processes of diiron cluster in wide pH aqueous media as revealed by post operando analysis.

The structural and functional investigation into an unusual nitrile synthase

The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.

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.

Synthesis of phosphine derivatives of [Fe2(CO)6(μ-sdt)] (sdt = SCH2SCH2S) and investigation of their proton reduction capabilities

The reactions of [Fe2(CO)6(μ-sdt)] (1) (sdt = SCH2SCH2S) with phosphine ligands have been investigated. Treatment of 1 with dppm (bis(diphenylphosphino)methane) or dcpm (bis(dicyclohexylphosphino)methane) affords the diphosphine-bridged products [Fe2(CO)4(μ-sdt)(μ-dppm)] (2) and [Fe2(CO)4(μ-sdt)(μ-dcpm)] (3), respectively. The complex [Fe2(CO)4(μ-sdt)(κ2-dppv)] (4) with a chelating diphosphine was obtained by reacting 1 with dppv (cis-1,2-bis(diphenylphosphino)ethene). Reaction of 1 with dppe (1,2-bis(diphenylphosphino)ethane) produces [{Fe2(CO)4(μ-sdt)}2(μ-κ1-dppe)] (5) in which the diphosphine forms an intermolecular bridge between two diiron cluster fragments. Three products were obtained when dppf (1,1′-bis(diphenylphosphino)ferrocene) was introduced to complex 1; they were [Fe2(CO)5(μ-sdt)(κ1-dppfO)] (6), the previously known [{Fe2(CO)5(μ-sdt)}2(μ-κ1-κ1-dppf)] (7), and [Fe2(CO)4(μ-sdt)(μ-dppf)] (8), with complex 8 being produced in highest yield. Single crystal X-ray diffraction analysis was performed on compounds 2, 3 and 8. All structures reveal the adoption of an anti-arrangement of the dithiolate bridges, while the diphosphines occupy dibasal positions. Infra-red spectroscopy indicates that the mono-substituted complexes 5, 6, and 7 are inert to protonation by HBF4.Et2O, but complexes 2, 3, 4 and [Fe2(CO)5(μ-sdt)(κ1-PPh3)] (9) show shifts of their ν(C-O) resonances that indicate that protons bind to the metal cores of the clusters. Addition of the one-electron oxidant [Cp2Fe]PF6 does not lead to any discernable shift in the IR resonances. The redox chemistry of the complexes was investigated by cyclic voltammetry, and the abilities of complexes to catalyze electrochemical proton reduction were examined.

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases.

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN- ligands. The biogenesis of this diiron site, termed [2FeH ], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2FeH ] precursor, termed [2FeP ], to the [FeFe]-hydrogenase. The binding site of [2FeP ] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2FeP ]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2FeP ]. Surprisingly, our results suggest that binding or transfer of [2FeP ] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.

Sulfur-Ligated [2Fe-2C] Clusters as Synthetic Model Systems for Nitrogenase.

Metal clusters featuring carbon and sulfur donors have coordination environments comparable to the active site of nitrogenase enzymes. Here, we report a series of di-iron clusters supported by the dianionic yldiide ligands, in which the Fe sites are bridged by two μ2-C atoms and four pendant S donors.The [L2Fe2] (L = {[Ph2P(S)]2C}2-) cluster is isolable in two oxidation levels, all-ferrous Fe2II and mixed-valence FeIIFeIII. The mixed-valence cluster displays two peaks in the Mössbauer spectra, indicating slow electron transfer between the two sites. The addition of the Lewis base 4-dimethylaminopyridine to the Fe2II cluster results in coordination with only one of the two Fe sites, even in the presence of an excess base. Conversely, the cluster reacts with 8 equiv of isocyanide tBuNC to give a monometallic complex featuring a new C-C bond between the ligand backbone and the isocyanide. The electronic structure descriptions of these complexes are further supported by X-ray absorption and resonant X-ray emission spectroscopies.

Time-Resolved Infrared Spectroscopy Reveals the pH-Independence of the First Electron Transfer Step in the [FeFe] Hydrogenase Catalytic Cycle.

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe−4S] electron relay covalently attached to the H2 binding site and a diiron cluster ligated by CO, CN–, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe−4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe−4S]H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

X-ray Crystal Structures of Methane Monooxygenase Hydroxylase Complexes with Variants of Its Regulatory Component: Correlations with Altered Reaction Cycle Dynamics.

Full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (αβγ)2 hydroxylase, sMMOH. Previous studies have shown that mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle. Here, X-ray crystal structures are reported for the sMMOH complex with two double variants within the core region of MMOB, DBL1 (N107G/S110A), and DBL2 (S109A/T111A), as well as two variants in the MMOB N-terminal region, H33A and H5A. DBL1 causes a 150-fold decrease in the formation rate constant of the reaction cycle intermediate P, whereas DBL2 accelerates the reaction of the dinuclear Fe(IV) intermediate Q with substrates larger than methane by three- to fourfold. H33A also greatly slows P formation, while H5A modestly slows both formation of Q and its reactions with substrates. Complexation with DBL1 or H33A alters the position of sMMOH residue R245, which is part of a conserved hydrogen-bonding network encompassing the active site diiron cluster where P is formed. Accordingly, electron paramagnetic resonance spectra of sMMOH:DBL1 and sMMOH:H33A complexes differ markedly from that of sMMOH:MMOB, showing an altered electronic environment. In the sMMOH:DBL2 complex, the position of M247 in sMMOH is altered such that it enlarges a molecular tunnel associated with substrate entry into the active site. The H5A variant causes only subtle structural changes despite its kinetic effects, emphasizing the precise alignment of sMMOH and MMOB required for efficient catalysis.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 μM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.

How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, the hallmark of chlorins and bacteriochlorins, producing protochlorophyllide a and contributing significantly to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in oxygenic phototrophs and in some purple bacteria. We overproduced the simplest form of the cyclase, AcsF from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the diiron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yield a k cat of 0.9 min-1, a K M of 7.0 µM for MgPME, and a K d for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as intermediates in the formation of the isocyclic ring, revealing the reaction chemistry that converts porphyrins to chlorins, and completing the work originated by Sam Granick in 1950.

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

Functional Characterization of COG1713 (YqeK) as a Novel Diadenosine Tetraphosphate Hydrolase Family.

Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap4A in B. subtilis YqeK shows a catalytic efficiency toward Ap4A similar to that of the symmetrically cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.IMPORTANCE Elevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

Substrate-Triggered Formation of a Peroxo-Fe2(III/III) Intermediate during Fatty Acid Decarboxylation by UndA

The iron-dependent oxidase UndA cleaves one C3-H bond and the C1-C2 bond of dodecanoic acid to produce 1-undecene and CO2. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3-H bond using a monoiron cofactor to activate O2 necessarily invoked unusual or potentially unfeasible steps. Here we present spectroscopic, crystallographic, and biochemical evidence that the cofactor of Pseudomonas fluorescens Pf-5 UndA is actually a diiron cluster and show that binding of the substrate triggers rapid addition of O2 to the Fe2(II/II) cofactor to produce a transient peroxo-Fe2(III/III) intermediate. The observations of a diiron cofactor and substrate-triggered formation of a peroxo-Fe2(III/III) intermediate suggest a small set of possible mechanisms for O2, C3-H and C1-C2 activation by UndA; these routes obviate the problematic steps of the earlier hypotheses that invoked a single iron.

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.

Using iron to generate a copper ligand

Biochemistry Many microbial enzymes are metal-dependent, and the microbe must acquire scarce metals from the environment. Microbes that use methane as a carbon source have a copper-dependent enzyme that oxidizes the methane. Peptides known as methanobactins (Mbns) acquire copper by using a pair of ligands comprising a nitrogen-containing ring and an adjacent thioamide. Kenney et al. describe the biosynthetic machinery that adds the copper-binding groups to a precursor peptide. This involves a complex of two homologs: MbnB, a member of a functionally uncharacterized protein family that includes a diiron cluster, and MbnC, which is even less well characterized. The iron cofactor is required for ligand synthesis. MbnB and MbnC homologs are encoded in many genomes, suggesting that they may have roles beyond Mbn biosynthesis. Science , this issue p. [1411][1] [1]: /lookup/doi/10.1126/science.aap9437

Diiron monooxygenases in natural product biosynthesis.

Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation of l-p-aminophenylalanine (l-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-β-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase. CmlA couples O2 activation and substrate hydroxylation via a structural change caused by formation of the l-PAPA-loaded CmlP:CmlA complex. The other new diiron family is typified by two enzymes, AurF and CmlI, which catalyze conversion of aryl-amine substrates to aryl-nitro products with incorporation of oxygen from O2. AurF from Streptomyces thioluteus catalyzes the formation of p-nitrobenzoate from p-aminobenzoate as a precursor to the biostatic compound aureothin, whereas CmlI from S. venezuelae catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. Both enzymes stabilize a novel type of peroxo-intermediate as the reactive species. The rare 6-electron N-oxygenation reactions of CmlI and AurF involve two progressively oxidized pathway intermediates. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis. CmlI ensures specificity by carrying out the multistep aryl-amine oxygenation without dissociating intermediate products.

Unprecedented (μ-1,1-Peroxo)diferric Structure for the Ambiphilic Orange Peroxo Intermediate of the Nonheme N-Oxygenase CmlI.

The final step in the biosynthesis of the antibiotic chloramphenicol is the oxidation of an aryl-amine substrate to an aryl-nitro product catalyzed by the N-oxygenase CmlI in three two-electron steps. The CmlI active site contains a diiron cluster ligated by three histidine and four glutamate residues and activates dioxygen to perform its role in the biosynthetic pathway. It was previously shown that the active oxidant used by CmlI to facilitate this chemistry is a peroxo-diferric intermediate (CmlIP). Spectroscopic characterization demonstrated that the peroxo binding geometry of CmlIP is not consistent with the μ-1,2 mode commonly observed in nonheme diiron systems. Its geometry was tentatively assigned as μ-η2:η1 based on comparison with resonance Raman (rR) features of mixed-metal model complexes in the absence of appropriate diiron models. Here, X-ray absorption spectroscopy (XAS) and rR studies have been used to establish a refined structure for the diferric cluster of CmlIP. The rR experiments carried out with isotopically labeled water identified the symmetric and asymmetric vibrations of an Fe-O-Fe unit in the active site at 485 and 780 cm-1, respectively, which was confirmed by the 1.83 Å Fe-O bond observed by XAS. In addition, a unique Fe···O scatterer at 2.82 Å observed from XAS analysis is assigned as arising from the distal O atom of a μ-1,1-peroxo ligand that is bound symmetrically between the irons. The (μ-oxo)(μ-1,1-peroxo)diferric core structure associated with CmlIP is unprecedented among diiron cluster-containing enzymes and corresponding biomimetic complexes. Importantly, it allows the peroxo-diferric intermediate to be ambiphilic, acting as an electrophilic oxidant in the initial N-hydroxylation of an arylamine and then becoming a nucleophilic oxidant in the final oxidation of an aryl-nitroso intermediate to the aryl-nitro product.

Synthetic and structural studies of the diiron toluenedithiolate carbonyl complexes with monophosphine ligands

Four diiron toluenedithiolate complexes 2–5 with monophosphine ligands are reported. Treatment of [μ-SC6H3(CH3)S-μ]Fe2(CO)6 (1) with tris(3-chlorophenyl)phosphine, tris(4-chlorophenyl)phosphine, tris(4-methylphenyl)phosphine or 2-(diphenylphosphino)benzaldehyde, and Me3NO∙2H2O in MeCN resulted in the formation of [μ-SC6H3(CH3)S-μ]Fe2(CO)5[P(3-C6H4Cl)3] (2), [μ-SC6H3(CH3)S-μ]Fe2(CO)5[P(4-C6H4Cl)3] (3), [μ-SC6H3(CH3)S-μ]Fe2(CO)5[P(4-C6H4CH3)3] (4), and [μ-SC6H3(CH3)S-μ]Fe2(CO)5[Ph2P(2-C6H4CHO)] (5) in 64–82% yields. Complexes 2–5 have been characterized by elemental analysis, IR, 1H NMR, 31P{1H} NMR, 13C{1H} NMR and further confirmed by single crystal X-ray diffraction analysis. The molecular structures show that 2–5 contain a butterfly diiron toluenedithiolate cluster coordinated by five terminal carbonyls and an apical monophosphine.

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI.

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O2, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under (18)O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single-turnover reaction between (18)O2-P and NH2-CAM is carried out in an (16)O2 atmosphere, CAM nitro groups contain both (18)O and (16)O, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.