Diiron Active Site Research Articles

Synthesis and Structural Characterization of a Non-Heme Iron Hyponitrite Complex.

Flavodiiron NO reductases (FNORs) are important enzymes in microbial pathogenesis, as they equip microbes with resistance to the human immune defense agent nitric oxide (NO). Despite much efforts, intermediates that would provide insight into how the non-heme diiron active sites of FNORs reduce NO to N2O could not be identified. Computations predict that iron-hyponitrite complexes are the key species, leading from NO to N2O. However, the coordination chemistry of non-heme iron centers with hyponitrite is largely unknown. In this study, we report the reactivity of two non-heme iron complexes with preformed hyponitrite. In the case of [Fe(TPA)(CH3CN)2](OTf)2, cleavage of hyponitrite and formation of an Fe2(NO)2 diamond core is observed. With less Lewis-acidic [Fe2(BMPA-PhO)2(OTf)2] (2), reaction with Na2N2O2 in polar aprotic solvent leads to the formation of a red complex, 3. X-ray crystallography shows that 3 is a tetranuclear iron-hyponitrite complex, [{Fe2(BMPA-PhO)2}2(μ-N2O2)](OTf)2, with a unique hyponitrite binding mode. This species provided the unique opportunity to us to study the interaction of hyponitrite with non-heme iron centers and the reactivity of the bound hyponitrite ligand. Here, either protonation or oxidation of 3 is found to induce N2O formation, supporting the hypothesis that hyponitrite is a viable intermediate in NO reduction.

Synthesis and Structural Characterization of a Non‐Heme Iron Hyponitrite Complex

Flavodiiron NO reductases (FNORs) are important enzymes in microbial pathogenesis, as they equip microbes with resistance to the human immune defense agent nitric oxide (NO). Despite much efforts, intermediates that would provide insight into how the non‐heme diiron active sites of FNORs reduce NO to N2O could not be identified. Computations predict that iron‐hyponitrite complexes are the key species, leading from NO to N2O. However, the coordination chemistry of non‐heme iron centers with hyponitrite is largely unknown. In this study, we report the reactivity of two non‐heme iron complexes with preformed hyponitrite. In the case of [Fe(TPA)(CH3CN)2](OTf)2, cleavage of hyponitrite and formation of an Fe2(NO)2 diamond core is observed. With less Lewis‐acidic [Fe2(BMPA‐PhO)2(OTf)2] (2), reaction with Na2N2O2 in polar aprotic solvent leads to the formation of a red complex, 3. X‐ray crystallography shows that 3 is a tetranuclear iron‐hyponitrite complex, [{Fe2(BMPA‐PhO)2}2(μ‐N2O2)](OTf)2, with a unique hyponitrite binding mode. This species provided the unique opportunity to us to study the interaction of hyponitrite with non‐heme iron centers and the reactivity of the bound hyponitrite ligand. Here, either protonation or oxidation of 3 is found to induce N2O formation, supporting the hypothesis that hyponitrite is a viable intermediate in NO reduction.

Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases

The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O2 molecule has bound to the diferrous diiron active site and before the OO bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both NN bond formation and NO bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual OO bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O2 reduction reaction in the family of heme‑copper oxidases.

DFT mechanistic study of biomimetic diiron complex catalyzed dehydrogenation: Unexpected Fe(III)Fe(III)-1,1-μ-hydroperoxy active species for hydride abstraction

The diiron active site is pivotal in catalyzing transformations in both biological and chemical systems. Recently, a range of biomimetic diiron catalysts have been synthesized, drawing inspiration from the active architecture of soluble methane monooxygenase (sMMO). These catalysts have been successfully deployed for the dehydrogenation of indolines, marking a significant advancement in the field. Using density functional theory (DFT) calculations, we have identified a novel mechanistic pathway that governs the dehydrogenation of indolines catalyzed by a biomimetic diiron complex. Specifically, this reaction is facilitated by the transfer of a hybrid atom from the C1 position of the substrate to the distal oxygen atom of the Fe(III)Fe(III)-1,1-μ-hydroperoxy active species. This transfer serves as the rate-limiting step for the heterolytic cleavage of the OO bond, ultimately generating the substrate cation. The mechanism we propose aligns well with mechanistic investigations incorporating both kinetic isotope effect (KIE) measurements and evaluations of stereochemical selectivity. This research contributes to the broader scientific understanding of catalysis involving biomimetic diiron complexes and offers valuable insights into the catalytic behaviors of non-heme diiron metalloenzymes.

Self-adaptive Metal-Organic Framework Assembles Di-iron Active Sites to Mimic Monooxygenases.

Despite significant efforts, it remains a challenge to design artificial enzymes that can mimic both structures and functions of natural enzymes. Here, we report the post-synthetic construction of binuclear iron catalysts in MOF-253 to mimic natural di-iron monooxygenases. The adjacent bipyridyl (bpy) linkers in MOF-253 can freely rotate to form the [(bpy)FeIII(μ2-OH)]2 active site in a self-adaptive fashion. The composition and structure of the [(bpy)FeIII(μ2-OH)]2 active sites in MOF-253 were characterized by a combination of inductively coupled plasma-mass spectrometry, thermogravimetric analysis, X-ray absorption spectrometry, and Fourier-transform infrared spectroscopy. The MOF-based artificial monooxygenase effectively catalyzed oxidative transformations of organic compounds, including C-H oxidation and alkene epoxidation reactions, using O2 as the only oxidant, which indicates the successful recapitulation of the structure and functions of natural monooxygenases using readily accessible MOFs. The di-iron system exhibited at least 27 times higher catalytic activity than the corresponding mononuclear control. DFT calculations showed that the binuclear system had a 14.2 kcal/mol lower energy barrier than the mononuclear system in the rate-determining C-H activation process, suggesting the importance of cooperativity of the iron centers in the [(bpy)FeIII(μ2-OH)]2 active site in the rate-determining step. The stability and recyclability of the MOF-based artificial monooxygenase were also demonstrated.

Structural basis for enzymatic terminal C–H bond functionalization of alkanes

Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo‐electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C–H bond toward a diiron active site. AlkG, an [Fe–4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C–H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.

Regulation of diiron active site of sMMO through regulatory and reductase components from Methylosinus sporium 5.

Methanotrophs are key archaea for the regulation of methane gas (CH4), one of the major sources of greenhouse gas, through the expression of sMMO and pMMO. The structures and functions of sMMO, a bacterial multicomponent monooxygenase (BMM) superfamily, are being widely investigated to understand O2 and C-H activations in diiron active sites, although elucidation of its mechanism needs to overcome various factors. Recent advances through structural and spectroscopic support demonstrate conformational changes by component interactions of regulatory (MMOB) and inhibitory enzymes. In this presentation, the allosteric effects of sMMO will be described based on the structural and spectroscopic studies. The hydroxylation of CH4 is accelerated by the interaction of MMOB, and it induces conformational changes for controlling substrates and product pathways. The structures of whole sequences of MMOR have not yet been reported, although structures of FAD-/NADH-binding domain and ferredoxin domain from NMR studies have been reported. The electronic structure of MMOR is investigated through electron paramagnetic resonance (EPR) studies, since it cannot provide its electronic structure with structural information. The annealing method from MMOR shows that Cys50 is first oxidized when it transfers electrons in the [2Fe-2S]+ domain and a neural form of FAD is a major form of cofactor.

Regioselectivity of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase by molecular dynamics.

The plant acyl-acyl carrier protein (ACP) desaturases are a family of soluble enzymes that convert saturated fatty-acyl ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction. These enzymes play a key role in biosynthesis of mono-unsaturated fatty acids in plants. The archetype of this group is the Δ9-18:0-ACP desaturase that introduces a cis double bond between carbons 9 and 10 to produce oleoyl-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). ACPs are central proteins in the fatty acid biosynthesis that deliver the substrate to the desaturase. They have been reported to show a varying degree of local dynamics and structural variability depending on the substrate length. It has been suggested that the substrate specific change in structure and dynamics is crucial for the decreasing enzymatic activity in the case of the Δ9-Desaturase for chains length shorter than 18. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position carbons 6 and 7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Electrode Integration of Synthetic Hydrogenase as Bioinspired and Noble Metal-Free Cathodes for Hydrogen Evolution

Diiron complexes mimicking the H-cluster of [FeFe]-hydrogenases have been extensively studied as (electro-)catalysts for proton reduction under homogeneous conditions. The incorporation of such complexes as “active sites” within macromolecular scaffolds such as organic polymers is receiving increasing attention as this strategy allows controlling the environment, that is, the outer coordination sphere, around the molecular catalytic center, to tune its performance as well as its stability. Here, we report on the synthesis and characterization of a library of metallo-copolymers featuring a bioinspired diiron active site and internal proton relays based on a previous report [Brezinski et al. Angew. Chem. Int. Ed. 2018, 57, 11898–11902]. The polymers are further functionalized with various amounts of pyrene groups for efficient noncovalent anchoring onto multi-walled carbon nanotubes (MWNTs), enabling the preparation of molecularly engineered electrode materials. The addition of pyrene anchors resulted in improved activity and stability, with a pyrene loading of about ∼8% corresponding to an optimized balance between polymer hydrophilicity and surface affinity. The best material displayed an average turnover frequency (TOFH2) of 4.3 ± 0.6 s–1 and a conservative turnover number for H2 production (TONH2) of 3.1 ± 0.4 × 105 after 20 h of continuous bulk electrolysis in aqueous conditions at 0.39 V overpotential. Interestingly, comparing such activities with an analogous diiron site deprived from polymeric scaffold revealed that latter could only show TONH2 of ∼4 ± 2 × 103 and TOFH2 of 0.06 ± 0.02 s–1 in 20 h under the same conditions. Post operando analysis of the modified electrodes suggests that electrode inactivation occurs via leaching of the diiron core from MWNT. In addition, a life cycle assessment was carried out to evaluate the performance of the engineered electrode materials not only from a technical perspective but also from an environmental point of view.

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A MnIII(μ-O)(μ-OH)FeIII species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(μ-O)(μ-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded MnIII(μ-OH)FeIII species. This final intermediate precedes formation of the MnIII/FeIII resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.

A suicide diiron oxygenase in p‐aminobenzoate biosynthesis in Chlamydia trachomatis

Folate is an essential one‐carbon carrier cofactor required for essential biological reactions such as amino acid and DNA biosynthesis. Folates are comprised of three chemical moieties: a pteridine ring, p‐aminobenzoate (pABA), and glutamate residues. Chlamydia trachomatis has been shown to synthesize folate de novo; however, it is missing several genes involved in the canonical folate biosynthesis pathway. Previous work has demonstrated that a single gene, ct610, functionally replaces the enzymes responsible for pABA production in the canonical biosynthesis pathway. However, CT610 does not use chorismate as the precursor to pABA as is used in the canonical pathway. Further experiments showed that isotopically labeled tyrosine was incorporated into the pABA molecule when synthesized by CT610 in vivo. However, in vitro experiments demonstrated that CT610 produces pABA in the presence of oxygen and a reducing agent without any added substrates, including tyrosine. This led to the hypothesis that CT610 is a self‐sacrificing “suicide” enzyme that donates an active site tyrosine residue in synthesizing pABA. CT610 has low sequence similarity to non‐heme diiron monooxygenases such as methane monooxygenase and class Ia ribonucleotide reductase, and the previously solved crystal structure of CT610 revealed a diiron active site. Here we describe our recent progress towards understanding this unique suicide mechanism for pABA biosynthesis. To determine the tyrosine residue sacrificed in the reaction, we generated five active site tyrosine to phenylalanine mutants and found that two of the variants completely abolished pABA production, suggesting that one of these residues is the precursor to pABA. Further, a conserved lysine residue is also essential for the reaction and may be a sacrificial amino group donor. We have confirmed the oxygenase activity of wild‐type CT610, which requires only a reducing agent and is not stimulated by the presence of tyrosine or other possible substrates. Further, we developed an Fe(II) reconstitution procedure, where the reconstituted enzyme exhibits a drastic increase in oxygenase activity. However, surprisingly, the reconstituted enzyme does not exhibit increased pABA synthase activity, although EDTA treatment of the as‐purified enzyme almost completely inhibits pABA production. This suggests that a metal other than iron is required for the reaction and thus current work is focused on testing other potential metal cofactors that may play a role in the novel suicide reaction.

Electron Transfer to Hydroxylase through Component Interactions in Soluble Methane Monooxygenase.

The hydroxylation of methane (CH4) is crucial to the field of environmental microbiology, owing to the heat capacity of methane, which is much higher than that of carbon dioxide (CO2). Soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily, is essential for the hydroxylation of specific substrates, including hydroxylase (MMOH), regulatory component (MMOB), and reductase (MMOR). The diiron active site positioned in the MMOH α-subunit is reduced through the interaction of MMOR in the catalytic cycle. The electron transfer pathway, however, is not yet fully understood due to the absence of complex structures with reductases. A type II methanotroph, Methylosinus sporium 5, successfully expressed sMMO and hydroxylase, which were purified for the study of the mechanisms. Studies on the MMOH-MMOB interaction have demonstrated that Tyr76 and Trp78 induce hydrophobic interactions through π-π stacking. Structural analysis and sequencing of the ferredoxin domain in MMOR (MMOR-Fd) suggested that Tyr93 and Tyr95 could be key residues for electron transfer. Mutational studies of these residues have shown that the concentrations of flavin adenine dinucleotide (FAD) and iron ions are changed. The measurements of dissociation constants (Kds) between hydroxylase and mutated reductases confirmed that the binding affinities were not significantly changed, although the specific enzyme activities were significantly reduced by MMOR-Y93A. This result shows that Tyr93 could be a crucial residue for the electron transfer route at the interface between hydroxylase and reductase.

An Overview of the Electron-Transfer Proteins That Activate Alkane Monooxygenase (AlkB).

Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron–sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research.

Fast Proton Transport in FeFe Hydrogenase via a Flexible Channel and a Proton Hole Mechanism

Di-iron hydrogenases are a class of enzymes that are capable of reducing protons to form molecular hydrogen with high efficiency. In addition to the catalytic site, these enzymes have evolved dedicated pathways to transport protons and electrons to the reaction center. Here, we present a detailed study of the most likely proton transfer pathway in such an enzyme using QM/MM molecular dynamics simulations. The protons are transported through a channel lined out from the protein exterior to the di-iron active site, by a series of hydrogen-bonded, weakly acidic or basic, amino acids and two incorporated water molecules. The channel shows remarkable flexibility, which is an essential feature to quickly reset the hydrogen-bond direction in the channel after each proton passing. Proton transport takes place via a “hole” mechanism, rather than an excess proton mechanism, the free energy landscape of which is remarkably flat, with a highest transition state barrier of only 5 kcal/mol. These results confirm our previous assumptions that proton transport is not rate limiting in the H2 formation activity and that cysteine C299 may be considered protonated at physiological pH conditions. Detailed understanding of this proton transport may aid in the ongoing attempts to design artificial biomimetic hydrogenases for hydrogen fuel production.

Understanding desaturation/hydroxylation activity of castor stearoyl Δ9-Desaturase through rational mutagenesis

A recently proposed reaction mechanism of soluble Δ9 desaturase (Δ9D) allowed us to identify auxiliary residues His203, Asp101, Thr206 and Cys222 localized near the di-iron active site that are supposedly involved in the proton transfer (PT) to and from the active site. The PT, along with the electron transfer (ET), seems to be crucial for efficient desaturation. Thus, perturbing the major PT chains is expected to impair the native reaction and (potentially) amplify minor reaction channels, such as the substrate hydroxylation. To verify this hypothesis, we mutated the four residues mentioned above into their counterparts present in a soluble methane monooxygenase (sMMO), and determined the reaction products of mutants. We found that the mutations significantly promote residual monohydroxylation activities on stearoyl-CoA, often at the expense of native desaturation activity. The favored hydroxylation positions are C9, followed by C10 and C11. Reactions with unsaturated substrate, oleoyl-CoA, yield erythro-9,10-diol, cis-9,10-epoxide and a mixture of allylic alcohols. Additionally, using 9- and 11-hydroxystearoyl-CoA, we showed that the desaturation reaction can proceed only with the hydroxyl group at position C11, whereas the hydroxylation reaction is possible in both cases, i.e. with hydroxyl at position C9 or C11. Despite the fact that the overall outcome of hydroxylation is rather modest and that it is mostly the desaturation/hydroxylation ratio that is affected, our results broaden understanding of the origin of chemo- and stereoselectivity of the Δ9D and provide further insight into the catalytic action of the NHFe2 enzymes.

Explorations of the nonheme high-valent iron-oxo landscape: crystal structure of a synthetic complex with an [FeIV2(μ-O)2] diamond core relevant to the chemistry of sMMOH.

Methanotrophic bacteria utilize methane monooxygenase (MMO) to carry out the first step in metabolizing methane. The soluble enzymes employ a hydroxylase component (sMMOH) with a nonheme diiron active site that activates O2 and generates a powerful oxidant capable of converting methane to methanol. It is proposed that the diiron(II) center in the reduced enzyme reacts with O2 to generate a diferric-peroxo intermediate called P that then undergoes O-O cleavage to convert into a diiron(IV) derivative called Q, which carries out methane hydroxylation. Most (but not all) of the spectroscopic data of Q accumulated by various groups to date favor the presence of an FeIV2(μ-O)2 unit with a diamond core. The Que lab has had a long-term interest in making synthetic analogs of iron enzyme intermediates. To this end, the first crystal structure of a complex with a FeIIIFeIV(μ-O)2 diamond core was reported in 1999, which exhibited an Fe⋯Fe distance of 2.683(1) Å. Now more than 20 years later, a complex with an FeIV2(μ-O)2 diamond core has been synthesized in sufficient purity to allow diffraction-quality crystals to be grown. Its crystal structure has been solved, revealing an Fe⋯Fe distance of 2.711(4) Å for comparison with structural data for related complexes with lower iron oxidation states.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Abstract Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Computational Study of the C5-Hydroxylation Mechanism Catalyzed by the Diiron Monooxygenase PtmU3 as Part of the Platensimycin Biosynthesis.

PtmU3 is a newly identified nonheme diiron monooxygenase, which installs a C-5 β-hydroxyl group into the C-19 CoA-ester intermediate involved in the biosynthesis of unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 possesses a noncanonical diiron active site architecture of a saturated six-coordinate iron center and lacks the μ-oxo bridge. Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes, how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown. In particular, the electronic characteristic of diiron is also unclear. To understand the catalytic mechanism of PtmU3, we constructed two reactant models in which both the Fe1II-Fe2III-superoxo and Fe1II-Fe2IV═O are considered to trigger the H-abstraction and performed a series of quantum mechanics/molecular mechanics calculations. Our calculation results reveal that PtmU3 is a special monooxygenase, that is, both atoms of the dioxygen molecule can be incorporated into two molecules of the substrate by the successive reactions. In the first-round reaction, PtmU3 uses the Fe1II-Fe2III-superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1II-Fe2IV═O complex. In the second-round reaction, the Fe1II-Fe2IV═O species is responsible for the hydroxylation of another molecule of the substrate. In the diiron center, Fe2 adopts the high spin state (S = 5/2) during the catalysis, whereas for Fe1, in addition to its structural role, it may also play an assistant role for Fe1 catalysis. In the two successive OH-installing steps, the H-abstraction is always the rate-liming step. E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization. Owing to the high reactivity of Fe1II-Fe2IV═O compared to Fe1II-Fe2III-superoxo, besides the C5-hydroxylation, the C3- or C18-hydroxylation was also calculated to be feasible.

A suicide diiron oxygenase in p ‐aminobenzoate biosynthesis in Chlamydia trachomatis

Folate is an essential cofactor required for several processes including DNA and amino acid biosynthesis. Folate molecules are made up of three parts: a pteridine ring, p-aminobenzoate (pABA), and a variable number of glutamate residues. Chlamydia trachomatis synthesizes folate de novo; however, several genes encoding enzymes required for the canonical folate biosynthesis pathway are missing, including PabA/B and PabC, which are normally required for pABA biosynthesis from chorismate. Previous studies have found that a single gene in C. trachomatis, CT610, functionally replaces the canonical pABA biosynthesis genes. Interestingly, CT610 does not use chorismate as a substrate. Instead, the CT610-route for pABA biosynthesis incorporates isotopically-labeled tyrosine into the synthesized pABA molecule. However, in vitro experiments revealed that CT610 produces pABA without any added substrates (including tyrosine) in the presence of a reducing agent and molecular oxygen. CT610 shares low sequence similarity to non-heme diiron oxygenases and the previously solved crystal structure revealed a diiron active site. Taken together, CT610 is proposed to be a self-sacrificing “suicide” enzyme that uses one of its active site tyrosine residues as a precursor to pABA in a reaction that requires O2 and a reduced diiron cofactor. Here, we discuss our recent progress towards uncovering the mechanism of CT610-catalyed pABA synthesis.

Biochemistry of aerobic biological methane oxidation.

Methanotrophic bacteria represent a potential route to methane utilization and mitigation of methane emissions. In the first step of their metabolic pathway, aerobic methanotrophs use methane monooxygenases (MMOs) to activate methane, oxidizing it to methanol. There are two types of MMOs: a particulate, membrane-bound enzyme (pMMO) and a soluble, cytoplasmic enzyme (sMMO). The two MMOs are completely unrelated, with different architectures, metal cofactors, and mechanisms. The more prevalent of the two, pMMO, is copper-dependent, but the identity of its copper active site remains unclear. By contrast, sMMO uses a diiron active site, the catalytic cycle of which is well understood. Here we review the current state of knowledge for both MMOs, with an emphasis on recent developments and emerging hypotheses. In addition, we discuss obstacles to developing expression systems, which are needed to address outstanding questions and to facilitate future protein engineering efforts.