Iron Enzymes Research Articles

Exploring the Reactivity of High Valent Iron Intermediates in Water

The exceptional reactivity observed in non‐heme iron enzymes can be attributed to their capability to access high‐valent iron oxygen species in their active site. Numerous inorganic model complexes have been reported to date, providing insights into the intricate structural and spectroscopic features of many iron‐containing enzymes and advancing our understanding of their enzymatic reaction pathways. While the reactivities of synthetic iron complexes have been evaluated using various oxidants, the investigation into the formation of reactive intermediates has primarily focused on acetonitrile. However, water, which serves as the medium in biological systems, has been less frequently employed in these studies. Motivated by this, we conducted a comprehensive study on the generation of key reactive species using various oxidants with a model complex, [(BnTPEN)Fe(II)(OTf)]+ (1) (where BnTPEN = N‐benzyl‐N,N,N‐tris(2‐pyridylmethyl)‐1,2‐diaminoethane) in water, which yielded important findings. In water, a quantitative yield of Fe(IV)=O species was achieved with the oxidant NaIO4. Additionally, we observed an equilibrium between side‐on Fe(III)‐OO and Fe(III)‐OOH, with the latter eventually converting to Fe(IV)=O. The insights gained from this study are likely to be relevant in the chemistry of other Fe(II) complexes with polypyridyl pentadentate ligands.

Computational Stabilization of a Non-Heme Iron Enzyme Enables Efficient Evolution of New Function.

Deep learning tools for enzyme design are rapidly emerging, and there is a critical need to evaluate their effectiveness in engineering workflows. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially relevant non-native functions. This superfamily has diverse catalytic functions and could provide a rich new source of biocatalysts for synthesis and industrial processes. Through systematic comparisons of directed evolution trajectories for a non-native, remote C(sp3)-H hydroxylation reaction, we demonstrate that the stabilized redesign can be evolved more efficiently than the wild-type enzyme. After three rounds of directed evolution, we obtained a 6-fold activity increase from the wild-type parent and an 80-fold increase from the stabilized variant. To generate the initial stabilized variant, we identified multiple structural and sequence constraints to preserve catalytic function. We applied these criteria to produce stabilized, catalytically active variants of a second Fe(II)/αKG enzyme, suggesting that the approach is generalizable to additional members of the Fe(II)/αKG superfamily. ProteinMPNN is user-friendly and widely accessible, and our results provide a framework for the routine implementation of deep learning-based protein stabilization tools in directed evolution workflows for novel biocatalysts.

What is the Origin of the Regioselective C3-Hydroxylation of L-Arg by the Nonheme Iron Enzyme Capreomycin C?

The nonheme iron dioxygenase capreomycin C (CmnC) hydroxylates a free L-arginine amino acid regio- and stereospecifically at the C3-position as part of the capreomycin antibiotics biosynthesis. Little is known on its structure, catalytic cycle and substrate specificity and, therefore, a comprehensive computational study was performed. A large QM cluster model of CmnC was created of 297 atoms and the mechanisms for C3-H, C4-H and C5-H hydroxylation and C3-C4 desaturation were investigated. All low-energy pathways correspond to radical reaction mechanisms with an initial hydrogen atom abstraction followed by OH rebound to form alcohol product complexes. The work is compared to alternative L-Arg hydroxylating nonheme iron dioxygenases and the differences in active site polarity are compared. We show that a tight hydrogen bonding network in the substrate binding pocket positions the substrate in an ideal orientation for C3-H activation, whereby the polar groups in the substrate binding pocket induce an electric field effect that guides the selectivity.

Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation.

Two of nature's recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides. The HExxH domain is a defining feature of zinc metalloproteases. Yet the HExxH motif-containing domain studied here catalyses β-hydroxylation and is an α-ketoglutarate non-haem iron enzyme. We determined the crystal structure for this HExxH protein at 2.8 Å, unveiling a distinct structural fold, thus expanding the family of α-ketoglutarate non-haem iron enzymes with a class that we propose to name αKG-HExxH. αKG-HExxH proteins represent a unique family of ribosomally synthesized and post-translationally modified peptide modifying enzymes that can furnish opportunities for genome mining, synthetic biology and enzymology.

Probing Ferryl Reactivity in a Nonheme Iron Oxygenase Using an Expanded Genetic Code.

The ability to introduce noncanonical amino acids as axial ligands in heme enzymes has provided a powerful experimental tool for studying the structure and reactivity of their FeIV=O ("ferryl") intermediates. Here, we show that a similar approach can be used to perturb the conserved Fe coordination environment of 2-oxoglutarate (2OG) dependent oxygenases, a versatile class of enzymes that employ highly-reactive ferryl intermediates to mediate challenging C-H functionalizations. Replacement of one of the cis-disposed histidine ligands in the oxygenase VioC with a less electron donating N δ-methyl-histidine (MeHis) preserves both catalytic function and reaction selectivity. Significantly, the key ferryl intermediate responsible for C-H activation can be accumulated in both the wildtype and the modified protein. In contrast to heme enzymes, where metal-oxo reactivity is extremely sensitive to the nature of the proximal ligand, the rates of C-H activation and the observed large kinetic isotope effects are only minimally affected by axial ligand replacement in VioC. This study showcases a powerful tool for modulating the coordination sphere of nonheme iron enzymes that will enhance our understanding of the factors governing their divergent activities.

Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function.

Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. For the Fe(II)/αKG enzyme tP4H, we performed site-saturation mutagenesis with both the wild-type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. ProteinMPNN is user-friendly and widely-accessible, and straightforward structural criteria were sufficient to obtain stabilized, catalytically-functional variants of the Fe(II)/αKG enzymes tP4H and GriE. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.

The spin-forbidden transition in iron(IV)-oxo catalysts relevant to two-state reactivity

Quintet oxoiron(IV) intermediates are often invoked in nonheme iron enzymes capable of performing selective oxidation, while most well-characterized synthetic model oxoiron(IV) complexes have a triplet ground state. These differing spin states lead to the proposal of a two-state reactivity model, where the complexes cross from the triplet to an excited quintet state. However, the energy of this quintet state has never been measured experimentally. Here, magnetic circular dichroism is used to assign the singlet and triplet excited states in a series of triplet oxoiron(IV) complexes. These transition energies are used to determine the energies of the quintet state via constrained fitting of 2p3d resonant inelastic x-ray scattering. This allowed for a direct correlation between the quintet energies and substrate C─H oxidation rates.

Observation of oxygenated intermediates in functional mimics of aminophenol dioxygenase

Aminophenol dioxygenases (APDO) are mononuclear nonheme iron enzymes that utilize dioxygen (O2) to catalyze the conversion of o-aminophenols to 2-picolinic acid derivatives in metabolic pathways. This study describes the synthesis and O2 reactivity of two synthetic models of substrate-bound APDO: [FeII(TpMe2)(tBu2APH)] (1) and [FeII(TpMe2)(tBuAPH)] (2), where TpMe2 = hydrotris(3,5-dimethylpyrazole-1-yl)borate, tBu2APH = 4,6-di-tert-butyl-2-aminophenolate, and tBuAPH2 = 4-tert-butyl-2-aminophenolate. Both Fe(II) complexes behave as functional APDO mimics, as exposure to O2 results in oxidative CC bond cleavage of the o-aminophenolate ligand. The ring-cleaved products undergo spontaneous cyclization to give substituted 2-picolinic acids, as verified by 1H NMR spectroscopy, mass spectrometry, and X-ray crystallography. Reaction of the APDO models with O2 at low temperature reveals multiple intermediates, which were probed with UV–vis absorption, electron paramagnetic resonance (EPR), Mössbauer (MB), and resonance Raman (rRaman) spectroscopies. The most stable intermediate at −70 °C in THF exhibits multiple isotopically-sensitive features in rRaman samples prepared with 16O2 and 18O2, confirming incorporation of O2-derived atom(s) into its molecular structure. Insights into the geometric structures, electronic properties, and spectroscopic features of the observed intermediates were obtained from density functional theory (DFT) calculations. Although functional APDO models have been previously reported, this is the first time that an oxygenated ligand-based radical has been detected and spectroscopically characterized in the ring-cleaving mechanism of a relevant synthetic system.

Substrate-Dependent Mechanism Switch in the Desaturation Reactions of the Mononuclear Nonheme Iron Enzyme PtlD

Substrate-Dependent Mechanism Switch in the Desaturation Reactions of the Mononuclear Nonheme Iron Enzyme PtlD

Publisher Correction: Engineering non-haem iron enzymes for enantioselective C(sp3)–F bond formation via radical fluorine transfer

Publisher Correction: Engineering non-haem iron enzymes for enantioselective C(sp3)–F bond formation via radical fluorine transfer

Biocatalytic enantioselective C(sp3)–H fluorination enabled by directed evolution of non-haem iron enzymes

Biocatalytic enantioselective C(sp3)–H fluorination enabled by directed evolution of non-haem iron enzymes

Non‐Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis

AbstractWe report the discovery and biosynthesis of new piperazine alkaloids‐arizonamides, and their derived compounds‐arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non‐heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3‐alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

Non-Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis.

We report the discovery and biosynthesis of new piperazine alkaloids-arizonamides, and their derived compounds-arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non-heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3-alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

Enhanced Reactivity through Equatorial Sulfur Coordination in Nonheme Iron(IV)-Oxo Complexes: Insights from Experiment and Theory.

Sulfur ligation in metalloenzymes often gives the active site unique properties, whether it is the axial cysteinate ligand in the cytochrome P450s or the equatorial sulfur/thiol ligation in nonheme iron enzymes. To understand sulfur ligation to iron complexes and how it affects the structural, spectroscopic, and intrinsic properties of the active species and the catalysis of substrates, we pursued a systematic study and compared sulfur with amine-ligated iron(IV)-oxo complexes. We synthesized and characterized a biomimetic N4S-ligated iron(IV)-oxo complex and compared the obtained results with an analogous N5-ligated iron(IV)-oxo complex. Our work shows that the amine for sulfur replacement in the equatorial ligand framework leads to a rate enhancement for oxygen atom and hydrogen atom transfer reactions. Moreover, the sulfur-ligated iron(IV)-oxo complex reacts through a different reaction mechanism as compared to the N5-ligated iron(IV)-oxo complex, where the former reacts through hydride transfer with the latter reacting via radical pathways. We show that the reactivity differences are caused by a dramatic change in redox potential between the two complexes. Our studies highlight the importance of implementing a sulfur ligand into the equatorial ligand framework of nonheme iron(IV)-oxo complexes and how it affects the physicochemical properties of the oxidant and its reactivity.

Engineering non-haem iron enzymes for enantioselective C(sp3)-F bond formation via radical fluorine transfer.

In recent years there has been a surge in the development of methods for the synthesis of organofluorine compounds. However, enzymatic methods for C-F bond formation have been limited to nucleophilic fluoride substitution. Here, we report the incorporation of iron-catalysed radical fluorine transfer, a reaction mechanism that is not used in naturally occurring enzymes, into enzymatic catalysis for the development of biocatalytic enantioselective C(sp 3)-F bond formation. Using this strategy, we repurposed (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE) to catalyse an N-fluoroamide directed C(sp 3)-H fluorination. Directed evolution has enabled SvHppE to be optimized, forming diverse chiral benzylic fluoride products with turnover numbers of up to 180 and with excellent enantiocontrol (up to 94% e.e.). Mechanistic investigations showed that the N-F bond activation is the rate-determining step, and the strong preference for fluorination in the presence of excess NaN3 can be attributed to the spatial proximity of the carbon-centered radical to the iron-bound fluoride.

Remediation of cadmium-contaminated soil by biochar-loaded nano-zero-valent iron and its microbial community responses

At present, cadmium (Cd)-contaminated soil has been increasingly serious, which is adverse to soil health. In this study, biochar (BC) and biochar-loaded nanoscale zero-valent iron (nZVI@BC) were synthesized for remediation of Cd-contaminated soil. The effects of various factors including reaction time, dosage, nZVI loading on Cd fractionation, soil available iron, pH, CEC, and soil enzyme activity were comprehensively discussed. It can be concluded that nZVI@BC exhibited more excellent immobilization effect of Cd, and was positively correlated with dosage and reaction time, but negatively related to nZVI loading. As a result, 5% nZVI@BC(1:3) performed the best. Soil available iron, pH, and CEC were significantly enhanced with increasing nZVI loading. The immobilization mechanism was analyzed, including ion exchange, physical adsorption, surface complexation, electrostatic attraction as well as co-precipitation. Besides, nZVI@BC remarkably promoted soil catalase activity, but inhibited urease and FDA hydrolase activities. Furthermore, the effects and differences on the diversity, abundance, and species composition of soil microbial communities were deeply explored. The order of bacterial community richness in each group was CK > Cd > BC > nZVI@BC, and the bacterial community diversity was CK > nZVI@BC > Cd > BC. The greatest difference of species abundance composition was observed between nZVI@BC treatment group and the control. Additionally, forty-three biomarkers were screened, which primarily belonged to Proteobacteria, Gemmatimonadetes, Firmicutes, and Actinobacteria. This study will provide the profound theoretical basis for nZVI@BC remediation of Cd-contaminated soil and its microbial community responses.

Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Catalytic divergencies in the mechanism of L-arginine hydroxylating nonheme iron enzymes.

Many enzymes in nature utilize a free arginine (L-Arg) amino acid to initiate the biosynthesis of natural products. Examples include nitric oxide synthases, which generate NO from L-Arg for blood pressure control, and various arginine hydroxylases involved in antibiotic biosynthesis. Among the groups of arginine hydroxylases, several enzymes utilize a nonheme iron(II) active site and let L-Arg react with dioxygen and α-ketoglutarate to perform either C3-hydroxylation, C4-hydroxylation, C5-hydroxylation, or C4-C5-desaturation. How these seemingly similar enzymes can react with high specificity and selectivity to form different products remains unknown. Over the past few years, our groups have investigated the mechanisms of L-Arg-activating nonheme iron dioxygenases, including the viomycin biosynthesis enzyme VioC, the naphthyridinomycin biosynthesis enzyme NapI, and the streptothricin biosynthesis enzyme OrfP, using computational approaches and applied molecular dynamics, quantum mechanics on cluster models, and quantum mechanics/molecular mechanics (QM/MM) approaches. These studies not only highlight the differences in substrate and oxidant binding and positioning but also emphasize on electronic and electrostatic differences in the substrate-binding pockets of the enzymes. In particular, due to charge differences in the active site structures, there are changes in the local electric field and electric dipole moment orientations that either strengthen or weaken specific substrate C-H bonds. The local field effects, therefore, influence and guide reaction selectivity and specificity and give the enzymes their unique reactivity patterns. Computational work using either QM/MM or density functional theory (DFT) on cluster models can provide valuable insights into catalytic reaction mechanisms and produce accurate and reliable data that can be used to engineer proteins and synthetic catalysts to perform novel reaction pathways.

Synthesis, Structure and Reactivity of a Mononuclear N,N,O-Bound Fe(II) α-Keto-Acid Complex.

A bulky, tridentate phenolate ligand (ImPh2 NNOtBu ) was used to synthesise the first example of a mononuclear, facial, N,N,O-bound iron(II) benzoylformate complex, [Fe(ImPh2 NNOtBu )(BF)] (2). The X-ray crystal structure of 2 reveals that the iron centre is pentacoordinate (τ=0.5), with a vacant site located cis to the bidentate BF ligand. The Mössbauer parameters of 2 are consistent with high-spin iron(II), and are very close to those reported for α-ketoglutarate-bound non-heme iron enzyme active sites. According to NMR and UV-vis spectroscopies, the structural integrity of 2 is retained in both coordinating and non-coordinating solvents. Cyclic voltammetry studies show that the iron centre has a very low oxidation potential and is more prone to electrochemical oxidation than the redox-active phenolate ligand. Complex 2 reacts with NO to form a S=3 /2 {FeNO}7 adduct in which NO binds directly to the iron centre, according to EPR, UV-vis, IR spectroscopies and DFT analysis. Upon O2 exposure, 2 undergoes oxidative decarboxylation to form a diiron(III) benzoate complex, [Fe2 (ImPh2 NNOtBu )2 (μ2 -OBz)(μ2 -OH)2 ]+ (3). A small amount of hydroxylated ligand was also observed by ESI-MS, hinting at the formation of a high-valent iron(IV)-oxo intermediate. Initial reactivity studies show that 2 is capable of oxygen atom transfer reactivity with O2 , converting methyl(p-tolyl)sulfide to sulfoxide.

The effects of different iron and phosphorus treatments on the formation and morphology of iron plaque in rice roots (Oryza sativa L).

Rice (Oryza sativa L.) is a pivotal cereal crop worldwide. It relies heavily on the presence of iron plaque on its root surfaces for optimal growth and enhanced stress resistance across diverse environmental conditions. To study the crystallographic aspects of iron plaque formation on rice roots, the concentrations of Fe2+ and PO4 3- were controlled in this study. The effects of these treatments were assessed through comprehensive analyzes encompassing root growth status, root surface iron concentration, root vitality, enzyme activities, and microstructural characteristics using advanced techniques such as root analysis, scanning electron microscopy (SEM), and ultrathin section transmission electron microscopy (TEM). The results demonstrated that an increase in the Fe2+ concentration or a decrease in the PO4 3- concentration in the nutrient solution led to improvements in various root growth indicators. There was an elevation in the DCB (dithionite-citrate-bicarbonate) iron content within the roots, enhanced root vitality, and a significant increase in the activities of the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes. Moreover, as the Fe2+ concentration increased, amorphous iron oxide minerals on the root surface were gradually transformed into ferrihydrite particles with sizes of approximately 200 nm and goethite particles with sizes of approximately 5 μm. This study showed that an increase in the Fe2+ concentration and a decrease in the PO4 3- concentration led to the formation of substantial iron plaque on the root surfaces. It is noteworthy that there was a distinct gap ranging from 0.5 to 3 μm between the iron plaque formed through PO4 3- treatment and the cellular layer of the root surface. This study elucidated the impacts of Fe2+ and PO4 3- treatments on the formation, structure, and morphology of the iron plaque while discerning variations in the spatial proximity between the iron plaque and root surface under different treatment conditions.