Non-heme Iron Research Articles

Trapping of a phenoxyl radical at a non-haem high-spin iron(II) centre.

The activation of dioxygen at haem and non-haem metal centres, and subsequent functionalization of unactivated C‒H bonds, has been a focal point of much research. In iron-mediated oxidation reactions, O2 binding at an iron(II) centre is often accompanied by an oxidation of the iron centre. Here we demonstrate dioxygen activation by sodium tetraphenylborate and protons in the presence of an iron(II) complex to form a reactive radical species, whereby the iron oxidation state remains unaltered in the presence of a highly oxidizing phenoxyl radical and O2. This complex, containing an unusual iron(II)-phenoxyl radical motif, represents an elusive example of a spectroscopically characterized oxygen-derived iron(II)-reactive intermediate during chemical and biological dioxygen activation at haem and non-haem iron active centres. The present report opens up strategies for the stabilization of a phenoxyl radical cofactor, with its full oxidizing capabilities, to act as an independent redox centre next to an iron(II) site during substrate oxidation reactions.

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.

Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.

The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.

Photo-reduction facilitated stachydrine oxidative N-demethylation reaction: A case study of Rieske non-heme iron oxygenase Stc2 from Sinorhizobium meliloti.

Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. They catalyze a diverse range of transformations in secondary metabolite biosynthesis and xenobiotic bioremediation. ROs typically shuttle electrons from NAD(P)H to the oxygenase component via reductase component(s). This chapter describes our recent biochemical characterization of stachydrine demethylase Stc2 from Sinorhizobium meliloti. In this work, the eosin Y/sodium sulfite pair serves as the photoreduction system to replace the NAD(P)H-reductase system. We describe Stc2 protein purification and quality control details as well as a flow-chemistry to separate the photo-reduction half-reaction and the oxidation half-reaction. Our study demonstrates that the eosin Y/sodium sulfite photo-reduction pair is a NAD(P)H-reductase surrogate for Stc2-catalysis in a flow-chemistry setting. Experimental protocols used in this light-driven Stc2 catalysis are likely to be applicable as a photo-reduction system for other redox enzymes.

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

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

Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

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

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

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

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

Catalytic reduction of oxygen to water by non-heme iron complexes: exploring the effect of the secondary coordination sphere proton exchanging site.

In this study, we prepared non-heme FeIII complexes (1, 2, and 3) of an N4 donor set of ligands (H2L, Me2L, and BPh2L). 1 is supported by a monoanionic bispyridine-dioxime ligand (HL). In 2 and 3, the primary coordination sphere of Fe remained similar to that in 1, except that the oxime protons of the ligand were replaced with two methyl groups and a bridging -BPh2 moiety, respectively. X-ray structures of the FeII complexes (1a and 3a) revealed similar Fe-N distances; however, they were slightly elongated in 2a. The FeIII/FeII potential of 1, 2, and 3 appeared at -0.31 V, -0.25 V, and 0.07 V vs. Fc+/Fc, respectively, implying that HL and Me2L have comparable donor properties. However, BPh2L is more electron deficient than HL or Me2L. 1 showed electrocatalytic oxygen reduction reaction (ORR) activity in acetonitrile in the presence of trifluoroacetic acid (TFAH) as the proton source at Ecat/2 = -0.45 V and revealed selective 4e-/4H+ reduction of O2 to H2O. 1 showed an effective overpotential (ηeff) of 0.98 V and turnover frequency (TOFmax) of 1.02 × 103 s-1. Kinetic studies revealed a kcat of 2.7 × 107 M-2 s-1. Strikingly, 2 and 3 remained inactive for electrocatalytic ORR, which established the essential role of the oxime scaffolds in the electrocatalytic ORR of 1. Furthermore, a chemical ORR of 1 has been investigated using decamethylferrocene as the electron source. For 1, a similar rate equation was noted to that of the electrocatalytic pathway. A kcat of 6.07 × 104 M-2 s-1 was found chemically. Complex 2, however, underwent a very slow chemical ORR. Complex 3 chemically enhances the 4e-/4H+ reduction of O2 and exhibits a TOF of 0.24 s-1 and a kcat value of 2.47 × 102 M-1 s-1. Based on the experimental observations, we demonstrate that the oxime backbone of the ligand in 1 works as a proton exchanging site in the 4e-/4H+ reduction of O2. The study describes how the ORR is affected by the tuning of the ligand scaffold in a family of non-heme Fe complexes.

Impact of carboxylate ligation on the C-H activation reactivity of a non-heme Fe(IV)O complex: a computational investigation.

A comprehensive DFT investigation has been presented to predict how a carboxylate-rich macrocycle would affect the reactivity of a non-heme Fe(IV)O complex towards C-H activation. The popular non-heme iron oxo complex [FeIV(O)(N4Py)]2+, (N4Py = N,N-(bis(2-pyridyl)methyl)N-bis(2-pyridylmethyl)amine) (1), has been selected here as the primary compound. It is transformed to the compound [FeIV(O)(nBu-P2DA)], where nBu-P2DA = N-(1',1'-bis(2-pyridyl)pentyl)iminodiacetate (2) after the replacement of two pyridine donors of N4Py with carboxylate groups. Two other complexes, namely 3 and 4, have been predicted sequentially substituting nitrogen with the carboxylate groups. Ethylbenzene and dihydrotoluene were chosen as substrates. In terms of C-H activation reactivity, an interesting pattern emerges: as the carboxylate group becomes more equatorially enriched, the reactivity increases, following the trend 1 < 2 < 3 < 4. This also aligns with available experimental reports related to complexes 1 and 2. Fe(IV)O complexes exhibit two-state reactivity (triplet and quintet), whereas the quintet state is more favourable due to the stabilization of the transition states through exchange interactions involving a greater number of unpaired electrons. A detailed analysis of the factors influencing reactivity has been performed, including distortion energy (which decreases for the transition state with the addition of carboxylate groups), the triplet-quintet oxidant energy gap (which consistently decreases as carboxylate group enrichment increases), steric factors, and quantum mechanical tunneling. This investigation provides a detailed explanation of the observed outcomes and predicts the higher reactivity of carboxylate-enriched Fe(IV)O complexes. After potential experimental verification, this could lead to the development of new, optimal catalysts for C-H activation.

Understanding the Chemistry of Nitrene and Highlighting its Remarkable Catalytic Capabilities as a Non-Heme Iron Enzyme

Understanding the Chemistry of Nitrene and Highlighting its Remarkable Catalytic Capabilities as a Non-Heme Iron Enzyme

Iron and cancer: overview of the evidence from population-based studies.

Iron is an essential nutrient required for various physiological processes in the body. However, iron imbalance can potentially contribute to initiating and promoting cancer development. Epidemiological studies have investigated the relationship between dietary iron intake and the risk of different types of cancer, yet, not all studies have consistently shown a significant association between dietary iron and cancer risk. Also, studies have shown different effects of dietary heme and non-heme iron intake on cancer risk. While some epidemiological studies suggest a possible link between high dietary iron (mainly heme-iron) intake and increased cancer risk, the evidence remains inconsistent. Moreover, multiple iron biomarkers, which can mirror physiological iron status, have demonstrated varied correlations with the risk of cancer, contingent upon the specific biomarker analyzed and the type of cancer being investigated. Here, we have investigated the current evidence on the potential relationship between dietary iron intake on one hand, and iron biomarkers on the other hand, with the risk of developing different types of cancer, including breast, prostate, lung, pancreatic, colon, colorectal, and liver cancers. Further research is warranted to better understand the complex relationship between dietary iron, physiological iron and cancer development. Future research should account for factors that affect and interact with dietary iron and physiological iron levels, such as genetic susceptibility, overall diet quality, and lifestyle habits.

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Whole-cell Rieske non-heme iron biocatalysts.

Rieske non-heme iron oxygenases (ROs) possess the ability to catalyze a wide range of reactions. Their ability to degrade aromatic compounds is a unique characteristic and makes ROs interesting for a variety of potential applications. However, purified ROs can be challenging to work with due to low stability and long, complex electron transport chains. Whole cell biocatalysis represents a quick and reliable method for characterizing the activity of ROs and harnessing their metabolic potential. In this protocol, we outline a step-by-step protocol for the overexpression of ROs for whole cell biocatalysis and characterization. We have utilized a caffeine-degrading, N-demethylation system, expressing the RO genes ndmA and ndmD, as an example of this method.

Dynamics of non-heme iron content in myocardium and heme oxygenase activity under hyperergic conditions

Dynamics of non-heme iron content in myocardium and heme oxygenase activity under hyperergic conditions

Combined intervention strategy for reversing iron-deficiency anaemia and deficiency in psychomotor development in chronic malnutrition.

Background: chronic iron-deficiency anaemia in children has a negative impact on neuronal and cognitive development. Despite current knowledge on this subject, in Bolivia iron intake along the psychomotor development stimulation as part of a comprehensive rehabilitation process for children with severe chronic malnutrition is not yet used. Objective: to evaluate the effect of a neurorestorative diet, consisting of iron supplements and other micronutrients, along with psychomotor stimulation in preschool children with chronic malnutrition, iron-deficiency anaemia and severe psychomotor delay. Patients and methods: twenty-four children between 1 and 56 months of age admitted to the integral nutritional recovery centre (INRC), Paediatric Hospital of Cochabamba, Bolivia were included. A strategy of intervention was applied consisting of nutritional replenishment through the administration of elaborated meals prepared from local foods with high heme and non-heme iron concentration, added with vegetables plus the administration of micronutrient´s supplementation and the psychomotor stimulation. Anthropometric indices, psychomotor and biochemical parameters were measured at four times points, during the hospitalisation period. Results: at the beginning, the anthropometric and psychomotor parameters were decreased (between -2 and -3 z score and below 50 % respectively). Combined strategy intervention with iron and other micronutrients together photons produced significant changes between the evaluated time points, both in anthropometric and psychomotor parameters, although these changes were less than expected. Conclusions: the combined strategy used in this study allowed recovery from the anaemia and minimal growth due to the low birth weight or chronic malnutrition. However, the intervention was insufficient to achieve a complete recovery.

Exploring the influence of H-bonding and ligand constraints on thiolate ligated non-heme iron mediated dioxygen activation.

Converting triplet dioxygen into a powerful oxidant is fundamentally important to life. The study reported herein quantitatively examines the formation of a well-characterized, reactive, O2-derived thiolate ligated FeIII-superoxo using low-temperature stopped-flow kinetics. Comparison of the kinetic barriers to the formation of this species via two routes, involving either the addition of (a) O2 to [FeII(S2 Me2N3(Pr,Pr))] (1) or (b) superoxide to [FeIII(S2 Me2N3(Pr,Pr))]+ (3) is shown to provide insight into the mechanism of O2 activation. Route (b) was shown to be significantly slower, and the kinetic barrier 14.9 kJ mol-1 higher than route (a), implying that dioxygen activation involves inner-sphere, as opposed to outer sphere, electron transfer from Fe(ii). H-bond donors and ligand constraints are shown to dramatically influence O2 binding kinetics and reversibility. Dioxygen binds irreversibly to [FeII(S2 Me2N3(Pr,Pr))] (1) in tetrahydrofuran, but reversibly in methanol. Hydrogen bonding decreases the ability of the thiolate sulfur to stabilize the transition state and the FeIII-superoxo, as shown by the 10 kJ mol-1 increase in the kinetic barrier to O2 binding in methanol vs. tetrahydrofuran. Dioxygen release from [FeIII(S2 Me2N3(Pr,Pr))O2] (2) is shown to be 24 kJ mol-1 higher relative to previously reported [FeIII(SMe2N4(tren))(O2)]+ (5), the latter of which contains a more flexible ligand. These kinetic results afford an experimentally determined reaction coordinate that illustrates the influence of H-bonding and ligand constraints on the kinetic barrier to dioxygen activation an essential step in biosynthetic pathways critical to life.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

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

Methods used to determine the structure of the oxygenase component of naphthalene 1,2 dioxygenase.

Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.