Nonheme Iron Halogenases Research Articles

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.

Aliphatic C[sbnd]H azidation by Mn based mimics of α-ketoglutarate dependent enzymes

α-Ketoglutarate dependent nonheme halogenases catalyze regioselective functionalization of aliphatic CH bonds via H abstraction by the ferryl site of the [X−FeIV=O] intermediate, followed by radical rebound to an adjacent halogen ligand X at the same Fe center. Herein we report biomimetic nonheme Mn complexes, possessing two labile cis-α coordination sites and capable of catalyzing the enantioselective azidation of C(sp3)−H groups. Proper ligand environment of the active sites imposes steric constraints on HO−Mn rebound, thus accounting for preferential C-radical rebound to the adjacent N3−Mn ligand. Such mechanism of selectivity control is analogous to that operating in the prototypical α-ketoglutarate-dependent nonheme iron halogenases.

Why Nonheme Iron Halogenases Do Not Fluorinate C-H Bonds: A Computational Investigation.

Selective halogenation is necessary for a range of fine chemical applications, including the development of therapeutic drugs. While synthetic processes to achieve C-H halogenation require harsh conditions, enzymes such as nonheme iron halogenases carry out some types of C-H halogenation, i.e., chlorination or bromination, with ease, while others, i.e., fluorination, have never been observed in natural or engineered nonheme iron enzymes. Using density functional theory and correlated wave function theory, we investigate the differences in structural and energetic preferences of the smaller fluoride and the larger chloride or bromide intermediates throughout the catalytic cycle. Although we find that the energetics of rate-limiting hydrogen atom transfer are not strongly impacted by fluoride substitution, the higher barriers observed during the radical rebound reaction for fluoride relative to chloride and bromide contribute to the difficulty of C-H fluorination. We also investigate the possibility of isomerization playing a role in differences in reaction selectivity, and our calculations reveal crucial differences in terms of isomer energetics of the key ferryl intermediate between fluoride and chloride/bromide intermediates. While formation of monodentate isomers believed to be involved in selective catalysis is shown for chloride and bromide intermediates, we find that formation of the fluoride monodentate intermediate is not possible in our calculations, which lack additional stabilizing interactions with the greater protein environment. Furthermore, the shorter Fe-F bonds are found to increase isomerization reaction barriers, suggesting that incorporation of residues that form a halogen bond with F and elongate Fe-F bonds could make selective C-H fluorination possible in nonheme iron halogenases. Our work highlights the differences between the fluoride and chloride/bromide intermediates and suggests potential steps toward engineering nonheme iron halogenases to enable selective C-H fluorination.

Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

Mechanistic Study of Chemoselectivity for Carbon Radical Hydroxylation versus Chlorination with FeIII (OH)(Cl) Complexes.

The FeIII (OH)(Cl) complex resembles the key intermediate proposed for the non-heme iron halogenases. Goldberg and co-workers reported that the FeIII (OH)(Cl) RC reacts with triphenylmethyl radical 1 to give an exclusive hydroxylation product. To understand the chemoselectivity of the reaction of RC with 1, density functional theory (DFT) calculations have been conducted. From RC, the competing pathways were identified as the OH-transfer, Cl-transfer, and isomerization pathways. The direct Cl-transfer is more favorable than direct OH-transfer by 2.8 kcal/mol. The hydrogen bonding interactions between the hydroxyl group and the pendent amine ligand impede the direct OH-transfer from RC. Compared with the direct Cl-transfer pathway, the isomerization pathways require lower barriers. In isomer RCiso2 , the equatorial hydroxyl group, which has smaller diabatic bond dissociation energy, prefers to transfer to form the hydroxylation product. In FeIII (Cl)2 RC2 and RC2iso , the equatorial chloride group also prefers to transfer to give the chlorination product.

Mechanistic Insights into Substrate Positioning That Distinguish Non-heme Fe(II)/α-Ketoglutarate-Dependent Halogenases and Hydroxylases

Non-heme iron halogenases and hydroxylases activate inert C–H bonds to selectively catalyze the functionalization of diverse biological products under physiological conditions. To better understand the differences in substrate positioning key to their divergent reactivities, we compiled available crystallographic and spectroscopic data, which revealed that hydroxylases prefer an acute oxo–Fe–H target angle while halogenases prefer a more obtuse angle. With molecular dynamics simulations guided by this experimental information, we simulated the representative hydroxylases TauD and VioC and the halogenases BesD and WelO5 with both acute and obtuse harmonic restraints. We identified key substrate interaction partners that maintain the angle of approach in the respective enzymes, such as Asp94 in TauD and His127 in BesD. Moreover, our simulations reveal that the protein environment in halogenases prevents the sampling of acute angles observed in hydroxylases and vice versa. To validate these classical observations, we optimized the structure with large-scale quantum mechanical (QM) simulations and confirmed that QM-derived substrate–enzyme hydrogen bond strengths were higher in the native configurations. We computed reaction barriers for the rate-limiting hydrogen atom transfer step and found them to be slightly lower from an acute angle regardless of the enzyme–substrate complex. Analysis of the halogenase reaction coordinate reveals the formation of hydrogen bonding networks between the Fe(III)-hydroxyl, monodentate succinate, and a member of the second coordination sphere that may inhibit the hydroxyl rebound.

Structural Insight into the Catalytic Mechanism of Non-Heme Iron Halogenase AdaV in 2′-Chloropentostatin Biosynthesis

Non-heme Fe2+ and 2-oxoglutarate (Fe2+/2OG)-dependent halogenases are a promising platform for biocatalytic halogenations owing to their ability to functionalize unactivated sp3 C–H bonds. To date, however, relatively few Fe2+/2OG-dependent halogenases have been identified that act on small stand-alone molecules. AdaV, a member of the carrier-protein-independent halogenases, selectively modifies the unactivated C2′ of the dAMP in 2′-chloropentostatin biosynthesis. In this study, we report the X-ray crystallographic structures of the AdaV complex with its substrate and various AdaV variants. The combined crystallographic and biochemical data clarify the molecular mechanism of AdaV for its substrate specificity and stereoselectivity. Moreover, we have engineered the AdaVQ203A/AdaVV269A variant to produce a mixture of halogenated and hydroxylated products and further engineered AdaVQ203A&V269A&G196D/E variants to merely keep hydroxylation activity. Remarkably, we have also proposed a dual-controlling mechanism for AdaV catalysis, in which G196 plays an important role in halogenation by creating an iron coordination site for chloride binding, while Q203&V269 serve to orient the Fe(III)-OH intermediate to constrain the oxygen rebounding onto the radical substrate. These results greatly expand the enzymatic repertoire regarding halogenated natural product biosynthesis and open the way for the rational and rapid discovery of more AdaV-related enzymes as toolkits for further synthetic biology uses.

Role of Secondary Coordination Sphere Residues in Halogenation Catalysis of Non-heme Iron Enzymes

Chemo- and regio-selective catalysis of the C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-Oxoglutarate (2OG)-dependent non-heme iron halogenases catalyze selective chlorination/bromination of C–H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well understood. In this work, we show that specific positioning of redox-active tyrosine residues in the SCS of non-heme iron halogenases has a huge impact on their structure, function, and reactivity. We discover that a tyrosine residue (F121Y) rationally incorporated to hydrogen bond to iron’s chloride ligand in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies demonstrate that DOPA modification in SyrB2 renders the enzyme non-functional. Bioinformatic analysis suggests that SyrB2-like halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Furthermore, molecular dynamics simulations in tandem with experimental demonstration of DOPA incorporation exclusively at position 121 enables us to uniquely identify that an axial-chloro haloferryl isomer is operant in SyrB2. We also identify conserved redox-inactive residues in the SCS of other 2OG-dependent non-heme iron halogenases to avoid DOPA-like unproductive oxidations. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases and will have significant implications toward the design of small-molecule and protein-based halogenation catalysts.

What Drives Radical Halogenation versus Hydroxylation in Mononuclear Nonheme Iron Complexes? A Combined Experimental and Computational Study.

Nonheme iron halogenases are unique enzymes in nature that selectively activate an aliphatic C–H bond of a substrate to convert it into C–X (X = Cl/Br, but not F/I). It is proposed that they generate an FeIII(OH)(X) intermediate in their catalytic cycle. The analogous FeIII(OH) intermediate in nonheme iron hydroxylases transfers OH• to give alcohol product, whereas the halogenases transfer X• to the carbon radical substrate. There remains significant debate regarding what factors control their remarkable selectivity of the halogenases. The reactivity of the complexes FeIII(BNPAPh2O)(OH)(X) (X = Cl, Br) with a secondary carbon radical (R•) is described. It is found that X• transfer occurs with a secondary carbon radical, as opposed to OH• transfer with tertiary radicals. Comprehensive computational studies involving density functional theory were carried out to examine the possible origins of this selectivity. The calculations reproduce the experimental findings, which indicate that halogen transfer is not observed for the tertiary radicals because of a nonproductive equilibrium that results from the endergonic nature of these reactions, despite a potentially lower reaction barrier for the halogenation pathway. In contrast, halogen transfer is favored for secondary carbon radicals, for which the halogenated product complex is thermodynamically more stable than the reactant complex. These results are rationalized by considering the relative strengths of the C–X bonds that are formed for tertiary versus secondary carbon centers. The computational analysis also shows that the reaction barrier for halogen transfer is significantly dependent on secondary coordination sphere effects, including steric and H-bonding interactions.

Exploring the Biocatalytic Potential of Fe/α-Ketoglutarate-Dependent Halogenases.

Freestanding Fe/α-ketoglutarate-dependent halogenases are oxidoreductases that catalyze the installation of halogen atoms into unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. Since their discovery in 2014, a small number of indole alkaloid and amino acid halogenases have been identified and characterized. First enzyme engineering examples suggest that the accessible substrate range of these enzymes may be expanded through the use of rational enzyme design and directed evolution. Structural investigations of non-heme iron halogenases acting on freestanding as well as tethered substrates are beginning to inform about the principles of the underlying halogenation mechanism.

Determining the Inherent Selectivity for Carbon Radical Hydroxylation versus Halogenation with FeIII(OH)(X) Complexes: Relevance to the Rebound Step in Non-heme Iron Halogenases.

The first structural models of the proposed cis-FeIII(OH)(halide) intermediate in the non-heme iron halogenases were synthesized and examined for their inherent reactivity with tertiary carbon radicals. Selective hydroxylation occurs for these cis-FeIII(OH)(X) (X = Cl, Br) complexes in a radical rebound-like process. In contrast, a cis-FeIII(Cl)2 complex reacts with carbon radicals to give halogenation. These results are discussed in terms of the inherent reactivity of the analogous rebound intermediate in both enzymes and related catalysts.

Chlorination versus hydroxylation selectivity mediated by the non-heme iron halogenase WelO5.

The selectivity of halogenation versus hydroxylation in α-KG de-pendent halogenases is vital to their function and has been widely studied, particularly using the halogenase SyrB2 as a model. WelO5, a new member of α-KG dependent halogenases, catalyzes the chlorination of 12-epi-fischerindole U in the welwitindolinone biosynthetic pathway. Herein, we give a detailed insight into the selectivity of WelO5 through combined quantum mechanical/molecular mechanical (QM/MM) calculations for the whole catalytic cycle. O2 activation leads to a Fe(iv)[double bond, length as m-dash]O moiety which adopts an equatorial conformation (in the plane consisting of His164, chloride and Fe atom), in contrast to axial conformation (perpendicular to the plane). Key to the conformational selectivity is a serine residue (Ser189) in the equatorial plane, that brings the precursor of the Fe(iv)[double bond, length as m-dash]O intermediate (a Fe(ii)-peracid complex) to the equatorial conformation through hydrogen bonding. Hydrogen abstraction of the substrate by the equatorial Fe(iv)[double bond, length as m-dash]O leads to a five-coordinated HO-Fe(iii)-Cl complex, where the hydroxyl ligand is still equatorial and thus relatively far from the substrate radical in the axial direction compared to the chloride ligand. This smoothly explains the extremely high selectivity of chlorination in WelO5 and provides a microscopic explanation for the experimental finding that S189A WelO5 ceases to display any chlorination selectivity versus hydroxylation. Notably, although Ser189 is vital for the selectivity of the enzyme, it is not part of the substrate binding pocket. Therefore, WelO5 serves as an excellent example how chemoselectivity can be achieved in directed evolution without the tedious redesign of the substrate binding pocket.

Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of a Pharmaceutically Relevant Compound.

Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.

The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: The Case of SyrB2/SyrB1

Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by the non-heme iron halogenase SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information on the dynamically associating protein complexes. Combining over 10 μs of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate–protein and protein–protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantetheine (PPant) delive...

Does Substrate Positioning Affect the Selectivity and Reactivity in the Hectochlorin Biosynthesis Halogenase?

In this work we present the first computational study on the hectochlorin biosynthesis enzyme HctB, which is a unique three-domain halogenase that activates non-amino acid moieties tethered to an acyl-carrier, and as such may have biotechnological relevance beyond other halogenases. We use a combination of small cluster models and full enzyme structures calculated with quantum mechanics/molecular mechanics methods. Our work reveals that the reaction is initiated with a rate-determining hydrogen atom abstraction from substrate by an iron (IV)-oxo species, which creates an iron (III)-hydroxo intermediate. In a subsequent step the reaction can bifurcate to either halogenation or hydroxylation of substrate, but substrate binding and positioning drives the reaction to optimal substrate halogenation. Furthermore, several key residues in the protein have been identified for their involvement in charge-dipole interactions and induced electric field effects. In particular, two charged second coordination sphere amino acid residues (Glu223 and Arg245) appear to influence the charge density on the Cl ligand and push the mechanism toward halogenation. Our studies, therefore, conclude that nonheme iron halogenases have a chemical structure that induces an electric field on the active site that affects the halide and iron charge distributions and enable efficient halogenation. As such, HctB is intricately designed for a substrate halogenation and operates distinctly different from other nonheme iron halogenases.

Group Transfer to an Aliphatic Bond: A Biomimetic Study Inspired by Nonheme Iron Halogenases

In this work, we predict a group-transfer reaction to an aliphatic substrate on a biomimetic nonheme iron center based on the structural and functional properties of nonheme iron halogenases. Transferring groups other than halogens to C–H bonds on the same catalytic center would improve the versatility and applicability of nonheme iron halogenases and enhance their use in biotechnology; however, few studies have been reported on this matter. Furthermore, very few biomimetic models are known that are able to transfer halogens or other groups to aliphatic C–H bonds. To gain insight into group transfer to an aliphatic C–H bond, we performed a detailed computational study on a biomimetic nonheme iron complex and studied the reactivity patterns with a model substrate (ethylbenzene). In particular, we investigated the reaction mechanisms of [FeIV(O)(TPA)X]+, TPA = tris(2-pyridylmethy1)amine, and X = Cl, NO2, N3 with ethylbenzene leading to 1-phenylethanol and 1-phenyl-1-X-ethane products. Interestingly, we find...

A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases

Enzymatic halogenation and haloperoxidation are unusual processes in biology; however, a range of halogenases and haloperoxidases exist that are able to transfer an aliphatic or aromatic C–H bond into C–Cl/C–Br. Haloperoxidases utilize hydrogen peroxide, and in a reaction with halides (Cl−/Br−), they react to form hypohalides (OCl−/OBr−) that subsequently react with substrate by halide transfer. There are three types of haloperoxidases, namely the iron-heme, nonheme vanadium, and flavin-dependent haloperoxidases that are reviewed here. In addition, there are the nonheme iron halogenases that show structural and functional similarity to the nonheme iron hydroxylases and form an iron(IV)-oxo active species from a reaction of molecular oxygen with α-ketoglutarate on an iron(II) center. They subsequently transfer a halide (Cl−/Br−) to an aliphatic C–H bond. We review the mechanism and function of nonheme iron halogenases and hydroxylases and show recent computational modelling studies of our group on the hectochlorin biosynthesis enzyme and prolyl-4-hydroxylase as examples of nonheme iron halogenases and hydroxylases. These studies have established the catalytic mechanism of these enzymes and show the importance of substrate and oxidant positioning on the stereo-, chemo- and regioselectivity of the reaction that takes place.

Aliphatic C-H Bond Halogenation by Iron(II)-α-Keto Acid Complexes and O2: Functional Mimicking of Nonheme Iron Halogenases.

α-Ketoglutarate-dependent nonheme halogenases catalyze the halogenation of aliphatic C-H bonds in the biosynthesis pathway of many natural products. An iron(IV)-oxo-halo species has been established as the active oxidant in the halogenation reactions. With an objective to emulate the function of the nonheme halogenases, two iron(II)-α-keto acid complexes, [(phdpa)Fe(BF)Cl] (1) and [(1,4-tpbd)Fe2(BF)2Cl2] (2) (where phdpa = N,N-bis(2-pyridylmethyl)aniline, 1,4-tpbd = N,N, N',N'-tetrakis(2-pyridylmethyl)benzene-1,4-diamine, and BF = benzoylformate), have been prepared. The iron complexes are capable of carrying out the oxidative halogenation of aliphatic C-H bonds using O2 as the terminal oxidant. Although the complexes are not selective toward C-H bond halogenation, they are the only examples of nonheme iron(II)-α-keto acid complexes mimicking the activity of nonheme halogenases. The dinuclear complex (2) exhibits enhanced reactivity toward C-H bond halogenation/hydroxylation.

Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2.

The ability of an FeIV═O intermediate in SyrB2 to perform chlorination versus hydroxylation was computationally evaluated for different substrates that had been studied experimentally. The π-trajectory for H atom abstraction (FeIV═O oriented perpendicular to the C-H bond of substrate) was found to lead to the S = 2 five-coordinate HO-FeIII-Cl complex with the C• of the substrate, π-oriented relative to both the Cl- and the OH- ligands. From this ferric intermediate, hydroxylation is thermodynamically favored, but chlorination is intrinsically more reactive due to the energy splitting between two key redox-active dπ* frontier molecular orbitals (FMOs). The splitting is determined by the differential ligand field effect of Cl- versus OH- on the Fe center. This makes chlorination effectively competitive with hydroxylation. Chlorination versus hydroxylation selectivity is then determined by the orientation of the substrate with respect to the HO-Fe-Cl plane that controls either the Cl- or the OH- to rebound depending on the relative π-overlap with the substrate C radical. The differential contribution of the two FMOs to chlorination versus hydroxylation selectivity in SyrB2 is related to a reaction mechanism that involves two asynchronous transfers: electron transfer from the substrate radical to the iron center followed by late ligand (Cl- or OH-) transfer to the substrate.

Electronic Structure of the Ferryl Intermediate in the α-Ketoglutarate Dependent Non-Heme Iron Halogenase SyrB2: Contributions to H Atom Abstraction Reactivity.

Low temperature magnetic circular dichroism (LT MCD) spectroscopy in combination with quantum-chemical calculations are used to define the electronic structure associated with the geometric structure of the Fe(IV)═O intermediate in SyrB2 that was previously determined by nuclear resonance vibrational spectroscopy. These studies elucidate key frontier molecular orbitals (FMOs) and their contribution to H atom abstraction reactivity. The VT MCD spectra of the enzymatic S = 2 Fe(IV)═O intermediate with Br(-) ligation contain information-rich features that largely parallel the corresponding spectra of the S = 2 model complex (TMG3tren)Fe(IV)═O (Srnec, M.; Wong, S. D.; England, J; Que, L; Solomon, E. I. Proc. Natl. Acad. Sci. USA 2012, 109, 14326-14331). However, quantitative differences are observed that correlate with π-anisotropy and oxo donor strength that perturb FMOs and affect reactivity. Due to π-anisotropy, the Fe(IV)═O active site exhibits enhanced reactivity in the direction of the substrate cavity that proceeds through a π-channel that is controlled by perpendicular orientation of the substrate C-H bond relative to the halide-Fe(IV)═O plane. Also, the increased intrinsic reactivity of the SyrB2 intermediate relative to the ferryl model complex is correlated to a higher oxyl character of the Fe(IV)═O at the transition states resulting from the weaker ligand field of the halogenase.