Unusual catalytic strategy by non-heme Fe(ii)/2-oxoglutarate-dependent aspartyl hydroxylase AspH.

Biocatalytic C-H oxidation reactions are of important synthetic utility, provide a sustainable route for selective synthesis of important organic molecules, and are an integral part of fundamental cell processes. The multidomain non-heme Fe(ii)/2-oxoglutarate (2OG) dependent oxygenase AspH catalyzes stereoselective (3R)-hydroxylation of aspartyl- and asparaginyl-residues. Unusually, compared to other 2OG hydroxylases, crystallography has shown that AspH lacks the carboxylate residue of the characteristic two-His-one-Asp/Glu Fe-binding triad. Instead, AspH has a water molecule that coordinates Fe(ii) in the coordination position usually occupied by the Asp/Glu carboxylate. Molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies reveal that the iron coordinating water is stabilized by hydrogen bonding with a second coordination sphere (SCS) carboxylate residue Asp721, an arrangement that helps maintain the six coordinated Fe(ii) distorted octahedral coordination geometry and enable catalysis. AspH catalysis follows a dioxygen activation-hydrogen atom transfer (HAT)-rebound hydroxylation mechanism, unusually exhibiting higher activation energy for rebound hydroxylation than for HAT, indicating that the rebound step may be rate-limiting. The HAT step, along with substrate positioning modulated by the non-covalent interactions with SCS residues (Arg688, Arg686, Lys666, Asp721, and Gln664), are essential in determining stereoselectivity, which likely proceeds with retention of configuration. The tetratricopeptide repeat (TPR) domain of AspH influences substrate binding and manifests dynamic motions during catalysis, an observation of interest with respect to other 2OG oxygenases with TPR domains. The results provide unique insights into how non-heme Fe(ii) oxygenases can effectively catalyze stereoselective hydroxylation using only two enzyme-derived Fe-ligating residues, potentially guiding enzyme engineering for stereoselective biocatalysis, thus advancing the development of non-heme Fe(ii) based biomimetic C-H oxidation catalysts, and supporting the proposal that the 2OG oxygenase superfamily may be larger than once perceived.

Catalytic Mechanism of Collagen Hydrolysis by Zinc(II)-Dependent Matrix Metalloproteinase-1.

Human matrix metalloproteinase-1 (MMP-1) is a zinc(II)-dependent enzyme that catalyzes collagenolysis. Despite the availability of extensive experimental data, the mechanism of MMP-1-catalyzed collagenolysis remains poorly understood due to the lack of experimental structure of a catalytically productive enzyme-substrate complex of MMP-1. In this study, we apply molecular dynamics and combined quantum mechanics/molecular mechanics to reveal the reaction mechanism of MMP-1 based on a computationally modeled structure of the catalytically competent complex of MMP-1 that contains a large triple-helical peptide substrate. Our proposed mechanism involves the participation of an auxiliary (second) water molecule (wat2) in addition to the zinc(II)-coordinated water (wat1). The reaction initiates through a proton transfer to Glu219, followed by a nucleophilic attack by a zinc(II)-coordinated hydroxide anion nucleophile at the carbonyl carbon of the scissile bond, leading to the formation of a tetrahedral intermediate (IM2). The process continues with a hydrogen-bond rearrangement to facilitate proton transfer from wat2 to the amide nitrogen of the scissile bond and, finally, C-N bond cleavage. The calculations indicate that the rate-determining step is the water-mediated nucleophilic attack with an activation energy barrier of 22.3 kcal/mol. Furthermore, the calculations show that the hydrogen-bond rearrangement/proton-transfer step can proceed in a consecutive or concerted manner, depending on the conformation of the tetrahedral intermediate, with the consecutive mechanism being energetically preferable. Overall, the study reveals the crucial role of a second water molecule and the dynamics for effective MMP-1-catalyzed collagenolysis.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

The front cover artwork is provided by Dr. Karabencheva-Christova's group at Michigan Technological University. The images show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. Read the full text of the Research Article at 10.1002/cphc.202200649.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

How Human TET2 Enzyme Catalyzes the Oxidation of Unnatural Cytosine Modifications in Double-Stranded DNA.

Methylation of cytosine bases is strongly linked to gene expression, imprinting, aging, and carcinogenesis. The Ten-eleven translocation (TET) family of enzymes, which are Fe(II)/2-oxoglutarate (2OG)-dependent enzymes, employ Fe(IV)=O species to dealkylate the lesioned bases to an unmodified cytosine. Recently, it has been shown that the TET2 enzyme can catalyze promiscuously DNA substrates containing unnatural alkylated cytosine. Such unnatural substrates of TET can be used as direct probes for measuring the TET activity or capturing TET from cellular samples. Herein, we studied the catalytic mechanisms during the oxidation of the unnatural C5-position modifications (5-ethylcytosine (5eC), 5-vinylcytosine (5vC) and 5-ethynylcytosine (5eyC)) and the demethylation of N4-methylated lesions (4-methylcytosine (4mC) and 4,4-dimethylcytosine(4dmC)) of the cytosine base by the TET2 enzyme using molecular dynamics (MD) and combined quantum mechanics and molecular mechanics (QM/MM) computational approaches. The results reveal that the chemical nature of the alkylation of the double-stranded (ds) DNA substrates induces distinct changes in the interactions in the binding site, the second coordination sphere, and long-range correlated motions of the ES complexes. The rate-determining hydrogen atom transfer (HAT) is faster in N4-methyl substituent substrates than in the C5-alkylations. Importantly, the calculations show the preference of hydroxylation over desaturation in both 5eC and 5vC substrates. The studies elucidate the post-hydroxylation rearrangements of the hydroxylated intermediates of 5eyC and 5vC to ketene and 5-formylmethylcytosine (5fmC), respectively, and hydrolysis of hemiaminal intermediate of 4mC to formaldehyde and unmodified cytosine proceed exclusively in aqueous solution outside of the enzyme environment. Overall, the studies show that the chemical nature of the unnatural alkylated cytosine substrates exercises distinct effects on the binding interactions, reaction mechanism, and dynamics of TET2.

Can Second Coordination Sphere and Long-Range Interactions Modulate Hydrogen Atom Transfer in a Non-Heme Fe(II)-Dependent Histone Demethylase?

Fe(II)-dependent oxygenases employ hydrogen atom transfer (HAT) to produce a myriad of products. Understanding how such enzymes use dynamic processes beyond the immediate vicinity of the active site to control the selectivity and efficiency of HAT is important for metalloenzyme engineering; however, obtaining such knowledge by experiments is challenging. This study develops a computational framework for identifying second coordination sphere (SCS) and especially long-range (LR) residues relevant for catalysis through dynamic cross-correlation analysis (DCCA) using the human histone demethylase PHF8 (KDM7B) as a model oxygenase. Furthermore, the study explores the mechanistic pathways of influence of the SCS and LR residues on the HAT reaction. To demonstrate the plausibility of the approach, we investigated the effect of a PHF8 F279S clinical mutation associated with X-linked intellectual disability, which has been experimentally shown to ablate PHF8-catalyzed demethylation. In agreement, the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies showed a change in the H31-14K9me2 substrate orientation and an increased HAT barrier. We systematically analyzed the pathways by which the identified SCS and LR residues may influence HAT by exploring changes in H3K9me2 substrate orientation, interdomain correlated motions, HAT transition state stabilization, reaction energetics, electron transfer mechanism, and alterations in the intrinsic electric field of PHF8. Importantly, SCS and LR variations decrease key motions of α9-α12 of the JmjC domain toward the Fe(IV)-center that are associated with tighter binding of the H31-14K9me2 substrate. SCS and LR residues alter the intrinsic electric field of the enzyme along the reaction coordinate and change the individual energetic contributions of residues toward TS stabilization. The overall results suggest that DCCA can indeed identify non-active-site residues relevant for catalysis. The substitutions of such dynamically correlated residues might be used as a tool to tune HAT in non-heme Fe(II)- and 2OG-dependent enzymes.

A synergy between the catalytic and structural Zn(II) ions and the enzyme and substrate dynamics underlies the structure-function relationships of matrix metalloproteinase collagenolysis.

Matrix metalloproteinases (MMPs) are Zn(II) dependent endopeptidases involved in the degradation of collagen. Unbalanced collagen breakdown results in numerous pathological conditions, including cardiovascular and neurodegenerative diseases and tumor growth and invasion. Matrix metalloproteinase-1 (MMP-1) is a member of the MMPs family. The enzyme contains catalytic and structural Zn(II) ions. Despite many studies on the enzyme, there is little known about the synergy between the two Zn(II) metal ions and the enzyme and substrate dynamics in MMP-1 structure-function relationships. We performed a computational study of the MMP-1•triple-helical peptide (THP) enzyme•substrate complex to provide this missing insight. Our results revealed Zn(II) ions' importance in modulating the long-range correlated motions in the MMP-1•THP complex. Overall, our results reveal the importance of the catalytic Zn(II) and the role of the structural Zn(II) ion in preserving the integrity of the enzyme active site and the overall enzyme-substrate complex synergy with the dynamics of the enzyme and the substrate. Notably, both Zn(II) sites participate in diverse networks of long-range correlated motions that involve the CAT and HPX domains and the THP substrate, thus exercising a complex role in the stability and functionality of the MMP-1•THP complex. Both the Zn(II) ions have a distinct impact on the structural stability and dynamics of the MMP-1•THP complex. The study shifts the paradigm from the "local role" of the Zn(II) ions with knowledge about their essential role in the long-range dynamics and stability of the overall enzyme•substrate (ES) complex.

Effects of the Nature of the Metal Ion, Protein and Substrate on the Catalytic Center in Matrix Metalloproteinase-1: Insights from Multilevel MD, QM/MM and QM Studies.

Matrix metalloproteinase-1 (MMP-1) is a Zn(II) dependent endopeptidase involved in the degradation of collagen, the most abundant structural protein in the extracellular matrix of connective tissues and the human body. Herein we performed a multilevel computational analysis including molecular dynamics (MD), combined quantum mechanics/molecular mechanics (QM/MM), and quantum mechanics (QM) calculations to characterize the structure and geometry of the catalytic Zn(II) within the MMP-1 protein environment in comparison to crystallographic and spectroscopic data. The substrate's removal fine-tuned impact on the conformational dynamics and geometry of the catalytic Zn(II) center was also explored. Finally, the study examined the effect of substituting catalytic Zn(II) by Co(II) on the overall structure and dynamics of the MMP-1 THP complex and specifically on the geometry of the catalytic metal center. Overall our QM/MM and QM studies were in good agreement with the MM description of the Zn(II) centers in the MD simulations.

Role of Structural Dynamics in Selectivity and Mechanism of Non-heme Fe(II) and 2-Oxoglutarate-Dependent Oxygenases Involved in DNA Repair.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic ß-hairpin, are involved in substrate binding in AlkBH2-dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2-dsDNA and AlkB-dsDNA; in AlkB-ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein-DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism.

Catalysis by the Non-Heme Iron(II) Histone Demethylase PHF8 Involves Iron Center Rearrangement and Conformational Modulation of Substrate Orientation.

PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-Nε-methylated K9 residue of histone H3. Altered PHF8 activity is linked to genetic diseases and cancer; thus, it is an interesting target for epigenetic modulation. We describe the use of combined quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of PHF8, including dioxygen activation, 2OG binding modes, and substrate demethylation steps. A PHF8 crystal structure manifests the 2OG C-1 carboxylate bound to iron in a nonproductive orientation, i.e., trans to His247. A ferryl-oxo intermediate formed by activating dioxygen bound to the vacant site in this complex would be nonproductive, i.e., "off-line" with respect to reaction with Nε-methylated K9. We show rearrangement of the "off-line" ferryl-oxo intermediate to a productive "in-line" geometry via a solvent exchange reaction (called "ferryl-flip") is energetically unfavorable. The calculations imply that movement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis proceeds with a low barrier, suggesting that two possible 2OG C-1 carboxylate geometries can coexist at room temperature. We explored alternative mechanisms for hydrogen atom transfer and show that second sphere interactions orient the Nε-methylated lysine in a conformation where hydrogen abstraction from a methyl C-H bond is energetically more favorable than hydrogen abstraction from the N-H bond of the protonated Nε-methyl group. Using multiple HAT reaction path calculations, we demonstrate the crucial role of conformational flexibility in effective hydrogen transfer. Subsequent hydroxylation occurs through a rebound mechanism, which is energetically preferred compared to desaturation, due to second sphere interactions. The overall mechanistic insights reveal the crucial role of iron-center rearrangement, second sphere interactions, and conformational flexibility in PHF8 catalysis and provide knowledge useful for the design of mechanism-based PHF8 inhibitors.

Catalysis by the JmjC histone demethylase KDM4A integrates substrate dynamics, correlated motions and molecular orbital control.

The N ε-methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(ii) and 2-oxoglutarate (2OG) -dependent JmjC domain enzymes are the largest family of histone N ε-methyl lysine demethylases (KDMs). The human KDM4 subfamily of JmjC KDMs is linked with multiple cancers and some of its members are medicinal chemistry targets. We describe the use of combined molecular dynamics (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) methods to study the mechanism of KDM4A, which catalyzes demethylation of both tri- and di-methylated forms of histone H3 at K9 and K36. The results show that the oxygen activation at the active site of KDM4A is optimized towards the generation of the reactive Fe(iv)-oxo intermediate. Factors including the substrate binding mode, correlated motions of the protein and histone substrates, and molecular orbital control synergistically contribute to the reactivity of the Fe(iv)-oxo intermediate. In silico substitutions were performed to investigate the roles of residues (Lys241, Tyr177, and Asn290) in substrate orientation. The Lys241Ala substitution abolishes activity due to altered substrate orientation consistent with reported experimental studies. Calculations with a macrocyclic peptide substrate analogue reveal that induced conformational changes/correlated motions in KDM4A are sequence-specific in a manner that influences substrate binding affinity. Second sphere residues, such as Ser288 and Thr289, may contribute to KDM4A catalysis by correlated motions with active site residues. Residues that stabilize key intermediates, and which are predicted to be involved in correlated motions with other residues in the second sphere and beyond, are shown to be different in KDM4A compared to those in another JmjC KDM (PHF8), which acts on H3K9 di- and mono-methylated forms, suggesting that allosteric type inhibition is of interest from the perspective of developing selective JmjC KDM inhibitors.

Structure-function relationships in KDM7 histone demethylases.

The demethylation of lysine residues of histone proteins is a key epigenetic mechanism in cells. The enzymes that catalyze these processes are called histone demethylases (KDMs). The largest family of KDMs is the Jumonji C (JmjC) domain-containing enzymes; these includes KDM2-7 subfamily of enzymes. The JmjC proteins are Fe(II) and 2-Oxoglutarate (2OG) - dependent dioxygenases that couple substrate oxidation to decarboxylation of 2OG to form succinate and CO2. The KDM7 subfamily of enzymes - PHF8 (KDM7B) and KIAA1718 (KDM7A) are human JmjC 2OG-dependent Nε-methyl lysine demethylases and are involved in demethylation of lysine residues in histones such as H3K27me2/1, H3K9me2/1 and H4K20me1. These enzymes are involved in multiple pathologic processes, including cancers and mental retardation. In this chapter, we present the current state of the art in the structural, biochemical and computational studies of KDM7 enzymes.

Conformational Dynamics Underlies Different Functions of Human KDM7 Histone Demethylases.

The human KDM7 subfamily histone H3 Nϵ-methyl lysine demethylases PHF8 (KDM7B) and KIAA1718 (KDM7A) have different substrate selectivities and are linked to genetic diseases and cancer. We describe experimentally based computational studies revealing that flexibility of the region linking the PHD finger and JmjC domains in PHF8 and KIAA1718 regulates interdomain interactions, the nature of correlated motions, and ultimately H3 binding and demethylation site selectivity. F279S an X-linked mental retardation mutation in PHF8 is involved in correlated motions with the iron ligands and second sphere residues. The calculations reveal key roles of a flexible protein environment in productive formation of enzyme-substrate complexes and suggest targeting the flexible KDM7 linker region is of interest from a medicinal chemistry perspective.

Conformational flexibility influences structure-function relationships in nucleic acid N-methyl demethylases.

N-Methylation of DNA/RNA bases can be regulatory or damaging and is linked to diseases including cancer and genetic disorders. Bacterial AlkB and human FTO are DNA/RNA demethylases belonging to the Fe(ii) and 2-oxoglutarate oxygenase superfamily. Modelling studies reveal conformational dynamics influence structure-function relationships of AlkB and FTO, e.g. why 1-methyladenine is a better substrate for AlkB than 6-methyladenine. Simulations show that the flexibility of the double stranded DNA substrate in AlkB influences correlated motions, including between the core jelly-roll fold and an active site loop involved in substrate binding. The FTO N- and C-terminal domains move in respect to one another in a manner likely important for substrate binding. Substitutions, including clinically observed ones, influencing catalysis contribute to the network of correlated motions in AlkB and FTO. Overall, the calculations highlight the importance of the overall protein environment and its flexibility to the geometry of the reactant complexes.

Combined Quantum Mechanics and Molecular Mechanics Studies of Enzymatic Reaction Mechanisms.

The combined quantum mechanics/molecular mechanics (QM/MM) methods have become a valuable tool in computational biochemistry and received versatile applications for studying the reaction mechanisms of enzymes. The approach combines the calculations of the electronic structure of the active site by QM, with modeling of the protein environment using MM force field, which allows the long-range electrostatics and steric effects on the enzyme reactivity to be accounted for. In this review, we review some key theoretical and computational aspects of the method and we also present some applications to particular enzymatic reactions such as tryptophan-7-halogenase, cyclooxygenase-1, and the epidermal growth factor receptor.

Integrating molecular probes and molecular dynamics to reveal binding modes of GLUT5 activatory and inhibitory ligands.

Fructose transporter GLUT5 is characterized by unusual substrate specificity and is linked to a variety of metabolic disorders. A series of high-affinity GLUT5-specific sugar-based probes - ManCous - have been very recently described and efficiently used as reporters of GLUT5 activity in cells. Here we present several 1 microsecond molecular dynamics (MD) simulations of GLUT5 and its complexes with fructose and two different ManCou probes, in a solvated cell membrane environment. These simulations show key molecular interactions within GLUT5 that promote the passage of the substrate through vs. blockage of the transport mechanism. Identification of these specific interactions and their long-range effects on GLUT5 structure and dynamics provides an essential basis for the future development of GLUT5-specific inhibitors.

Structural Insights from Molecular Dynamics Simulations of Tryptophan 7-Halogenase and Tryptophan 5-Halogenase.

Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenases-PrnA and PyrH-was investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site.

Conformational Dynamics of Matrix Metalloproteinase-1·Triple-Helical Peptide Complexes.

Matrix metalloproteinase-1 (MMP-1) is a zinc-dependent protease that catalyzes hydrolysis of interstitial collagens. A previously reported X-ray crystallographic structure revealed specific interactions between a triple-helical peptide (THP) model of interstitial collagen and the hemopexin-like (HPX) and catalytic (CAT) domains of MMP-1. An NMR-based structure of MMP-1 in a complex with a different THP was also solved, where docking was used to model the MMP-1·THP interactions and develop a mechanism for the early stages of collagenolysis. To provide greater insight into and reveal specific details of the collagenolytic mechanism, molecular dynamics (MD) studies of the MMP-1·THP NMR-derived and X-ray crystallographic complexes were performed and compared. The "open/extended" conformation of the NMR-derived MMP-1·THP complex was found to lead to a catalytically productive complex. The X-ray crystallographic MMP-1·THP complex was initially in a "closed/collapsed" conformation, and did not yield a productive complex. The NMR-derived structure of the MMP-1·THP complex possessed many more atomistic interactions between MMP-1 and the THP compared with the X-ray crystallographic structure of the MMP-1·THP complex, and also had greater participation of MMP-1 in the local unwinding/destabilization of the THP. The atomistic interactions support the favorable energetics of the initial step of collagenolysis originating from the NMR-derived MMP-1·THP complex structure.

Mechanistic Insights into the Reaction of Chlorination of Tryptophan Catalyzed by Tryptophan 7-Halogenase.

Tryptophan 7-halogenase catalyzes chlorination of free tryptophan to 7-chlorotryptophan, which is the first step in the antibiotic pyrrolnitrin biosynthesis. Many biologically and pharmaceutically active natural products contain chlorine and thus, an understanding of the mechanism of its introduction into organic molecules is important. Whilst enzyme-catalyzed chlorination is accomplished with ease, it remains a difficult task for the chemists. Therefore, utilizing enzymes in the synthesis of chlorinated organic compounds is important, and providing atomistic mechanistic insights about the reaction mechanism of tryptophan 7-halogenase is vital and timely. In this work, we examined a mechanism for the reaction of tryptophan chlorination, performed by tryptophan 7-halogenase, by calculating potential energy and free energy surfaces using two different Combined Quantum Mechanical/Molecular Mechanical (QM/MM) methods both employing Density Functional Theory (DFT) for the QM region. Both computational strategies agree on the nature of the rate-limiting step and provided close results for the reaction barriers of the two reaction steps. The calculations for both the potential energy and the free energy profiles showed very similar geometric features and hydrogen bonding interactions for the characterized stationary points.