An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase.

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H2 O2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2 O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H2 O2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2 O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.

Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant.

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C-H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV-visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.

Reply to Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1''.

The high-resolution X-ray crystal structure of the ternary complex FtmOx1 ⋅ 2OG ⋅ fumitremorgin B and the catalytic mechanism were recently reported by us (DOI 10.1002/anie.202112063). In their Correspondence, Zhang, Costello, Liu et al. criticize our work in several aspects. Herein, we address these questions one by one. These structural clarifications and new computational results further support the CarC-like mechanistic model.

Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1".

In their recent Angewandte Chemie publication (doi: 10.1002/anie.202112063), Cen, Wang, Zhou et al. reported the crystal structure of a ternary complex of the non-heme iron endoperoxidase FtmOx1 (PDB entry 7ETK). The biochemical data assessed in this study were from a retracted study (doi: 10.1038/nature15519) by Zhang, Liu, Zhang et al.; no additional biochemical data were included, yet there was no discussion on the source of the biochemical data in the report by Cen, Wang, Zhou et al. Based on this new crystal structure and subsequent QM/MM-MD calculations, Cen, Wang, Zhou et al. concluded that their work provided evidence supporting the CarC-like mechanistic model for FtmOx1 catalysis. However, the authors did not accurately describe either the CarC-like model or the COX-like model, and they did not address the differences between them. Further, and contrary to their interpretations in the manuscript, the authors' data are consistent with the COX-like model once the details of the CarC-like and COX-like models have been carefully analyzed.

Combined spectroscopic and structural approaches to explore the mechanism of histidine-ligated heme-dependent aromatic oxygenases.

The emergence of histidine-ligated heme-dependent aromatic oxygenases (HDAOs) has greatly enriched heme chemistry, and more studies are required to appreciate the diversity found in His-ligated heme proteins. This chapter describes recent methods in probing the HDAO mechanisms in detail, along with the discussion on how they can benefit structure-function studies of other heme systems. The experimental details are centered on studies of TyrHs, followed by explanation of how the results obtained would advance the understanding of the specific enzyme and also HDAOs. Spectroscopic methods, namely, electronic absorption and EPR spectroscopies, and X-ray crystallography are valuable techniques commonly used to characterize the properties of the heme center and the nature of heme-based intermediate. Herein, we show that the combination of these tools are extremely powerful, not only because one can acquire electronic, magnetic, and conformational information from different phases, but also because of the advantages brought by spectroscopic characterization on crystal samples.

Coordination Dynamics of Iron is a Key Player in the Catalysis of Non-heme Enzymes.

Mononuclear nonheme iron enzymes catalyze a large variety of oxidative transformations responsible for various biosynthesis and metabolism processes. Unlike their P450 counterparts, non-heme enzymes generally possess flexible and variable coordination architecture, which can endow rich reactivity for non-heme enzymes. This Concept highlights that the coordination dynamics of iron can be a key player in controlling the activity and selectivity of non-heme enzymes. In ergothioneine synthase EgtB, the coordination switch of the sulfoxide radical species enables the efficient and selective C-S coupling reaction. In iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases, the conformational flip of ferryl-oxo intermediate can be extensively involved in selective oxidation reactions. Especially, the five-coordinate ferryl-oxo species may allow the substrate coordination via O or N atom, which may facilitate the C-O or C-N coupling reactions via stabilizing the transition states and inhibiting the unwanted hydroxylation reactions.

Caffeine Biodegradation by Cytochrome P450 1A2. What Determines the Product Distributions?

Caffeine is a natural compound found in plant seeds that after consumption by humans effects the central nervous system as well as the cardiovascular system. In general, the cytochrome P450 enzymes in the liver are involved in the biodegradation of caffeine, which gives paraxanthine, theobromine and theophylline products. There has been debate for many years why multiple products are obtained and how their distributions are determined. To this end we performed a high-level computational study using a combination of molecular dynamics and quantum mechanical approaches. A series of quantum chemical cluster models on the mechanism of caffeine activation by P450 model complexes give hydrogen atom abstraction barriers that predicts the correct ordering and statistical distribution of products. Our studies highlight that second-coordination sphere effects and thermochemical properties of the substrate determine the product distributions.

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.

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions.

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H2 O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H2 O2 complex which would be more abundant in the peroxide-driven reactions.

Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light.

Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H2 O2 ). Here, we use the T252E-CYP199A4 variant to study peroxide-driven oxidation activity by using H2 O2 and urea-hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H2 O2 in the presence of substrate that can undergo carbon-hydrogen abstraction. This peroxygenase variant could efficiently catalyse O-demethylation and an enantioselective epoxidation reaction (94 % ee). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H2 O2 generating system was used to support efficient product formation with this peroxygenase enzyme.

A comparison of the bacterial CYP51 cytochrome P450 enzymes from Mycobacterium marinum and Mycobacterium tuberculosis.

Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1.

The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owing to the lack of a reliable complex structure of FtmOx1 with fumitremorgin B. Herein we provide the X-ray crystal structure of the ternary complex FtmOx1⋅2OG⋅fumitremorgin B at a resolution of 1.22 Å. Our structures show that the binding of fumitremorgin B induces significant compression of the active pocket and that Y68 is in close proximity to C26 of the substrate. Further MD simulation and QM/MM calculations support a CarC-like mechanism, in which Y68 acts as the H atom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis.

Repurposed and artificial heme enzymes for cyclopropanation reactions.

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed.

Design and Engineering of an Efficient Peroxidase Using Myoglobin for Dye Decolorization and Lignin Bioconversion.

The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-ß-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α-Helical Metalloprotein.

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.

How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by Nδ-Methyl Histidine Affect Its Properties and Functions? A Computational Study.

Heme peroxidases have important functions in nature related to the detoxification of H2O2. They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H2O2 complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome c peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered Nδ-methyl histidine-ligated cytochrome c peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially Nδ-methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome c peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the Nδ-methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the Nδ-methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by Nδ-methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on Nδ-methyl histidine-ligated cytochrome c peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.

Bioengineering of Cytochrome P450 OleTJE: How Does Substrate Positioning Affect the Product Distributions?

The cytochromes P450 are versatile enzymes found in all forms of life. Most P450s use dioxygen on a heme center to activate substrates, but one class of P450s utilizes hydrogen peroxide instead. Within the class of P450 peroxygenases, the P450 OleTJE isozyme binds fatty acid substrates and converts them into a range of products through the α-hydroxylation, ß-hydroxylation and decarboxylation of the substrate. The latter produces hydrocarbon products and hence can be used as biofuels. The origin of these product distributions is unclear, and, as such, we decided to investigate substrate positioning in the active site and find out what the effect is on the chemoselectivity of the reaction. In this work we present a detailed computational study on the wild-type and engineered structures of P450 OleTJE using a combination of density functional theory and quantum mechanics/molecular mechanics methods. We initially explore the wild-type structure with a variety of methods and models and show that various substrate activation transition states are close in energy and hence small perturbations as through the protein may affect product distributions. We then engineered the protein by generating an in silico model of the double mutant Asn242Arg/Arg245Asn that moves the position of an active site Arg residue in the substrate-binding pocket that is known to form a salt-bridge with the substrate. The substrate activation by the iron(IV)-oxo heme cation radical species (Compound I) was again studied using quantum mechanics/molecular mechanics (QM/MM) methods. Dramatic differences in reactivity patterns, barrier heights and structure are seen, which shows the importance of correct substrate positioning in the protein and the effect of the second-coordination sphere on the selectivity and activity of enzymes.

Redox reactions of heme proteins with flavonoids.

Proteins containing heme groups perform a variety of important functions in living organisms. The heme groups are involved in catalyzing oxidation/reduction reactions, in electron transfer, and in binding small molecules, like oxygen or nitric oxide. Flavonoids, low molecular weight plant polyphenols, are ubiquitous components of human diet. They are also components of many plant extracts used in herbal medicine as well as of food supplements. Due to their relatively low reduction potential, flavonoids are prone to oxidation. This paper provides a review of redox reactions of various heme proteins, including catalase, some peroxidases, cytochrome P450, cytochrome c, myoglobin, and hemoglobin with flavonoids. Potential biological significance of these reactions is discussed, in particular when flavonoids are delivered to the body at pharmacological doses.

Recent advances in heme biocatalysis engineering.

Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L-1 . This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.

The ins and outs of iron: Escorting iron through the mammalian cytosol.

Mammalian cells contain thousands of metalloproteins and have evolved sophisticated systems for ensuring that metal cofactors are correctly assembled and delivered to their proper destinations. Equally critical in this process are the strategies to avoid the insertion of the wrong metal cofactor into apo-proteins and to avoid the damage that redox-active metals can catalyze in the cellular milieu. Iron and zinc are the most abundant metal cofactors in cells and iron cofactors include heme, iron-sulfur clusters, and mono- and dinuclear iron centers. Systems for the intracellular trafficking of iron cofactors are being characterized. This review focuses on the trafficking of ferrous iron cofactors in the cytosol of mammalian cells, a process that involves specialized iron-binding proteins, termed iron chaperones, of the poly rC-binding protein family.