Enzyme Heme Research Articles

Machine-Learning Prediction of Protein Function from the Portrait of Its Intramolecular Electric Field.

We introduce a machine learning framework designed to predict enzyme functionality directly from the heterogeneous electric fields inherent to protein active sites. We apply this method to a curated data set of heme-iron oxidoreductases, spanning three enzyme classes: monooxygenases, peroxidases, and catalases. Conventional analysis, focused on simplistic, point electric fields along the Fe-O bond, is shown to be inadequate for accurate activity prediction. Our model demonstrates that the enzyme's heterogeneous 3-D electric field, alone, can accurately predict its function, without relying on additional protein-specific information. Through feature selection, we uncover key electric field components that not only validate previous studies but also underscore the crucial role of multiple components beyond the traditionally emphasized electric field along the Fe-O bond in heme enzymes. Furthermore, by integrating protein dynamics, principal component analysis, clustering, and QM/MM calculations, we reveal that while dynamic complexities in protein structures can obscure predictions, the model still retains its accuracy. This research significantly advances our understanding of how protein scaffolds possess signature electric fields tailored to their functions at the active site. Moreover, it presents a novel electrostatics-based tool to harness these signature electric fields for predicting enzyme function.

Biochemical and kinetic properties of three indoleamine 2,3-dioxygenases of Aspergillus fumigatus: mechanism of increase in the apparent Km by ascorbate.

Indoleamine 2,3-dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L-Trp) to form N-formyl-kynurenine. Similar to other heme proteins, IDO only binds to O2 when the heme iron is ferrous (FeII), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The FeII-O2 IDOs of A. fumigatus, particularly FeII-O2 IDOγ, have relatively long half-lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed-competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The FeII-O2 IDO can repeat the dioxygenase reaction as long as it reacts with L-Trp; however, substrate-free FeII-O2 IDO is converted into inactive FeIII-IDO by autoxidation. Thus, L-Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L-Trp, appears to function as a competitive (or mixed-competitive) inhibitor of IDOs.

Heme‐Enzymatic Biocatalysis for C−C or C−N Bond Formation

AbstractThe C−C or C−N bond formation is critical in the synthesis of pharmaceuticals and other value‐added products; however, traditional metal‐catalysed synthesis has brought about environmental and resource issues. A plethora of engineered heme‐dependent enzymes, such as cytochrome P450, have exhibited enormous potential in biocatalysis for C−C or C−N bond formation. With the development of computational and spectroscopic methods, the mechanisms underlying heme‐catalysed C−C or C−N bond formation have been extensively investigated. In the presence of carbene or nitrene precursor, an active iron porphyrin carbene (IPC) or iron porphyrin nitrene (IPN) is formed, which subsequently reacts with a second substrate to form new C−C or C−N bonds. Apart from the widely studied IPC/IPN‐facilitated catalytic pathway, halide‐initiated radical cyclization pathway and Cpd‐I‐catalysed diradical pathway have also been proposed. These mechanistic insights have enabled rational engineering and de novo design of heme enzymes. This review summarises recent mechanistic advances in heme enzymatic C−C or C−N bond formation and presents successful applications of mechanism‐based enzyme design. It would shed light on the development of tailored biocatalysts for the synthesis of complex but valuable industrial products.

Biomimetic Electronic Communication of Iodine Doped Single-Atom Fe Site for Highly Active and Stable Dopamine Oxidation.

Rational design of highly active and stable catalysts for dopamine oxidation is still a great challenge. Herein, inspired by the catalytic pocket of natural enzymes, an iodine (I)-doped single Fe-site catalyst (I/FeSANC) is synthesized to mimic the catalytic center of heme enzymes in both geometrical and electronic structures, aiming to enhance dopamine (DA) oxidation. Experimental studies and theoretical calculations show that electronic communication between I and FeN5 effectively modulates the electronic structure of the active site, greatly optimizing the overlap of Fe 3d and O 2p orbitals, thereby enhancing OH adsorption. In addition, the electronic communication induced by iodine doping attenuates the attack of proton hydrogen on the active center, thereby enhancing the stability of I/FeSANC. This work provides new insights into the design of highly active and stable single-atom catalysts and enhances the understanding of catalytic mechanisms for DA oxidation at the atomic scale.

Secondary Sphere Lewis Acid Activated Heme Superoxo Adducts Mimic Crucial Non‐Covalent Interactions in IDO/TDO Heme Dioxygenases

Heme enzymes play a central role in a medley of reactivities within a wide variety of crucial biological systems. Their active sites are highly decorated with pivotal evolutionarily optimized non‐covalent interactions that precisely choreograph their biological functionalities with specific regio‐, stereo‐, and chemo‐selectivities. Gaining a clear comprehension of how such weak interactions within the active sites control reactivity offers powerful information to be implemented into the design of future therapeutic agents that target these heme enzymes. To shed light on such critical details pertaining to tryptophan dioxygenating heme enzymes, this study investigates the indole dioxygenation reactivities of Lewis acid‐activated heme superoxo model systems, wherein an unprecedented kinetic behavior is revealed. In that, the activated heme superoxo adduct is observed to undergo indole dioxygenation with the intermediacy of a non‐covalently organized precursor complex, which forms prior to the rate‐limiting step of the overall reaction landscape. Spectroscopic and theoretical characterization of this precursor complex draws close parallels to the ternary complex of heme dioxygenases, which has been postulated to be of crucial importance for successful 2,3‐dioxygenative cleavage of indole moieties.

Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes

Heme-containing enzymes, critical across life's domains and promising for industrial use, face stability challenges. Despite the demand for robust industrial biocatalysts, the mechanisms underlying the thermal stability of heme enzymes remain poorly understood. Addressing this, our research utilizes a ‘keystone cofactor heme-interaction approach’ to enhance ligand binding and improve the stability of lignin peroxidase (LiP). We engineered mutants of the white-rot fungus PcLiP (Phanerochaete chrysosporium) to increase thermal stability by 8.66 °C and extend half-life by 29 times without losing catalytic efficiency at 60 °C, where typically, wild-type enzymes degrade. Molecular dynamics simulations reveal that an interlocked cofactor moiety contributes to enhanced structural stability in LiP variants. Additionally, a stability index developed from these simulations accurately predicts stabilizing mutations in other PcLiP isozymes. Using milled wood lignin, these mutants achieved triple the conversion yields at 40 °C compared to the wild type, offering insights for more sustainable white biotechnology through improved enzyme stability.

Exploring the Bonding Nature of Iron(IV)-Oxo Species through Valence Bond Calculations and Electron Density Analysis.

Compound I (Cpd I) plays a pivotal role in substrate transformations within the catalytic cycle of cytochrome P450 enzymes (P450s). A key constituent of Cpd I is the iron(IV)-oxo unit, a structural motif also found in other heme enzymes and nonheme enzymes. In this study, we performed ab initio valence bond (VB) calculations, employing the valence bond self-consistent field (VBSCF) and breathing orbital valence bond (BOVB) methods, to unveil the bonding nature of this vital "Fe(IV)═O″ unit in bioinorganic chemistry. Comparisons were drawn with the triplet O2 molecule, which shares some electronic characteristics with iron(IV)-oxo. Additionally, Cpd I models of horseradish peroxidase (HRP) and catalase (CAT) were analyzed to assess the proximal ligand effect on the electronic structure of iron(IV)-oxo. Our VB analysis underscores the significant role of noncovalent resonance effects in shaping the iron(IV)-oxo bonding. The resonance stabilization within the π and σ frameworks occurs to comparable degrees, with additional stabilization resulting from resonance between VB structures from these frameworks. Furthermore, we elucidated the substantial influence of proximal and equatorial ligands in modulating the relative significance of different VB structures. Notably, in the presence of these ligands, iron(IV)-oxo is better described as iron(III)-oxyl or iron(II)-oxygen, displaying weak covalent character but enhanced by resonance effects. Although both species exhibit diradicaloid characters, resonance stabilization in iron(IV)-oxo is weaker than in O2. Further exploration using the Laplacian of electron density shows that, unlike O2, which exhibits a charge concentration region between its two oxygen atoms, iron(IV)-oxo species display a charge depletion region.

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

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

Iron and cardiovascular health: A review.

Iron is an essential element for the biological processes of living organisms, including the production of crucial oxygen-carrying proteins, formation of heme enzymes, and playing roles in electron transfer and oxidation-reduction reactions. It plays a significant role in various cardiovascular functions, including bioenergetics, electrical activity, and programmed cell death. Minor deficiencies of iron have been found to have negative impact on cardiovascular function in patients with heart failure (HF). The contractility of human cardiomyocytes is impaired by iron deficiency (ID), which results in reduced mitochondrial function and lower energy production, ultimately leading to cardiac function impairment, contributing to significant morbidity and mortality in patients with HF. This review discusses iron homeostasis within the human body, as well as ID pathophysiology and its role in HF. Focusing on therapeutic approaches including iron supplementation and/or repletion in patients with ID and HF, comparing results from recent clinical trials. Intravenous (IV) iron therapy has shown promising results in treating ID in HF patients. Large, randomized trials and meta-analysis, like Ferinject Assessment in patients with ID and chronic HF, AFFIRM-AHF, IRONMAN, and HEART-FID have demonstrated the efficacy of IV iron supplementation with IV ferric carboxymaltose or IV ferric derisomaltose in reducing hospitalizations and improving quality of life in patients with Heart Failure with reduced ejection fraction (HFrEF), New York Heart Association (NYHA) II-III. However, survival and mortality have demonstrated no improvement during acute exacerbations of HF or in outpatient management. The potential benefits of IV iron across the entire HF spectrum and its interaction with other HF therapies remain areas of interest for further research.

Iron and ferritin deficiency in women with hypothyroidism and chronic lymphocytic thyroiditis - systematic review.

Iron is one of the essential microelements necessary for maintaining the body's homeostasis. It serves various roles, including being a crucial component in the proper structure of many enzymes and supporting the transport of oxygen and electrons. Its deficiency can lead to anaemia, which is a common clinical condition often associated with thyroid diseases. Iron deficiency is one of the most common nutritional deficiencies, and its prevalence is strongly associated with socioeconomic status. It is the primary cause of anaemia in 42% of children and 50% of women. Importantly, iron deficiency is placed among the top 5 causes of disability in women. Thyroid peroxidase (TPO) is an enzyme essential for the production of thyroid hormones, and iron is a key factor in its proper functioning. Therefore, in the case of iron deficiency, the activity of this enzyme is also reduced. Iron is also a factor that is important in epigenetic modification processes, and its deficiency may contribute to genomic changes potentially promoting the development of autoimmune thyroid diseases. Adequate supplementation in patients with Hashimoto's disease is one of the crucial elements of effective therapy. In addition to iodine, selenium, and magnesium supplementation, attention should be paid to proper iron intake. Iron is an element that is a component of the heme enzyme- thyroid peroxidase, which owes its activity to the binding of haem, and its function is the production of thyroid hormones. Iron can be delivered to the body in haem and non-haem forms. The haem form is found particularly in haemoglobin-rich red meat, but also in eggs, fish, and poultry. On the other hand, non-haem iron can be found in legumes, grains, fruits, and vegetables. Our study aimed to gather and summarise knowledge from scientific literature regarding iron deficiency anaemia and its association with hypothyroidism in women, as well as the possible mechanisms and pathogenesis of these conditions. The paper also aims to highlight that considering the high risk of iron deficiency, assessing iron status along with ferritin should be an integral part of additional diagnostic measures in cases of hypothyroidism, particularly Hashimoto's disease.

Catalytic Differences between Flavohemoglobins of Giardia intestinalis and E. coli.

The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Sorghum drought tolerance is enhanced by cerium oxide nanoparticles via stomatal regulation and osmolyte accumulation

Sorghum [Sorghum bicolor (L.) Moench] yield is limited by the coincidence of drought during its sensitive stages. The use of cerium oxide nanoparticles in agriculture is minimal despite its antioxidant properties. We hypothesize that drought-induced decreases in photosynthetic rate in sorghum may be associated with decreased tissue water content and organelle membrane damage. We aimed to quantify the impact of foliar application of nanoceria on transpiration rate, accumulation of compatible solutes, photosynthetic rate and reproductive success under drought stress in sorghum. In order to ascertain the mechanism by which nanoceria mitigate drought-induced inhibition of photosynthesis and reproductive success, experiments were undertaken in a factorial completely randomized design or split-plot design. Foliar spray of nanoceria under progressive soil drying conserved soil moisture by restricting the transpiration rate than water spray, indicating that nanoceria exerted strong stomatal control. Under drought stress at the seed development stage, foliar application of nanoceria at 25 mg L−1 significantly improved the photosynthetic rate (19%) compared to control by maintaining a higher tissue water content (18%) achieved by accumulating compatible solutes. The nanoceria-sprayed plants exhibited intact chloroplast and thylakoid membranes because of increased heme enzymes [catalase (53%) and peroxidase (45%)] activity, which helped in the reduction of hydrogen peroxide content (74%). Under drought, compared to water spray, nanoceria improved the seed-set percentage (24%) and individual seed mass (27%), eventually causing a higher seed yield. Thus, foliar application of nanoceria at 25 mg L−1 under drought can increase grain yield through increased photosynthesis and reproductive traits.

Myeloperoxidase induces monocyte migration and activation after acute myocardial infarction.

Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.

Axial Ligation Impedes Proton-Coupled Electron-Transfer Reactivity of a Synthetic Compound-I Analogue.

The nature of the axial ligand in high-valent iron-oxo heme enzyme intermediates and related synthetic catalysts is a critical structural element for controlling proton-coupled electron-transfer (PCET) reactivity of these species. Herein, we describe the generation and characterization of three new 6-coordinate, iron(IV)-oxo porphyrinoid-π-cation-radical complexes and report their PCET reactivity together with a previously published 5-coordinate analogue, FeIV(O)(TBP8Cz+•) (TBP8Cz = octakis(p-tert-butylphenyl)corrolazinato3-) (2) (Cho, K. A high-valent iron-oxo corrolazine activates C-H bonds via hydrogen-atom transfer. J. Am. Chem. Soc. 2012, 134, 7392-7399). The new complexes FeIV(O)(TBP8Cz+•)(L) (L = 1-methyl imidazole (1-MeIm) (4a), 4-dimethylaminopyridine (DMAP) (4b), cyanide (CN-)(4c)) can be generated from either oxidation of the ferric precursors or by addition of L to the Compound-I (Cpd-I) analogue at low temperatures. These complexes were characterized by UV-vis, electron paramagnetic resonance (EPR), and Mössbauer spectroscopies, and cryospray ionization mass spectrometry (CSI-MS). Kinetic studies using 4-OMe-TEMPOH as a test substrate indicate that coordination of a sixth axial ligand dramatically lowers the PCET reactivity of the Cpd-I analogue (rates up to 7000 times slower). Extensive density functional theory (DFT) calculations together with the experimental data show that the trend in reactivity with the axial ligands does not correlate with the thermodynamic driving force for these reactions or the calculated strengths of the O-H bonds being formed in the FeIV(O-H) products, pointing to non-Bell-Evans-Polanyi behavior. However, the PCET reactivity does follow a trend with the bracketed reduction potential of Cpd-I analogues and calculated electron affinities. The combined data suggest a concerted mechanism (a concerted proton electron transfer (CPET)) and an asynchronous movement of the electron/proton pair in the transition state.

Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Xenobiotics Triggering Acute Intermittent Porphyria and Their Effect on Mouse Brain Respiratory Complexes.

Heme enzyme dysfunction causes a group of diseases called porphyrias. Particularly, a decrease in porphobilinogen deaminase, involved in the third step of heme biosynthesis, leads to acute intermittent porphyria (AIP). Considering our previous works demonstrating the multiplicity of brain metabolisms affected by porphyrinogenic agents, this study aimed to elucidate whether they cause any alteration on the mitochondrial respiratory chain. The activities of respiratory chain complexes (I to IV) were measured in encephalon mitochondria of CF1 male mice receiving volatile anesthetics: isoflurane (2 mL/kg) and sevoflurane (1.5 mL/kg), ethanol (30%), allylisopropylacetamide (AIA) (350 mg/kg), and barbital (167 mg/kg). Moreover, they were compared versus animals with pathological levels of 5-aminolevulinic acid (ALA, 40 mg/kg). Complex I-III activity was induced by isoflurane and decreased by AIA, ethanol, and ALA. Complex II-III activity was increased by sevoflurane and decreased by isoflurane and AIA. Complex II activity was increased by sevoflurane and barbital and decreased by AIA, ethanol, and ALA. Complex IV activity was increased by barbital and ALA and decreased by sevoflurane. The damage to the respiratory chain by ALA could be reflecting the pathophysiological condition of patients with AIP. Better understanding the broad effect of porphyrinogenic drugs and the mechanisms acting on the onset of AIP is vital in translational medicine.

Activity assays of NnlA homologs suggest the natural product N-nitroglycine is degraded by diverse bacteria.

Linear nitramines (R-N(R')NO2; R' = H or alkyl) are toxic compounds, some with environmental relevance, while others are rare natural product nitramines. One of these natural product nitramines is N-nitroglycine (NNG), which is produced by some Streptomyces strains and exhibits antibiotic activity towards Gram-negative bacteria. An NNG degrading heme enzyme, called NnlA, has recently been discovered in the genome of Variovorax sp. strain JS1663 (Vs NnlA). Evidence is presented that NnlA and therefore, NNG degradation activity is widespread. To achieve this objective, we characterized and tested the NNG degradation activity of five Vs NnlA homologs originating from bacteria spanning several classes and isolated from geographically distinct locations. E. coli transformants containing all five homologs converted NNG to nitrite. Four of these five homologs were isolated and characterized. Each isolated homolog exhibited similar oligomerization and heme occupancy as Vs NnlA. Reduction of this heme was shown to be required for NnlA activity in each homolog, and each homolog degraded NNG to glyoxylate, NO2- and NH4+ in accordance with observations of Vs NnlA. It was also shown that NnlA cannot degrade the NNG analog 2-nitroaminoethanol. The combined data strongly suggest that NnlA enzymes specifically degrade NNG and are found in diverse bacteria and environments. These results imply that NNG is also produced in diverse environments and NnlA may act as a detoxification enzyme to protect bacteria from exposure to NNG.

Spectroelectrochemistry for determination of the redox potential in heme enzymes: Dye-decolorizing peroxidases

Dye-decolorizing peroxidases (DyPs) are heme-containing enzymes that are structurally unrelated to other peroxidases. Some DyPs show high potential for applications in biotechnology, which critically depends on the stability and redox potential (E°') of the enzyme. Here we provide a comparative analysis of UV–Vis- and surface-enhanced resonance Raman-based spectroelectrochemical methods for determination of the E°' of DyPs from two different organisms, and their variants generated targeting E°' upshift. We show that substituting the highly conserved Arginine in the distal side of the heme pocket by hydrophobic amino acid residues impacts the heme architecture and redox potential of DyPs from the two organisms in a very distinct manner. We demonstrate the advantages and drawbacks of the used spectroelectrochemical approaches, which is relevant for other heme proteins that contain multiple heme centers or spin populations.

Molecular Mechanics with Generalized Born Surface Area (MMGBSA) Calculations and Docking Studies Unravel some Antimalarial Compounds Using Heme O Synthase as Therapeutic Target

AbstractThe enzyme Heme O Synthase (HOS) is essential for producing heme A and heme O, which are critical for defense against reactive oxygen species, drug detoxification, gas synthesis, transport, and electron transport in Plasmodium species. It has become vital to discover inhibitory molecules/compounds/medicines that target the synthesis of heme due to the emergence of drug‐resistant strains of Plasmodium falciparum. Therefore, in this study, we employed molecular mechanics with Generalized Born surface area (MMGBSA) calculations and docking studies to investigate potential antimalarial compounds targeting HOS from antimalarial botanicals. Screening these compounds, we have identified 2 compounds; Meliantrol and Tamarixetin with better binding affinities (−8.4 Kcal/mol and −8.3 Kcal/mol respectively) than the current standard inhibitor(Inabenfide) of HOS (−8.0 Kcal/mol). The MMGBSA calculations provided insight into the thermodynamics of the binding process and helped identify key interactions responsible for the stability of the HOS‐ligand complex. In addition, the 2 compounds were further screened comparatively with the standard HOS inhibitor considering their protein‐ligand interaction profile and ADMET profile and these 2 selected compounds outperformed Inabenfide. Our results predict that these compounds are potential drug candidates with domiciled therapeutic functions against Malaria therefore, open doors for more experimental validations for drug development.

Application of engineered heme enzymes based on myoglobin with high peroxidase activity for efficient degradation of various emerging pollutants

Emerging pollutants in the aquatic environment from pharmaceutical and personal care products (PPCPs) attract much attention due to their environmental risk and toxicological properties, posing a threat to human health and safety. In this study, we fulfilled the direct and efficient degradation of PPCPs (i.e. diclofenac sodium, 2-mercaptobenzothiazole, paracetamol, and furosemide) using engineered H2O2-dependent heme enzymes based on myoglobin (Mb) with high peroxidase activity. Especially, the triple mutant F43Y/P88W/F138W Mb could efficiently degrade diclofenac sodium, 2-mercaptobenzothiazole, paracetamol, and furosemide with a degradation rate of ∼98.5%, ∼99%, ∼98%, and ∼91%, respectively. These activities are much higher than other reported native enzymes, such as laccase and horseradial peroxidase. As further analyzed by ESI-MS, we identified multiple degradation products of those four contaminants and thus proposed possible degradation mechanisms. This study indicates that enzyme-based degradation of emerging pollutants is a promising environmental remediation approach.