Active Site Research Articles

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Alkene Isomerization Using a Heterogeneous Nickel-Hydride Catalyst.

Transition metal-catalyzed alkene isomerization is an enabling technology used to install an alkene distal to its original site. Due to their well-defined structure, homogeneous catalysts can be fine-tuned to optimize reactivity, stereoselectivity, and positional selectivity, but they often suffer from instability and nonrecyclability. Heterogeneous catalysts are generally highly robust but continue to lack active-site specificity and are challenging to rationally improve through structural modification. Known single-site heterogeneous catalysts for alkene isomerization utilize precious metals and bespoke, expensive, and synthetically intense supports. Additionally, they generally have mediocre reactivity, inspiring us to develop a heterogeneous catalyst with an active site made from readily available compounds made of Earth-abundant elements. Previous work demonstrated that a very active homogeneous catalyst is formed upon protonation of Ni[P(OEt)3]4 by H2SO4, generating a [Ni-H]+ active site. This catalyst is incredibly active, but also decomposes readily, which severely limits its utility. Herein we show that by using a solid acid (sulfated zirconia, SZO300), not only is this decomposition prevented, but high activity is maintained, improved selectivity is achieved, and a broader scope of functional groups is tolerated. Preliminary mechanistic experiments suggest that the catalytic reaction likely goes through an intermolecular, two-electron pathway. A detailed kinetic study comparing the state-of-the-art Ni and Pd isomerization catalysts reveals that the highest activity and selectivity is seen with the Ni/SZO300 system. The reactivity of Ni/SZO300, is not limited to alkene isomerization; it is also a competent catalyst for hydroalkenylation, hydroboration, and hydrosilylation, demonstrating the broad application of this heterogeneous catalyst.

Enhanced Electron Delocalization Induced by Ferromagnetic Sulfur doped C 3 N 4 Triggers Selective H 2 O 2 Production.

For the 2D metal-free carbon catalysts, the atomic coplanar architecture enables a large number of pzorbitals tooverlap laterally,thus formingπ-electron delocalization, and the delocalization degree of the central atom dominates the catalytic activity. Herein, designing sulfur-doped defect-rich graphitic carbon nitride (S-Nv-C3N4) materials as a model, we proposea strategy to promote localized electronpolarization by enhancing the ferromagnetism of ultra-thin 2D carbon nitride nanosheets.The introduction of sulfur (S)further promotes localized ferromagnetic coupling,thereby inducing long-range ferromagnetic ordering and accelerating the electron interface transport. Meanwhile, the hybridization of sulfur atoms breaks the symmetry and integrity of the unit structure, promoteselectron enrichment and stimulating electron delocalization at the active site. This optimization enhances the *OOH desorption, providing a favorable kinetic pathway for the production of hydrogen peroxide (H2O2). Consequently, S-Nv-C3N4exhibits high selectivity (>95%)and achieves a superb H2O2production rate,approaching4374.8ppm duringcontinuous electrolysisover 300-hour. According to theoreticalcalculationand in-situspectroscopy,the ortho-S configurationcan provide ferromagnetic perturbation incarbon active centers, leading to the electron delocalization, which optimizes the OOH* adsorption during the catalytic process.

Thermodynamics of Proton-Coupled Electron Transfer at Tricopper μ-Oxo/Hydroxo/Aqua Complexes.

Multicopper oxidases (MCOs) utilize a tricopper active site to reduce dioxygen to water through 4H+ 4e- proton-coupled electron transfer (PCET). Understanding the thermodynamics of PCET at a tricopper cluster is essential for elucidating how MCOs harness the oxidative power of O2 while mitigating oxidative damage. In this study, we determined the O-H bond dissociation free energies (BDFEs) and pKa values of a series of tricopper hydroxo and tricopper aqua complexes as synthetic models of the tricopper site in MCOs. Tricopper intermediates on the path of alternating electron and proton transfer (ET-PT-ET-PT-ET) have modest BDFE(O-H) values in the range of 53.0-57.1 kcal/mol. In contrast, those not on the path of ET-PT-ET-PT-ET display much higher (78.1 kcal/mol) or lower (44.7 kcal/mol) BDFE(O-H) values. Additionally, the pKa of bridging OH and OH2 motifs increase by 8-16 pKa units per oxidation state. The same oxidation state changes have a lesser impact on the pKa of N-H motif in the secondary coordination sphere, with an increase of ca. 5 pKa units per oxidation state. The steeper pKa increase of the tricopper center promotes proton transfer from the secondary coordination sphere. Overall, our study shed light on the PCET pathway least prone to decomposition, elucidating why tricopper centers are an optimal choice for promoting efficient oxygen reduction reaction.

Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM) of Piperine

In this article, we introduce a proof-of-concept strategy, Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM), to expedite the discovery of drug metabolites. The use of a bioactive natural product, piperine, that has a well-curated metabolite profile but an unpredictable computational metabolism (Biotransformer v3.0) was selected. We developed an electrochemical reaction to oxidize piperine into a range of metabolites, which were detected by LC-MS. A series of chemically plausible metabolites were predicted based on ion fragmentation patterns. These metabolites were docked into the active site of CYP3A4 using Autodock4.2. From the clustered low-energy profile of piperine in the active site, it can be inferred that the most likely metabolic position of piperine (based on intermolecular distances to the Fe-oxo active site) is the benzo[d][1,3]dioxole motif. The metabolic profile was confirmed by comparison with the literature, and the electrochemical reaction delivered plausible metabolites, vide infra, thus, demonstrating the power of the hyphenated technique of tandem electrochemical detection and computational evaluation of binding poses. Taken together, we outline a novel approach where diverse data sources are combined to predict and confirm a metabolic outcome for a bioactive structure.

Discovery of allosteric regulators with clinical potential to stabilize alpha-L-iduronidase in mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 μM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment.

Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution.

Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559-31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

Boosting potassium and sodium-ion storage performance by in-situ self-assembly of a Co-based π-d conjugated coordination polymer on graphene nanosheets

To obtain anode materials with good electrochemical performances in both potassium ion batteries (PIBs) and sodium-ion batteries (SIBs), a Co-based π-d conjugated coordination polymer (Co-HHTP) is in-situ assembled on graphene (G) nanosheets using a facile one-step solvothermal method. The resulting Co-HHTP/G composite shows improved electrochemical performances over pure Co-HHTP. After 300 cycles of using as an anode of PIBs/SIBs for the first time, the reversible specific capacity of the Co-HHTP/G composite is found to be 381/277 mAh g−1 at 100 mA g−1. After undergoing 500 cycles at 1000 mA g−1, the material's reversible specific capacities remain at 234/164 mAh g−1 for PIBs/SIBs. The improved performances of the Co-HHTP/G composite electrode are due to the incorporation of graphene. This incorporation not only reduces structural stacking and promotes active site utilization, but also increases electrical conductivity and structural stability. According to the ex-situ X-ray photoelectron spectrometer (XPS) spectra, the Co-HHTP/G's energy storage mechanism throughout the process of potassisation/depotassisation is found to be linked to the aromatic rings presented in the organic ligands.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

Comparison of the Mechanisms of Hydrolysis of Organophosphates with Good and Poor Leaving Group by Phosphotriesterase from Pseudomonas diminuta

Abstract Combined quantum mechanics/molecular mechanics approaches are used to determine the mechanisms of organophosphate hydrolysis in an active site of Pseudomonas diminuta phosphotriesterase. For a substrate with a good leaving group, the reaction proceeds through two elementary stages with low energy barriers, and a gain in energy is observed. With a poor leaving group, only the formation of an unstable reaction intermediate is possible, and hydrolysis is incomplete. A comparison of the resulting reaction mechanisms explains the experimental kinetic data, according to which the enzyme hydrolyzes only substrates with good leaving groups.

Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review

Synthetic polymers, commonly known as plastics, are currently present in all aspects of our lives. Although they are useful, they present the problem of what to do with them after their lifespan. There are currently mechanical and chemical methods to treat plastics, but these are methods that, among other disadvantages, can be expensive in terms of energy or produce polluting gases. A more environmentally friendly alternative is recycling, although this practice is not widespread. Based on the practice of the so-called circular economy, many studies are focused on the biodegradation of these polymers by enzymes. Using enzymes is a harmless method that can also generate substances with high added value. Novel and enhanced plastic-degrading enzymes have been obtained by modifying the amino acid sequence of existing ones, especially on their active site, using a wide variety of genetic approaches. Currently, many studies focus on the common aim of achieving strains with greater hydrolytic activity toward a different range of plastic polymers. Although in most cases the depolymerization rate is improved, more research is required to develop effective biodegradation strategies for plastic recycling or upcycling. This review focuses on a compilation and discussion of the most important research outcomes carried out on microbial biotechnology to degrade and recycle plastics.

High‐Density Rare‐Earth Single‐Atom‐Triggered Unconventional Transition of Adsorption Configuration on La1Pd Monatomic Alloy Metallene for Sustainable Electrocatalytic Alkynol Semi‐Hydrogenation

AbstractElectrocatalytic alkynol semi‐hydrogenation for the high‐value chemicals alkenol with mild conditions and carbon‐free emission is a potentially green and sustainable alternative to conventional thermocatalytic routes, which generally involves the design of electrocatalysts with high activity and high selectivity. Here, the rare‐earth single‐atom (Ln = La, Nd, Pr) coordinated Pd metallene (Ln1Pdene) is reported for electrocatalytic 2‐methyl‐3‐butyn‐2‐ol (MBY) semi‐hydrogenation reaction (MBY ESHR) to synthesis of 2‐methyl‐3‐buten‐2‐ol (MBE). Typically, in alkaline medium containing 0.1 m MBY, MBY conversion and MBE selectivity of La1Pdene are as high as ≈97% and ≈95%, respectively, with excellent electrocatalytic stability. Meanwhile, in situ infrared spectra reveal the conversion of MBY to MBE during the dynamic electrocatalytic process. Theoretical calculations reveal that the interaction between La single‐atom and Pd host triggers an unconventional transformation of the intermediate MBE* adsorption configuration during electrocatalytic hydrogenation, achieving the optimal desorption energy between La1Pd and target product and optimizing the reaction energy barriers to inhibit the over‐hydrogenation of MBE. Moreover, La single‐atom as the adsorption active site of the hydrogen supplier H2O effectively reduces the competition adsorption between the reactants MBY and H2O, rendering La single‐atom and Pd host as the synergistic co‐catalytic active sites to promote the electrocatalytic MBY semi‐hydrogenation reaction.

Investigating the Inhibitory Factors of Sucrose Hydrolysis in Sugar Beet Molasses with Yeast and Invertase

Sugar beet molasses is a low-value byproduct from the sugar industry. It contains significant amounts of sucrose (approx. 50% (w/w)), which can be used for many different applications, for example, as feedstock for the production of fuel (as ethanol) and biobased chemicals such as 5-hydoxymethyl furfural (HMF). To produce platform chemicals, sucrose is hydrolyzed into its monomeric C6 sugars: glucose and fructose. When comparing the hydrolysis rates of molasses with a pure sucrose solution, the specific reaction rate is much slower (Qp/x,60min = 93 and 70 gprod L−1 h−1 gcell−1 for pure sucrose and crude molasses, respectively) at the same sucrose concentration (300 g/L) and process conditions. To clarify why molasses inhibits the enzymatic hydrolysis rate, the influence of its viscosity and inorganic and organic composition was investigated. Also, the effects of molasses and treated molasses on pure enzymes, invertase (from Saccharomyces cerevisiae, 0.05 mg/mL), compared with hydrolysis using whole cells of Baker’s yeast (3 mg/mL), were tested. The results indicate an inhibitory effect of potassium (Qp/x,60min = 76 gprod L−1 h−1 gcell−1), generally at high salt concentrations (Qp/x,60min = 67 gprod L−1 h−1 gcell−1), which could be correlated to the solution’s high salt concentrations and possibly the synergistic effects of different ions when applying concentrations that were four times that in the molasses. Also, the viscosity and sucrose purity seem to have an effect, where pure sucrose solutions and thick juice from the sugar mill yielded higher hydrolysis rates (Qp/x,60min = 97 gprod L−1 h−1 gcell−1) than molasses-type solutions with a higher viscosity (Qp/x,60min = 70–74 gprod L−1 h−1 gcell−1). Attempting to further understand the effects of different components on the invertase activity, an in silico investigation was performed, indicating that high salt concentrations affected the binding of sucrose to the active site of the enzyme, which can result in a lower reaction rate. This knowledge is important for future scale-up of the hydrolysis process, since reduced hydrolysis rates require larger volumes to provide a certain productivity, requiring larger process equipment and thereby higher investment costs.

Revealing the Origin of Graphene Anchored Single Palladium Atom for Electrochemical CO2 Reduction to Syngas with Tunable CO/H2 Ratios

Pd based catalysts are rare metal‐based catalyst to yield tunable CO/H2 ratios for Fischer‐Tropsch synthesis. How to achieve the co‐production of CO and H2 with as little Pd as possible is extremely meaningful for Cn industry. Recent experiment revealed single Pd atom anchored on graphene exhibits high activity for CO2 electroreduction to syngas, yet the origin of activity and controllable CO/H2 ratios, especially the exact Pd coordination structure, remains elusive. Here we employ grand‐canonical density functional theory to show that Pd‐N1, rather than the commonly accepted Pd‐N4, serves as the active center, and the charge‐carrying capability is an effective descriptor. The site with more Pd‑C coordination can better submerge in graphene's delocalized π electrons for higher charge‐carrying capacity to carry excess charges that occupy Pd 4dz2 orbital and promote electron injection. Importantly, the tunable CO/H2 ratio can be explained with difference in charge‐carrying capability of transition state for *COOH and *H2 formation. This work solves the puzzle of coordinating structure of Pd active site and demonstrates the important role of charge‐carrying capability in electrochemical process, which shall provide a reference for further exploration of efficient electrocatalysts.

Influence of Oxygen Vacancies in La0.4Sr0.6FeO3-δ Perovskite Oxide Nanoparticles for the Oxygen Evolution Reaction

Oxygen vacancies are important factors to tune the performance of oxide catalysts for the oxygen evolution reaction (OER). However, it remains challenging how to control the concentration of the oxygen vacancies and decouple their effects on OER from other factors. In this work, we use a specific composition/phase La0.4Sr0.6FeO3- d perovskite oxide as an example to tailor the oxygen vacancies of their nanoparticulate using a modified molten salt synthesis and ozone treatment. The OER performance of the resulting La0.4Sr0.6FeO3- d nanoparticles with different degree of oxygen vacancies ranging from 0 to 25% are compared. The OER reactivity increases with increased oxygen vacancies in the materials, but the change is nonlinear. The OER stability, on the other hand, has a bimodal distribution. The activities of fully oxidized sample and the most oxygen-deficient sample degrade slower than the two samples in the middle. The results suggest that it is feasible to design catalysts with oxygen vacancy to be the most OER active and stable. Further investigation into the oxygen vacancy – active site relation would offer guiding principle to design and synthesize high OER performance oxide catalysts.

Cerium-doped Nickel SulfideNanospheres as EfficientCatalysts for Overall Water Splitting.

The development of non-precious metal electrocatalysts with excellent activity and durability for electrochemical water splitting has always been a goal. Transition metal sulfides are attractive electrocatalysts for both hydrogen evolution reaction (HER) and oxygen evolution reaction(OER). In this article, we designed and constructed efficient catalysts with multiple synergistic interactions and synthesized Ce-NiS2@NFnanosphere using a solvothermal method. Ce-NiS2@NF exhibits excellent HER performance, OER performance, and overall water splitting capability in alkaline electrolytes, demonstrating good stability. The addition of Ce influences the activity of the catalysts, attributed to the synergistic interactions creating more active sites and higher intrinsic activity through the introduction of Ce heteroatoms. Additionally, the self-supported conductive substrate promotes electron transfer, enhancing the intrinsic activity and active site density of the catalyst.This study provides an in-depth investigation into structural design and performance enhancement, offering ideas for designing efficient catalysts for overall water electrolysis. This work provides an in-depth study in terms of structural design performance enhancement and provides ideas for designing efficient alkaline bifunctional catalysts. Valuable insights have been provided in elucidating the intrinsic mechanism of the catalytic activity of cerium-doped nickel sulfidenanospheres,thus providing new guidance in the field of energy conversion technology.

Alkylation of Phenol with Methanol over ZSM‐5 Zeolite

AbstractThe catalytic performance of different molecular sieves in the alkylation of phenol with methanol to produce p‐cresol was evaluated, and the acidity and structure of the catalysts were characterized using N2 adsorption–desorption and Fourier transform infrared spectroscopy techniques. It was shown that the catalytic activity of molecular sieves was closely related to their acidic site number, specific surface area, and pore structure. Since Zeolite ZSM‐5 (Zeolites Socony Mobil No. 5) has a relatively small amount of strong acid as well as a small pore size, it is favorable for the diffusion of p‐cresol out of the pores. Therefore, focusing on ZSM‐5, the effects of operating conditions such as weight hourly space velocity (WHSV), phenol/methanol (P/M) ratio, and reaction temperature on the catalytic performance were investigated. The results showed that increasing the WHSV, P/M ratio, and reaction temperature improved the selectivity to cresol. In addition, X‐ray diffraction and Thermal Gravity/Differential Thermal Gravity characterization methods were also carried out for ZSM‐5, and it was found that the deactivation of ZSM‐5 was due to coke deposition and pore blockage on the active site and that the selectivity to cresol could be restored by regenerating the catalyst and covering the strong acid site, which resulted in the reduction of the strong acid site to increase the selectivity of the product.

Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

Nitrobindins (Nbs) are all-β-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

Investigation of Seasonal Dynamism of Peroxidase from Strains of Aspergillus sp.

Peroxidases A and B production were carried out from Aspergillus tamari and fumigates respectively isolated from petroleum hydrocarbon spilled soil. Physicochemical properties of the respective soil showed pH of 4.45 and 6.5 for soils from point 1 and II respectively and higher conductivity of 613 and 1013 (Ω-1 Cm-1), respectively when compared with the control sample. Dissolved mineral of Cl-, SO4, K, Ca, Mg in the respective soil samples from the petroleum spilled sites was significantly high when compared with the control experiment except for soil sample I which showed a relative low phosphate concentration of 1,23 in the presence of the control experiment, respectively. TOC and TOM contents were 87.91, 119.04; 108.13 and 146.42 mg/g for soil sample I, and III, respectively. In all the tested parameters, the experimented soils were significantly high than the control soil sample. Molecular tests (18S rDNA.) were used to identify the pure isolates of Aspergillia. Studies on the effect of the incubation period on the production of peroxidase from strains of ofAspergillus tamarrii sp. and Aspergillusshowed that the highest peroxidase (A and B) activity representing peroxidase from dry and wet conditions were obtained on the day 6th and 5th of the fermentation time figure peroxidase A activity peaked at pH 5 while that of peroxidase B peaked at pH 6.0; proteins with highest peroxidase activity was peak precipitated at 60% and 80% saturation of the salt for peroxidase A and B, respectively. The gel chromatogram showed single almost superimposed peaks of enzyme activity for peroxidase A and B respectively. Peroxidase A and B activity peaked at pH 4.5 and 5.0. Optimum temperature for the enzyme activity was at 50 and 60°C respectively. Km and V max of 3.45mM and 280 μmole/min; 2.44mM and 305μmole/min were extrapolated from the reciprocal curve of Lineweaver-burke at various concentrations of 2,6 DMP for peroxidase A. Fe, Ca, Co and Mn selected as their notable impact in the active site of peroxidase guided the selected were assayed in the presence of the enzymes, respectively. The stability curve obtained for the peroxidases was single biphasic which represents the first order; Peroxidase B maintained greater stability than A at its optimum pH and pH 7. The enzymes maintained greater than 50% of their activity after 30 min of incubation as activity progressively decreased up to 40% after 60 min of incubation. Thermal stability of peroxidase A and B at their respective optimum temperatures (50 and 60°C) and at 70°C showed a biphasic stability curve of peroxidase A but single phase in peroxidase B. Peroxidase B maintained greater stability than A at its optimum temperature and at 70°C. The enzymes maintained greater than 50% of their activity after 60 min of incubation. Stability curve of peroxidase A and B at 70°C showed a maximum activity of the enzymes after 30 min of incubation. However, Peroxidase B maintained greater stability than A after 60 min of incubation. Peroxidase B maintained 57.89% of its activity after 60 min while peroxidase A from fig 19 showed a residual activity of 41.2%.

Promoting Electrochemical CO2 Reduction to Formate via Sulfur‐Assisted Electrolysis

AbstractElectrochemical CO2 reduction reaction (CO2RR) provides a renewable approach to transform CO2 to produce chemicals and fuels. Unfortunately, it faces the challenges of sluggish CO2 activation and slow water dissociation. This study reports the modification of Bi‐based electrocatalyst by S, which leads to a remarkable enhancement in activity and selectivity during electrochemical CO2 reduction to formate. Based on comprehensive in situ examinations and kinetic evaluations, it is observed that the presence of S species over Bi catalyst can significantly enhance its interaction with K+(H2O)n, facilitating fast dissociation of water molecules to generate protons. Further in situ attenuated total reflectance surface‐enhanced infrared absorption spectroscopy (ATR‐SEIRAS) and in situ Raman spectroscopy measurements reveal that S modification is able to decrease the oxidation state of Bi active site, which can effectively enhance CO2 activation and facilitate HCOO* intermediate formation while suppressing competing hydrogen evolution reaction. Consequently, the S‐modified Bi catalyst achieves impressive electrochemical CO2RR performance, reaching a formate Faradaic efficiency (FEformate) of 91.2% at a formate partial current density of ≈135 mA cm−2 and a potential of −0.8 V versus RHE in an alkaline electrolyte.