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Single-atom catalysts (SACs) have developed as a potential advanced variety of catalysts wherein copper SACs (Cu SACs) have appeared as a rising star in catalysis in which promising catalytic activity is achieved by a well-adjusted d-band center. Herein, the most recent advancements in the synthesis of copper SACs are highlighted. Various electrocatalytic applications such as carbon dioxide reduction reaction, oxygen reduction reaction, nitrogen reduction reaction, biotherapeutic applications, coupling reactions, hydroxylation, hydrogenation, photocatalytic reactions, and other miscellaneous reactions of Cu SACs coordinated into different supports have been thoroughly reviewed, and the stability and electrocatalytic performance of Cu SACs in fuel cells, zinc–CO2 batteries, and zinc–air batteries have been discussed. This review summarizes the study of most recent developments in the synthesis and applications of Cu SACs. [ABSTRACT FROM AUTHOR]

Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N -acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2 H 2 O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.

Nitenpyram, taking the place of imidacloprid, is a widely used neonicotinoid insecticide to control Nilaparvata lugens in Asia. Two P450s, CYP4CE1 and CYP6ER1, are key factors in the metabolic resistance against nitenpyram and imidacloprid. In this study, we found that CYP4CE1 expression was strongly associated with nitenpyram resistance in 8 field-collected populations, whereas CYP6ER1 expression correlated with imidacloprid resistance. Hence, we focused on nitenpyram metabolism by CYP4CE1, due to that imidacloprid metabolism by CYP6ER1 has intensively investigated. Mass spectrometry analysis revealed that recombinant CYP4CE1 metabolized nitenpyram into three products, N-desmethyl nitenpyram, hydroxy-nitenpyram, and N-desmethyl hydroxy-nitenpyram, with a preference for hydroxylation. In contrast, CYP6ER1 metabolized nitenpyram into a single product, N-desmethyl nitenpyram. These results provide new insights into the specific catalytic mechanisms of P450 enzymes in neonicotinoid metabolism and underscore the importance of different catalytic reactions in neonicotinoid insecticide resistance.

N -Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1- exo -acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H · abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H · from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2 H 2 O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Peatland wildfires contribute significantly to the atmospheric release of light-absorbing organic carbon, often referred to as brown carbon. In this study, we examine the presence of nitrogen-containing organic compounds (NOCs) within marine aerosols across the Western Pacific Ocean, which are influenced by peatland fires from Southeast Asia. Employing ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in electrospray ionization (ESI) positive mode, we discovered that NOCs are predominantly composed of reduced nitrogenous bases, including CHN+ and CHON+ groups. Notably, the count of NOC formulas experiences a marked increase within plumes from peatland wildfires compared to those found in typical marine air masses. These NOCs, often identified as N-heterocyclic alkaloids, serve as potential light-absorbing chromophores. Furthermore, many NOCs demonstrate pyrolytic stability, engage in a variety of substitution reactions, and display enhanced hydrophilic properties, attributed to chemical processes such as methoxylation, hydroxylation, methylation, and hydrogenation that occur during emission and subsequent atmospheric aging. During the daytime atmospheric transport, aging of aromatic N-heterocyclic compounds, particularly in aliphatic amines prone to oxidation and reactions with amine, was observed. The findings underscore the critical role of peatland wildfires in augmenting nitrogen-containing organics in marine aerosols, underscoring the need for in-depth research into their effects on marine ecosystems and regional climatic conditions.

The antioxidant N -(1,3-Dimethylbutyl)- N '-phenyl- p -phenylenediamine (6PPD) and its oxidized quinone product 6PPD-quinone (6PPD-Q) in rubber have attracted attention due to the ecological risk that they pose. Both 6PPD and 6PPD-Q have been detected in various environments that humans cohabit. However, to date, a clear understanding of the biotransformation of 6PPD-Q and a potential biomarker for exposure in humans are lacking. To address this issue, this study presents a comprehensive analysis of the extensive biotransformation of 6PPD-Q across species, encompassing both in vitro and in vivo models. We have tentatively identified 17 biotransformation metabolites in vitro , 15 in mice in vivo , and confirmed the presence of two metabolites in human urine samples. Interestingly, different biotransformation patterns were observed across species. Through semiquantitative analysis based on peak areas, we found that almost all 6PPD-Q underwent biotransformation within 24 h of exposure in mice, primarily via hydroxylation and subsequent glucuronidation. This suggests a rapid metabolic processing of 6PPD-Q in mammals, underscoring the importance of identifying effective biomarkers for exposure. Notably, monohydroxy 6PPD-Q and 6PPD-Q-O-glucuronide were consistently the most predominant metabolites across our studies, highlighting monohydroxy 6PPD-Q as a potential key biomarker for epidemiological research. These findings represent the first comprehensive data set on 6PPD-Q biotransformation in mammalian systems, offering insights into the metabolic pathways involved and possible exposure biomarkers.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin ( 2 ) and 1β-hydroxy-bufalin ( 3 ), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Angucyclines are an important group of microbial natural products that display tremendous chemical diversity. Classical angucyclines are composed of a tetracyclic benz[ a ]anthracene scaffold with one ring attached at an angular orientation. However, in atypical angucyclines, the polyaromatic aglycone is cleaved at A-, B-, or C-rings, leading to structural rearrangements and enabling further chemical variety. Here, we have elucidated the branching points in angucycline biosynthesis leading toward cleavage of the C-ring in lugdunomycin and thioangucycline biosynthesis. We showed that 12-hydroxylation and 6-ketoreduction of UWM6 are shared steps in classical and C-ring-cleaved angucycline pathways, although the bifunctional 6-ketoreductase LugOIIred harbors additional unique 1-ketoreductase activity. We identified formation of the key intermediate 8- O -methyltetrangomycin by the LugN methyltransferase as the branching point toward C-ring-cleaved angucyclines. The final common step in lugdunomycin and thioangucycline biosynthesis is quinone reduction, catalyzed by the 7-ketoreductases LugG and TacO, respectively. In turn, the committing step toward thioangucyclines is 12-ketoreduction catalyzed by TacA, for which no orthologous protein exists on the lugdunomycin pathway. Our results confirm that quinone reductions are early tailoring steps and, therefore, may be mechanistically important for subsequent C-ring cleavage. Finally, many of the tailoring enzymes harbored broad substrate promiscuity, which we utilized in combinatorial enzymatic syntheses to generate the angucyclines SM 196 A and hydranthomycin. We propose that enzyme promiscuity and the competition of many of the enzymes for the same substrates lead to a branching biosynthetic network and formation of numerous shunt products typical for angucyclines rather than a canonical linear metabolic pathway.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152Δ matAB that could produce aklavinone, 9- epi -aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152Δ matAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models ( A , B , and C ) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C 4 -H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C , the dipole moment is positioned along the C 4 -H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C 4 -H hydroxylation selectivity. Furthermore, an active site Tyr 233 residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr 233 phenol loses a proton to the nearby Asp 179 residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C 4 -H bond of the substrate and guides the selectivity to the C 4 -hydroxylation of the substrate.

Fluorinated liquid crystal monomers (FLCMs) have been suggested as emerging contaminants, raising global concern due to their frequent occurrence, potential toxic effects, and endurance capacity in the environment. However, the environmental fate of the FLCMs remains unknown. To fill this knowledge gap, we investigated the aerobic microbial transformation mechanisms of an important FLCM, 4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3, 5-difluoro-4'-propylbiphenyl (DTMDPB), using an enrichment culture termed as BG1. Our findings revealed that 67.5 ± 2.1% of the initially added DTMDPB was transformed in 10 days under optimal conditions. A total of 14 microbial transformation products obtained due to a series of reactions (e.g., reductive defluorination, ether bond cleavage, demethylation, oxidative hydroxylation and aromatic ring opening, sulfonation, glucuronidation, O -methylation, and thiolation) were identified. Consortium BG1 harbored essential genes that could transform DTMDPB, such as dehalogenation-related genes [e.g., glutathione S-transferase gene ( GST ), 2-haloacid dehalogenase gene (2-HAD), nrdB , nuoC , and nuoD ]; hydroxylating-related genes hcaC , ubiH , and COQ7 ; aromatic ring opening-related genes ligB and catE ; and methyltransferase genes ubiE and ubiG . Two DTMDPB-degrading strains were isolated, which are affiliated with the genus Sphingopyxis and Agromyces . This study provides a novel insight into the microbial transformation of FLCMs. The findings of this study have important implications for the development of bioremediation strategies aimed at addressing sites contaminated with FLCMs.

Boron nitride nanosheets (BNNSs) are widely used as fillers to increase the thermal conductivity of a substrate. However, the solvent resistance of BNNSs limits their widespread application. In this paper, hydroxyl functionalized boron nitride nanosheets (OH-BNNSs) were prepared through a one-step aqueous shear exfoliation process. The dangling bonds were formed during the exfoliation process and were attacked by water molecules, and thereby hydroxyl functionalization of BNNSs was achieved. The OH-BNNSs were added to poly-(vinyl alcohol) (PVA) to prepare a nanocomposite with high thermal conductivity. The high-speed dispersive homogenizer was selected as the shear-exfoliating device, water as solvent, and sodium dodecyl sulfate (SDS) as surfactant. The effects of operating conditions on the concentration of few-layer OH-BNNSs were systematically studied. Under the optimized conditions, 0.88 mg/mL of OH-BNNSs was obtained. The as-obtained OH-BNNSs were filled in PVA to obtain composite film. The in-plane thermal diffusivity of OH-BNNSs-PVA films reached 9.03 mm2/s, which was 40-times the in-plane thermal diffusivity of the pure PVA film and 1.6-times the in-plane thermal diffusivity of the PVA composites filled by BNNSs prepared from ultrasonic exfoliation. The reasons were attributed to better dispersibility in PVA aqueous solution for OH-BNNSs. This paper provided a simple, scalable, and environmentally friendly preparation method for OH-BNNSs. [ABSTRACT FROM AUTHOR]

We report and analyze chemoselectivity in the gas phase reactions of cycloalkenes (cyclohexene, cycloheptene, cis-cyclooctene, 1,4-cyclohexadiene) with a non-heme iron(IV)-oxo complex [(PyTACN)Fe(O)(Cl)]+, which models the active species in iron-dependent halogenases. Unlike in the halogenases, we did not observe any chlorination of the substrate. However, we observed two other reaction pathways: allylic hydrogen atom transfer (HAT) and alkene epoxidation. The HAT is clearly preferred in the case of 1,4-cyclohexadiene, both pathways have comparable reaction rates in reaction with cyclohexene, and epoxidation is strongly favored in reactions with cycloheptene and cis-cyclooctene. This preference for epoxidation differs from the reactivity of iron(IV)-oxo complexes in the condensed phase, where HAT usually prevails. To understand the observed selectivity, we analyze effects of the substrate, spin state, and solvation. Our DFT and CASPT2 calculations suggest that all the reactions occur on the quintet potential energy surface. The DFT-calculated energies of the transition states for the epoxidation and hydroxylation pathways explain the observed chemoselectivity. The SMD implicit solvation model predicts the relative increase of the epoxidation barriers with solvent polarity, which explains the clear preference of HAT in the condensed phase. [ABSTRACT FROM AUTHOR]

Unspecific peroxygenases (UPOs) are emerging as promising biocatalysts for selective oxyfunctionalization of unactivated C-H bonds. However, their potential in large-scale synthesis is currently constrained by suboptimal chemical selectivity. Improving the selectivity of UPOs requires a deep understanding of the molecular basis of their catalysis. Recent molecular simulations have sought to unravel UPO's selectivity and inform their design principles. However, most of these studies focused on substrate-binding poses. Few researchers have investigated how the reactivity of CpdI, the principal oxidizing intermediate in the catalytic cycle, influences selectivity in a realistic protein environment. Moreover, the influence of protein electrostatics on the reaction kinetics of CpdI has also been largely overlooked. To bridge this gap, we used multiscale simulations to interpret the regio- and enantioselective hydroxylation of the n -heptane substrate catalyzed by Agrocybe aegerita UPO ( Aae UPO). We comprehensively characterized the energetics and kinetics of the hydrogen atom-transfer (HAT) step, initiated by CpdI, and the subsequent oxygen rebound step forming the product. Notably, our approach involved both free energy and potential energy evaluations in a quantum mechanics/molecular mechanics (QM/MM) setting, mitigating the dependence of results on the choice of initial conditions. These calculations illuminate the thermodynamics and kinetics of the HAT and oxygen rebound steps. Our findings highlight that both the conformational selection and the distinct chemical reactivity of different substrate hydrogen atoms together dictate the regio- and enantio-selectivity. Building on our previous study of CpdI's formation in Aae UPO, our results indicate that the HAT step is the rate-limiting step in the overall catalytic cycle. The subsequent oxygen rebound step is swift and retains the selectivity determined by the HAT step. We also pinpointed several polar and charged amino acid residues whose electrostatic potentials considerably influence the reaction barrier of the HAT step. Notably, the Glu196 residue is pivotal for both the CpdI's formation and participation in the HAT step. Our research offers in-depth insights into the catalytic cycle of Aae UPO, which will be instrumental in the rational design of UPOs with enhanced properties.

Paclitaxel (PTX) is heralded as one of the most successful natural-product drugs for the treatment of refractory cancers. In humans, the hepatic metabolic transformation of PTX is primarily mediated by two cytochrome P450 enzymes (P450s): CYP3A4 and CYP2C8. The impact of P450 metabolism on the anticancer effectiveness of PTX is significant. However, the precise mechanism underlying selective P450-catalyzed reactions in PTX metabolism remains elusive. To address this knowledge gap, we conducted molecular docking and molecular dynamics simulations using multiple crystal structures of CYP3A4, which originally contained other ligands. These methods enabled us to determine the most plausible binding structure of PTX within the enzyme. By further employing hybrid quantum mechanics and molecular mechanics calculations, we successfully identified two primary pathways for the reaction between compound I (Cpd I) of CYP3A4 and PTX. One of these pathways involves the formation of an epoxide, while the other proceeds through a ketone intermediate.

Achieving single-step syntheses of a set of related compounds divergently and selectively from a common starting material affords substantial efficiency gains when compared with preparing those same compounds by multiple individual syntheses. In order for this approach to be realized, complementary reagent systems must be available; here, a panel of engineered P450 BM3 enzymes is shown to fulfill this remit in the selective C-H hydroxylation of cyclobutylamine derivatives at chemically unactivated sites. The oxidations can proceed with high regioselectivity and stereoselectivity, producing valuable bifunctional intermediates for synthesis and applications in fragment-based drug discovery. The process also applies to bicyclo[1.1.1]pentyl (BCP) amine derivatives to achieve the first direct enantioselective functionalization of the bridging methylenes and open a short and efficient route to chiral BCP bioisosteres for medicinal chemistry. The combination of substrate, enzyme, and reaction engineering provides a powerful general platform for small-molecule elaboration and diversification.

Fe II /α-ketoglutarate-dependent dioxygenases (Fe/αKG) make up a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/αKG homologues catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis; however, we have identified that this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over β-hydroxylation with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologues and provided insight into substrate recognition and reactivity trends. Investigation of this substrate-dependent chemistry improves our understanding of kainoid synthases and their biocatalytic application.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae , the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase ( hpaB ) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Polychlorinated biphenyls (PCBs) are environmental contaminants that can cause neurotoxicity. PCBs, such as PCB 95 (2,2',3,5',6-pentachlorobiphenyl), can be metabolized by cytochrome P450 enzymes into neurotoxic metabolites. To better understand how the metabolism of PCB 95 affects neurotoxic outcomes, we conducted a study on the disposition of PCB 95 in transgenic mouse models. The mice were given a single oral dose of PCB 95 (1.0 mg/kg) and were euthanized 24 h later for analysis. PCB 95 levels were highest in adipose tissue, followed by the liver, brain, and blood. Adipose tissue levels were significantly higher in wild-type (WT) mice than in Cyp2abfgs-null (KO) or CYP2A6-transgenic (KI) mice. We also observed genotype-dependent differences in the enrichment of aS-PCB 95 in female mice, with a less pronounced enrichment in KO than WT and KI mice. Ten hydroxylated PCB 95 metabolites were detected in blood and tissue across all exposure groups. The metabolite profiles differed across tissues, while sex and genotype-dependent differences were less pronounced. Total OH-PCB levels were highest in the blood, followed by the liver, adipose tissue, and brain. Total OH-PCB blood levels were lower in KO than in WT mice, while the opposite trend was observed in the liver. In male mice, total OH-PCB metabolite levels were significantly lower in KI than in WT mice in blood and the liver, while the opposite trend was observed in female mice. In conclusion, the study highlights the differences in the atropselective disposition of PCB 95 and its metabolites in different types of mice, demonstrating the usefulness of these transgenic mouse models for characterizing the role of PCB metabolism in PCB neurotoxicity.

Poly(etheretherketone) (PEEK) is regarded as an attractive orthopedic material because of its good biocompatibility and mechanical properties similar to natural bone. The efficient activation methods for the surfaces of PEEK matrix materials have become a hot research topic. In this study, a method using a femtosecond laser (FSL) followed by hydroxylation was developed to achieve efficient bioactivity. It produces microstructures, amorphous carbon, and grafted -OH groups on the PEEK surface to enhance hydrophilicity and surface energy. Both experimental and simulation results show that our modification leads to a superior ability to induce apatite deposition on the PEEK surface. The results also demonstrate that efficient grafting of C-OH through FSL-hydroxylation can effectively enhance cell proliferation and osteogenic differentiation compared to other modifications, thus improving osteogenic activity. Overall, FSL hydroxylation treatment is proved to be a simple, efficient, and environmentally friendly modification method for PEEK activation. It could expand the applications of PEEK in orthopedics, as well as promote the surface modification and structural design of other polymeric biomaterials to enhance bioactivity.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis , AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O 2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

γ- and δ-lactones are valuable flavor and fragrance compounds. Their synthesis depends on the availability of suitable hydroxy fatty acid precursors. Three short unspecific peroxygenases were identified that selectively hydroxylate the C4 and C5 positions of C8-C12 fatty acids to yield after lactonization the corresponding γ- and δ-lactones. A preference for C4 over C5 hydroxylation gave γ-lactones as the major products. Overoxidation of the hydroxy fatty acids was addressed via the reduction of the resulting oxo acids using an alcohol dehydrogenase in a bienzymatic cascade reaction.

Hyoscyamine 6β-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted. [ABSTRACT FROM AUTHOR]

Nucleation and biomineralization of apatite on titanium-based material surfaces is crucial to improve surface biocompatibility, osseointegration, and rapid bone ingrowth in biomedical applications such as joint replacements. This work is designed to provide new insights on the molecular processes in the initial nucleation of calcium phosphate on TiO2 surface by means of classical molecular dynamics (MD) simulations. Aggregation of calcium and phosphate ions in pure aqueous solution was studied, and the free energies during ion adsorption was investigated by the calculation of PMFs. The MD results suggest surface hydroxylation rate and nanotopographies of TiO2 substrate contribute significantly to the initial nucleation of calcium phosphate. Our simulations suggest that surface hydroxyls on TiO2 provide active sites for the aggregation of calcium phosphate. Both calcium ions and phosphate ions could bind to the hydroxylated TiO2 surface directly or indirectly via the first water layer. Surface nanotopographies (e.g., grooves or ridges) seem to be able to restrict the diffusion of calcium ions and phosphate ions, hence offering more opportunities for adsorption by trapping ions inside channels. Therefore, it can be inferred that apatite may be formed more favorably on porous or concave surfaces. [ABSTRACT FROM AUTHOR]

[Fe(β-BPMCN)(CH3CN)2]2+ (1, BPMCN = N,N'-bis(pyridyl-2-methyl)-N,N'-dimethyl-trans-1,2-diaminocyclo- hexane) is a relatively poor catalyst for cyclohexane oxidation by H2O2 and cannot perform benzene hydroxylation. However, addition of Sc3+ activates the 1/H2O2 reaction mixture to be able to hydroxylate cyclohexane and benzene within seconds at -40 °C. A metastable S = 1/2 FeIII-(η¹- OOH) intermediate 2 is trapped at -40 °C, which undergoes rapid decay upon addition of Sc3+ at rates independent of [substrate] but linearly dependent on [Sc3+]. HClO4 elicits comparable reactivity as Sc3+ at the same concentration. We thus postulate that these additives both facilitate O-O bond heterolysis of 2 to form a common highly electrophilic FeV=O oxidant that is comparably reactive to the fastest nonheme highvalent iron-oxo oxidants found to date. [ABSTRACT FROM AUTHOR]

Catalytic activation of hydroxyl functionalities is of great interest for the production of pharmaceuticals and commodity chemicals. Here, 2-alkoxycarbonyl- and 2-phenoxycarbonyl-phenylboronic acid were identified as efficient catalysts for the direct and chemoselective activation of oxime N-OH bonds in the Beckmann rearrangement. This classical organic reaction provides a unique approach to prepare functionalized amide products that may be difficult to access using traditional amide coupling between carboxylic acids and amines. Using only 5 mol % of boronic acid catalyst and perfluoropinacol as an additive in a polar solvent mixture, the operationally simple protocol features mild conditions, a broad substrate scope, and a high functional group tolerance. A wide variety of diaryl, aryl-alkyl, heteroaryl-alkyl, and dialkyl oximes react under ambient conditions to afford high yields of amide products. Free alcohols, amides, carboxyesters, and many other functionalities are compatible with the reaction conditions. Investigations of the catalytic cycle revealed a novel boron-induced oxime transesterification providing an acyl oxime intermediate involved in a fully catalytic nonself-propagating Beckmann rearrangement mechanism. The acyl oxime intermediate was prepared independently and was subjected to the reaction conditions. It was found to be self-sufficient; it reacts rapidly, unimolecularly without the need for free oxime. A series of control experiments and 18O labeling studies support a true catalytic pathway involving an ionic transition structure with an active and essential role for the boronyl moiety in both steps of transesterification and rearrangement. According to 11B NMR spectroscopic studies, the additive perfluoropinacol provides a transient, electrophilic boronic ester that is thought to serve as an internal Lewis acid to activate the ortho-carboxyester and accelerate the initial, rate-limiting step of transesterification between the precatalyst and the oxime substrate. [ABSTRACT FROM AUTHOR]

Diastereoselective α-hydroxylation using molecular oxygen has been achieved with chiral α-alkyl N-tert-butanesulfinyl imidates and α-aryl N'-tert-butanesulfinyl amidines. The aza-enolates generated from deprotonation of imidates/amidines can be intercepted by O2 with excellent diastereocontrol and subsequently transformed into α-hydroxylation products in the presence of the reductant trimethyl phosphite. [ABSTRACT FROM AUTHOR]

A direct double functionalization involving both difluorination and hydroxylation of enamides is reported. With the appropriate combination of an electrophilic fluorinating reagent and H2O, the most convenient and ecofriendly hydroxylating agent, the preparation of 3-(difluoroalkyl)-3-hydroxyisoindolin-1-ones was achieved under basic or Brønsted acidic conditions. Suitable conditions for trifluorination as well as C(sp²)-H fluorination were also identified. Subsequent asymmetric functionalization of the obtained gem-difluorinated products has also been demonstrated. [ABSTRACT FROM AUTHOR]

The detachment process of an oil molecular layer situated above a horizontal substrate was often described by a three-stage process. In this mechanism, the penetration and diffusion of water molecules between the oil phase and the substrate was proposed to be a crucial step to aid in removal of oil layer/drops from substrate. In this work, the detachment process of a two-dimensional alkane molecule layer from a silica surface in aqueous surfactant solutions is studied by means of molecular dynamics (MD) simulations. By tuning the polarity of model silica surfaces, as well as considering the different types of surfactant molecules and the water flow effects, more details about the formation of water molecular channel and the expansion processes are elucidated. It is found that for both ionic and nonionic type surfactant solutions, the perturbation of surfactant molecules on the two-dimensional oil molecule layer facilitates the injection and diffusion of water molecules between the oil layer and silica substrate. However, the water channel formation and expansion speed is strongly affected by the substrate polarity and properties of surfactant molecules. First, only for the silica surface with relative stronger polarity, the formation of water molecular channel is observed. Second, the expansion speed of the water molecular channel upon the ionic surfactant (dodecyl trimethylammonium bromide, DTAB and sodium dodecyl benzenesulfonate, SDBS) flooding is more rapidly than the nonionic surfactant system (octylphenol polyoxyethylene(10) ether, OP-10). Third, the water flow speed may also affect the injection and diffusion of water molecules. These simulation results indicate that the water molecular channel formation process is affected by multiple factors. The synergistic effects of perturbation of surfactant molecules and the electrostatic interactions between silica substrate and water molecules are two key factors aiding in the injection and diffusion of water molecules and helpful for the oil detachment from silica substrate. [ABSTRACT FROM AUTHOR]

The self-assembly synthesis of Cu(II)-silsesquioxane involving 2,9-dimethyl-1,10-phenanthroline (neocuproine) as an additional N ligand at copper atoms was performed. The reaction revealed an unprecedented aerobic hydroxylation of only one of the two methyl groups in neocuproine to afford the corresponding geminal diol. The produced derivative of oxidized neocuproine acts as a two-centered N,O ligand in the assembly of the hexacopper cage product [Cu6(Ph5Si5O10)2•(C14H11N2O2)2] (1), coordinating two of the six copper centers in the product. Two siloxanolate ligands [PhSi(O)O]5 in the cis configuration coordinate to the rest of the copper(II) ions. Compound 1 is a highly efficient homogeneous precatalyst in the oxidation of alkanes and alcohols with peroxides. [ABSTRACT FROM AUTHOR]

Hydroxylation of arylboronic acids to aryl alcohols was realized by a scalable electrocatalytic method. The present electrochemical hydroxylation employs low-cost methyl viologen as an organic cathodic electrocatalyst and involves O2 as a green and sustainable reactant. The electrochemical kinetic studies shown here can be a powerful tool to gain rich mechanistic and kinetic information and thus an in-depth understanding of the electrocatalytic mechanism. [ABSTRACT FROM AUTHOR]

We use ambient-pressure X-ray photoelectron spectroscopy (APXPS) to study chemical changes, including hydroxylation and water adsorption, at copper oxide surfaces from ultrahigh vacuum to ambient relative humidities of ~5%. Polycrystalline CuO and Cu2O surfaces were prepared by selective oxidation of metallic copper foils. For both oxides, hydroxylation occurs readily, even at high-vacuum conditions. Hydroxylation on both oxides plateaus near ~0.01% relative humidity (RH) at a coverage of ~1 monolayer. In contrast to previous studies, neither oxide shows significant accumulation of molecular water; rather, both surfaces show a high affinity for adventitious carbon contaminants. Results of isobaric and isothermic experiments are compared, and the strengths and potential drawbacks of each method are discussed. We also provide critical evaluations of the effects of the hot filament of the ion pressure gauge on the reactivity of gas-phase species, the peak fitting procedure on the quantitative analysis of spectra, and rigorous accounting of carbon contamination on data analysis and interpretation. This work underscores the importance of considering experimental design and data analysis protocols during APXPS experiments with water vapor in order to minimize misinterpretations arising from these factors. [ABSTRACT FROM AUTHOR]

Water adsorption and reaction on pure and Ni-modified CoOOH nanowires were investigated using ambient pressure photoemission spectroscopy (APPES). The unique capabilities of APPES enable us to observe water dissociation and monitor formation of surface species on pure and Ni-modified CoOOH under elevated pressures and temperatures for the first time. Over a large range of pressures (UHV to 1 Torr), water dissociates readily on the pure and Ni-modified CoOOH surfaces at 27 °C. With an increase in H2O pressure, a greater degree of surface hydroxylation was observed for all samples. At 1 Torr H2O, ratios of different oxygen species indicate a transformation of CoOOH to CoOxHy in pure and Ni-modified CoOOH. In temperature dependent studies, desorption of weakly bound water and surface dehydroxylation were observed with increasing temperature. Larger percentages of surface hydroxyl groups at higher temperatures were observed on Ni-modified CoOOH compared to pure CoOOH, which indicates an increased stability of surface hydroxyl groups on these Ni-modified surfaces. [ABSTRACT FROM AUTHOR]

The interaction of water vapor with a single crystal ZnO(1010) surface was investigated using synchrotron-based ambient pressure X-ray photoelectron spectroscopy (APXPS). Two isobaric experiments were performed at 0.3 and 0.07 Torr water vapor pressure at sample temperatures ranging from 750 to 295 K up to a maximum of 2% relative humidity (RH). Below 10-4 % RH the ZnO(1010) interface is covered with ~0.25 monolayers of OH groups attributed to dissociation at nonstoichiometric defect sites. At ~10-4 % RH there is a sharp onset in increased surface hydroxylation attributed to reaction at stoichiometric terrace sites. The surface saturates with an OH monolayer ~0.26 nm thick and occurs in the absence of any observable molecularly bound water, suggesting the formation of a 1 x 1 dissociated monolayer structure. This is in stark contrast to ultrahigh vacuum experiments and molecular simulations that show the optimum structure is a 2 x 1 partially dissociated H2O/OH monolayer. The sharp onset to terrace site hydroxylation at ~10-4 % RH for ZnO(1010) contrasts with APXPS observations for MgO(100) which show a sharp onset at 10-2 % RH. A surface thermodynamic analysis reveals that this shift to lower RH for ZnO(1010) compared to MgO(100) is due to a more favorable Gibbs free energy for terrace site hydroxylation. [ABSTRACT FROM AUTHOR]

Based on the observation that an increased substrate size is paralleled by an enhanced diastereoselectivity, a late-stage dihydroxylation protocol toward the 21mer CMP (collagen model peptide) Ac-(Pro-Hyp-Gly)3-Pro-Dyp-Gly-(Pro-Hyp-Gly)3-NH2 is presented. C3 and C4 hydroxylation have a converse effect on the triple-helical stability of collagen. Their combined influence on the melting temperature was studied by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

The detection of tyrosinase, a biomarker for melanoma, is of great significance. Herein, a fluorescent tyrosinase probe, with resorufin as the fluorophore and m-tolylboronic acid pinacol ester as the receptor, is proposed. The response relies on the tyrosinase-catalyzed hydroxylation of phenylboronic acid pinacol ester at an adjacent position followed by 1,6-rearrangement elimination to release resorufin. This probe well quantifies tyrosinase in the range from 1 to 100 U mL-1 with a detection limit of 0.5 U mL-1. Importantly, the probe exhibits high selectivity for tyrosinase over other biological substances including reactive oxygen species. In addition, it is successfully applied to the imaging of tyrosinase in cells. This probe provides a novel platform for selective detection of tyrosinase in biosystems. [ABSTRACT FROM AUTHOR]

A mechanistic study of H2O2-dependent C-H bond hydroxylation by myoglobin reconstituted with a manganese porphycene was carried out. The X-ray crystal structure of the reconstituted protein obtained at 1.5 Å resolution reveals tight incorporation of the complex into the myoglobin matrix at pH 8.5, the optimized pH value for the highest turnover number of hydroxylation of ethylbenzene. The protein generates a spectroscopically detectable two-electron oxidative intermediate in a reaction with peracid, which has a half-life up to 38 s at 10 °C. Electron paramagnetic resonance spectra of the intermediate with perpendicular and parallel modes are silent, indicating formation of a low-spin MnV-oxo species. In addition, the MnV-oxo species is capable of promoting the hydroxylation of sodium 4-ethylbenzenesulfonate under single turnover conditions with an apparent second-order rate constant of 2.0 M-1 s-1 at 25 °C. Furthermore, the higher bond dissociation enthalpy of the substrate decreases the rate constant, in support of the proposal that the H-abstraction is one of the rate-limiting steps. The present engineered myoglobin serves as an artificial metalloenzyme for inert C-H bond activation via a high-valent metal species similar to the species employed by native monooxygenases such as cytochrome P450. [ABSTRACT FROM AUTHOR]

Understanding the interaction between oxides and water is critical for designing many of their functionalities, including the electrocatalysis of molecular oxygen reduction. In this study, we probed the hydroxylation of model (001)-oriented La1-xSrxMnO3 (LSMO) perovskite surfaces, where the electronic structure and manganese valence were controlled by five substitution levels of lanthanum with strontium, using ambient-pressure X-ray photoelectron spectroscopy in a humid environment. The degree of hydroxyl formation on the oxide surface correlated with the proximity of the valence band center relative to the Fermi level. LSMO perovskites with a valence band center closer to the Fermi level were more reactive toward water, forming more hydroxyl species at a given relative humidity. More hydroxyl species correlate with greater electron-donating character to the surface free energy in wetting and reduce the activity to catalyze oxygen reduction reaction (ORR) kinetics in a basic solution. New strategies for designing more active catalysts should include design of electronically conducting oxides with lower valence band centers relative to the Fermi level at ORR-relevant potentials. [ABSTRACT FROM AUTHOR]

Under solventless grinding conditions, mechanochemical ruthenium-catalyzed hydroarylations of alkynes with acetanilides lead to trisubstituted alkenes. Only catalytic amounts of pivalic acid or copper acetate are required, and without the need for external heating, the reaction times are shorter than those of their solution-based counterpart. Mechanochemical oxidative annulations through palladium-catalyzed intramolecular amination convert the products into N-acetylindoles. [ABSTRACT FROM AUTHOR]

The direct Pd-catalyzed ortho C-H hydroxylation of benzaldehydes was achieved using 4-chloroanthranilic acid as the transient directing group, 1-fluoro-2,4,6-trimethylpyridnium triflate as the bystanding oxidant, and p-toluenesulfonic acid as the putative oxygen nucleophile. The unusual C-H chlorination and polyfluoroalkoxylation reactions signaled the importance of external nucleophiles to the outcome of Pd(IV) reductive eliminations. [ABSTRACT FROM AUTHOR]

A concise synthesis of (+)-pancratistatin and (+)-7-deoxypancratistatin from benzene using an enantioselective, dearomative carboamination strategy has been achieved. This approach, in combination with the judicious choice of subsequent olefin-type difunctionalization reactions, permits rapid and controlled access to a hexasubstituted core. Finally, minimal use of intermediary steps as well as direct, late stage C-7 hydroxylation provides both natural products in six and seven operations. [ABSTRACT FROM AUTHOR]

Heme proteins utilize the heme cofactor, an iron porphyrin, to perform a diverse range of reactions including dioxygen binding and transport, electron transfer, and oxidation/oxygenations. These reactions share several key metalloporphyrin intermediates, typically derived from dioxygen and its congeners such as hydrogen peroxide. These species are composed of metal-dioxygen, metal-superoxo, metal-peroxo, and metal-oxo adducts. A wide variety of synthetic metalloporphyrinoid complexes have been synthesized to generate and stabilize these intermediates. These complexes have been studied to determine the spectroscopic features, structures, and reactivities of such species in controlled and well-defined environments. In this Review, we summarize recent findings on the reactivity of these species with common porphyrinoid scaffolds employed for biomimetic studies. The proposed mechanisms of action are emphasized. This Review is organized by structural type of metal-oxygen intermediate and broken into subsections based on the metal (manganese and iron) and porphyrinoid ligand (porphyrin, corrole, and corrolazine). [ABSTRACT FROM AUTHOR]

In one yearly cycle (2016), D4 and DS were detected in biogas samples (n = 36, 0.105-2.33 mg/m3) from a Chinese municipal landfill, while D4-D6 were detected in influents/effluents of leachate storage pond (n = 72, < LOQ303 μg/L). Mass loads of cVMS in both biogas (591-6575 mg/d) and leachate influents (659-5760 mg/d) increased from January to July (summer), and then decreased from July to December (winter). Removal experiments indicated that 1) hydrolysis and volatilization were predominant removal mechanism for D4 and DS, respectively, in leachate storage pond, responsible for their more significant removal (94.5-100%) in August; 2) indirect phototransformation (t1/2 = 25.5-87.0 days), such as hydroxylation by OH radical generated in leachates, was the predominant (50.0-75.5%) removal pathway for D6, which led to the largest removal efficiencies (65.2-73.7%) in June, the month with the largest sun light intensity and highest photosensitizer (e.g., Ee2+. and NO3−) concentrations. Monohydroxylated products of DS and D6, D4TOH and DSTOH, were detected in leachate effluents (39.6-187 ng/L) during May-July. Compared to DS and D6, volatilization half-lives of D4TOH (86.3 days) and DSTOH (177 days) in leachates were 2.9 and 1.4 times longer, while their hydrolysis half-lives (7.50 days for D4TOH and 21.5 days for DSTOH) were 7.1 and 10 times shorter, respectively. [ABSTRACT FROM AUTHOR]

Carbon-supported ferric oxide nanoparticles (Fe2O3 NPs) were constructed via directly carbonizing mesoporous poly(ionic liquid) with [Fe(CN)6]3- anions, which was prepared through the free-radical self-polymerization of ionic liquid monomer 1-allyl-3-vinylimidazolium chloride in a soft-template route and successive ion exchange with potassium ferricyanide. The unique mesoporous ionic networks enabled the molecular dispersion of ferric precursors and therefore resulted in highly dispersed Fe2O3 NPs on carbon. The catalyst exhibited high yield, remarkable turnover number, and good reusability in the reductant-free aerobic oxidation of benzene to phenol with O2. The synergistic effect of Fe2O3 NPs and carbon accounted for the high performance. This work delivered the first efficient and environmentally friendly heterogeneous catalyst for reductant-free benzene hydroxylation with O2. [ABSTRACT FROM AUTHOR]

Water present in confining geometries plays key roles in many systems of scientific and technological relevance. Prominent examples are living cells and nanofluidic devices. Despite its importance, a complete understanding of the dynamics of water in nanoscale confinement remains elusive. In this work, we use molecular dynamics (MD) simulation to investigate the diffusive dynamics of water monolayers confined in chemically heterogeneous silica slit pores. The effect of chemical heterogeneity is systematically investigated through the fraction fSiOH of randomly distributed surface sites that possess hydroxyl functional groups. Partial hydroxylation results in heterogeneous surfaces comprising nanoscale hydrophobic and hydrophilic regions. We find that the in-plane diffusivity of water increases monotonically with fSiOH; at low surface hydroxylation (fSiOH ⩽ 50%), slow water dynamics arise due to the formation of icelike structures in the hydrophobic regions, while at fSiOH ⩾ 75%, surface-water H-bonds in the hydrophilic regions result in faster dynamics. We show that surface patterning with ordered hydrophobic and hydrophilic "stripes" can be used to induce one-dimensional diffusion, with water diffusing through the slit pore preferentially along the direction of the hydrophilic surface patterns. [ABSTRACT FROM AUTHOR]

A base-promoted cascade reaction for the regiospecific synthesis of substituted 3-hydroxyisoindolinones under transition-metal-free conditions is developed. The base-mediated C--C bond coupling and N-α-sp³C-H bond hydroxylation are involved in this procedure, which features high regioselectivity, efficiency, and environmental friendliness. Various substituted 3-hydroxyisoindolinones, including some bioactive molecules, were provided in up to 93% yield for 28 examples. [ABSTRACT FROM AUTHOR]

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metalhydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process. [ABSTRACT FROM AUTHOR]

A stable insoluble P450 BM3 system that can be easily separated from the reaction medium for recycling, while using less-expensive cofactors (NADH and BNAH) than costly NADPH, can represent a promising biocatalyst in industrial applications. In this context, the present work investigates the immobilization of double mutant cytochrome P450 BM3 (R966D/W1046S) by adsorption and cross-linking-adsorption on Ni2+-functionalized magnetic nanoparticles (MNPs). By oxidizing NADH or BNAH, the immobilized BM3 succeeded in hydroxylating the substrates (10-pNCA and myristic acid) to a similar degree as the free enzyme. The adsorbed enzyme showed 88% hydroxylation residual activity after five reaction cycles (five continuous days), which was increased to 100% by cross-linking the adsorbed enzyme. In addition, the cross-linked-adsorbed enzyme kept 41% of its initial activity toward NADH after one month of storage at 4 °C, while the free enzyme showed only 31% residual activity after 1 week and was inactive afterward. The results of this work highlighted that the appropriate choice of the enzyme-support-cofactor system can result in an active, stable, and recyclable biocatalyst, which could attract growing industry interest. [ABSTRACT FROM AUTHOR]