Engineered P450s can catalyze some non-natural reactions with high efficiency and excellent selectivity, such as the carbine transfer, nitrene transfer, C-H insertion, and C-H amination, opening alternative routes for sustainable production of chemicals. Recent experiments revealed that two engineered cytochrome P450 enzymes (P450-CIS and P411-CIS) show different efficiencies and stereoselectivities in the olefin cyclopropanation, but key factors that affect the activity remain unclear. In this work, both quantum mechanics (QM) and QM/molecular mechanics (MM) methods were employed to explore the catalytic reactions and selectivity of these two engineered cytochrome P450 enzymes. On the basis of our results, the cyclopropanation of styrene is suggested to mainly occur on the open-shell singlet (OSS) and triplet state surfaces, which contain two elementary steps. The reactive iron(III)-porphyrin carbene (IPC) radical first attacks the terminal alkenyl group of styrene to form a C-radical intermediate, which then undergoes a cyclization reaction affording the cyclopropanation products. Importantly, it is found that the stereoselectivity of cyclopropanations is elucidated only if considering the real protein environment, and the stereoselectivity is determined by multiple factors, such as the relative orientation of IPC to styrene, the binding affinity of the substrate, and the reaction barriers of rate-limiting steps. It is the enzymatic environment that makes the reaction highly stereoselective, which provides useful clues for designing whole-cell catalysts for non-natural chemical reactions.

The proton/electron transfer reactions between cysteine residue (Cys) and tyrosinyl radical (Tyr(•)) are an important step for many enzyme-catalyzed processes. On the basis of the statistical analysis of protein data bank, we designed three representative models to explore the possible proton/electron transfer mechanisms from Cys to Tyr(•) in proteins. Our ab initio calculations on simplified models and quantum mechanical/molecular mechanical (QM/MM) calculations on real protein environment reveal that the direct electron transfer between Cys and Tyr(•) is difficult to occur, but an inserted water molecule can greatly promote the proton/electron transfer reactions by a double-proton-coupled electron transfer (dPCET) mechanism. The inserted H2O plays two assistant roles in these reactions. The first one is to bridge the side chains of Tyr(•) and Cys via two hydrogen bonds, which act as the proton pathway, and the other one is to enhance the electron overlap between the lone-pair orbital of sulfur atom and the π-orbital of phenol moiety and to function as electron transfer pathway. This water-mediated dPCET mechanism may offer great help to understand the detailed electron transfer processes between Tyr and Cys residues in proteins, such as the electron transfer from Cys439 to Tyr730(•) in the class I ribonucleotide reductase.

Carbapenem antibiotics possess a broad spectrum of antibacterial activity and high resistance to hydrolytic inactivation by β-lactamases. Carbapenem synthase (CarC), an iron(II) and 2-(oxo)glutarate-dependent oxygenase, catalyzes the epimerization and desaturation of (3S,5S)-carbapenam to produce (5R)-carbapenem in the last step of the simple carbapenem biosynthesis. Recently, the complete crystal structure of CarC was reported, allowing us to perform accurate quantum mechanics/molecular mechanics (QM/MM) calculations to explore the detailed reaction mechanism. We first analyzed the dioxygen binding site on metal and identified that the Fe IV -oxo species has two potential orientations with either the oxo group trans to His101 or trans to His251. The former is energetically unstable, which can rapidly isomerize into the latter by rotation of the oxo group. Arg279 plays important roles in regulating the dioxygen binding and assisting the isomerization of Fe IV -oxo species. The calculation results clearly support the stepwise C5-epimerization and C2/3-desaturation processes, involving two complete oxidative cycles. The epimerization process converts (3S,5S)-carbapenam to the initial product (3S,5R)-carbapenam, undergoing H5 atom abstraction by Fe IV =O species, inversion of C5-radical and reconstitution of the inverted C5-H bond by Tyr165. In the desaturation process, (3S,5R)-carbapenam rebinds the CarC active site with a new orientation compared to (3S,5S)-carbapenam does in the epimerization. In addition, the desaturation across C2-C3 occurs without involving any active site residue other than the Fe IV =O center. Whereas Tyr165 is not involved in the desaturation reaction, it plays a key role in binding (3S,5R)-carbapenam.(3S,5R)-carbapenam is a substrate superior to its epimer (3S,5S)-carbapenam for CarC to produce (5R)-carbapenem by efficient desaturation. Besides, the substrate hydroxylations compete with the target epimerization and desaturation reactions.

AsqJ from Aspergillus nidulans is a nonheme FeII/α-ketoglutarate-dependent dioxygenase that catalyzes the conversion of benzodiazepinedione into 4′-methoxyviridicatin, which is a key step in the biosynthesis of quinolone alkaloids. A series of recent experiments have demonstrated that AsqJ is able to perform the decoupled desaturation and epoxidation reactions. Herein, on the basis of the published crystal structures, combined quantum mechanics and molecular mechanics (QM/MM) calculations have been performed to explore both the desaturation and epoxidation processes. Our calculations reveal that the quintet state of the FeIV–O complex is the ground state, and the catalytic reaction occurs on the quintet-state surface. The FeIV–oxo species should first undergo an isomerization to initiate the reactions. In the desaturation process, the abstraction of the first hydrogen atom is suggested to follow the σ-channel mechanism. This step is calculated to be rate-limiting with an energy barrier of 19.3 kcal/mol. The abstraction of the second hydrogen atom is found to be quite easy. After the desaturation process, the regenerated FeIV–oxo species first attacks the CC bond of the desaturated intermediate to form a carbon-based radical intermediate, corresponding to an energy barrier of 18.1 kcal/mol, then the radical intermediate completes the ring closure with a barrier of 3.9 kcal/mol. Besides, the calculations using the substrate analogous that lacks the N4-methyl reveal that the H atom abstraction by FeIV–oxo is still accessible, which suggests that the absence of N4-methyl does not affect the desaturation process itself but may influence the other processes that occur prior to the desaturation.

Tobacco-specific N'-nitrosonornicotine (NNN), a genotoxic nitrosamine classified as Group 1 carcinogen, is also present in atmospheric particulate matter and has even been detected as a new disinfection byproduct in wastewaters. NNN generally requires metabolic activation by cytochrome P450 enzymes to exert its genotoxicity, but the respective biotransformation pathways have not been described in detail. In this work, we performed density functional theory (DFT) calculations to unravel possible NNN activation pathways including α-hydroxylation, β-hydroxylation, pyridine N-oxidation, and norcotinine formation. The results reveal an initial rate-determining H-atom abstraction step for α-hydroxylation, followed by an unexpected kinetic competition between denitrosation and OH rebound, leading to ( iso-)myosmine as a detoxified product and α-hydroxyNNNs as the precursor of carcinogenic diazohydroxides, respectively. Further detoxification routes are given by β-hydroxylation with relative high reaction barrier and N-oxidation with comparable barrier to the toxifying α-hydroxylation. Moreover, we show for the first time how norcotinine can be generated as a minor NNN metabolite that is formed from iso-myosmine through a unique porphyrin-assisted H atom 1,2-transfer mechanism. These results demonstrate that the carcinogenic potential of NNN is subject to a kinetic competition between activating and deactivating metabolic routes, and identify respective biomarkers to inform about the individual risk associated with NNN exposure.

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Microplastics have become an emerging concerned global environmental pollution problem. their strong adsorption towards the coexisting organic pollutants can cause additional environmental risks. therefore, the adsorption capacity and mechanisms are necessary information for the comprehensive environmental assessments of both microplastics and organic pollutants. to overcome the lack of adsorption information, five quantitative structure-property relationship (QSPR) models were developed for predicting the microplastic/water partition coefficients (log K d) of organics between polyethylene/seawater, polyethylene/freshwater, polyethylene/pure water, polypropylene/seawater, and polystyrene/seawater. All the QSPR models show good fitting ability (R 2 = 0.811-0.939), predictive ability (Q 2 ext = 0.835-0.910, RMSE ext = 0.369-0.752), and robustness (Q cv 2 = 0.882-0.957). They can be used to predict the K d values of organic pollutants (such as polychlorinated biphenyls, chlorobenzene, polycyclic aromatic hydrocarbons, antibiotics perfluorinated compounds, etc.) under different pH conditions. the hydrophobic interaction has been indicated as an important mechanism for the adsorption of organic pollutants to microplastics. in sea waters, the role of hydrogen bond interaction in adsorption is considerable. For polystyrene, π-π interaction contributes to the partitioning. The developed models can be used to quickly estimate the adsorption capacity of organic pollutants on microplastics in different types of water, providing necessary information for ecological risk studies of microplastics. Microplastics, defined as plastics with particle size < 5 mm, have become one of the most prominent global environmental pollution problems 1,2. They may originate directly from industrial and personal products, or from the degradation of large-size plastics 3. For environmental management, we can ban the direct sources of microplastics to a certain extent. However, the wide application of plastic products in daily life makes hundreds of millions of tons of plastic waste, which definitely become the precursors of microplastics, be discharged into the environment each year 4. As a result, microplastics have been detected in waste water 5,6 , natural water 7,8 , and even in drinking water 9. At present, the pollution of microplastics has become a persistent environmental problem that needs to be urgently addressed. Therefore, comprehensive and accurate assessment of their environmental risks (e.g., environmental behavior and ecotoxicity) is particularly important for developing effective environmental policies. Previous studies proved that the large specific surface area makes microplastics show high adsorption capacity to the coexisting organic pollutants, such as polycyclic aromatic hydrocarbons 10 , polychlorinated biphenyls 11 , etc. Some ionizable organic pollutants (e.g., antibiotics) also can be adsorbed on microplastics 12. The adsorption interaction may further alter the behavior and toxicity of both microplastics and organic ...

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