O–O Bond Formation and Oxygen Release in Photosystem II Are Enhanced by Spin-Exchange and Synergetic Coordination Interactions

The photosystem II (PSII)-catalyzed water oxidation is crucial for maintaining life on earth. Despite the extensive experimental and computational research that has been conducted over the past two decades, the mechanisms of O–O bond formation and oxygen release during the S3 ∼ S0 stage remain disputed. While the oxo-oxyl radical coupling mechanism in the “open-cubane” S4 state is widely proposed, recent studies have suggested that O–O bond formation may occur from either the high-spin water-unbound S4 state or the “closed-cubane” S4 state. To gauge the various mechanisms of O–O bond formation proposed recently, the comprehensive QM/MM and QM calculations have been performed. Our studies show that both the nucleophilic O–O coupling from the Mn4 site of the high-spin water-unbound S4 state and the O5–O6 or O5–OW2 coupling from the “closed-cubane” S4 state are unfavorable kinetically and thermodynamically. Instead, the QM/MM studies clearly favor the oxyl-oxo radical coupling mechanism in the “open-cubane” S4 state. Furthermore, our comparative research reveals that both the O–O bond formation and O2 release are dictated by (a) the exchange-enhanced reactivity and (b) the synergistic coordination interactions from the Mn1, Mn3, and Ca sites, which partially explains why nature has evolved the oxygen-evolving complex cluster for the water oxidation

Biodegradation of 2,5-Dihydroxypyridine by 2,5-Dihydroxypyridine Dioxygenase and Its Mutants: Insights into O–O Bond Activation and Flexible Reaction Mechanisms from QM/MM Simulations

2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 is a mononuclear non-heme iron oxygenase responsible for the biodegradation of 2,5-dihydroxypyridine (DHP) to N-formylmaleamic acid (NFM). Here, extensive quantum mechanical–molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations are used to elucidate the degradation mechanism of DHP by wild-type NicX and its H105F variant (NicXH105F) and the roles of key residues. In particular, NicX and NicXH105F can catalyze the ring opening degradation of DHP to NFM, but flexible mechanisms are adopted therein. Both reactions of NicX and NicXH105F are initiated by the attack of FeIII superoxide species onto the substrate, during which a proton-coupled electron transfer (PCET) process is involved. For wild-type NicX, the PCET reaction is mediated by the adjacent His105, while the further proton transfer from His105 to the peroxo species can remarkably enhance the following O–O cleavage. However, for the NicXH105F mutant, a water molecule replaces the role of residue His105, which not only stabilizes the substrate binding via a H bonding network but also functions as a base to mediate the PCET process. For the NicXH105A mutant, MD simulations show that the disruption of the H bonding network can displace the substrate binding, leading to the loss of enzyme activity. These findings can expand our understanding of the PCET-mediated O–O bond activation and the flexible catalytic routes in various mutants, which have general implications on enzyme catalysis

Molecular Dynamics and QM/MM Calculations Predict the Substrate-Induced Gating of Cytochrome P450 BM3 and the Regio- and Stereoselectivity of Fatty Acid Hydroxylation

Theory predicts herein enzymatic activity from scratch. We show that molecular dynamics (MD) simulations and quantum-mechanical/molecular mechanics (QM/MM) calculations of the fatty acid hydroxylase P450 BM3 predict the binding mechanism of the fatty acid substrate and its enantio/regioselective hydroxylation by the active species of the enzyme, Compound I. The MD simulations show that the substrate’s entrance involves hydrogen-bonding interactions with Pro25, Glu43, and Leu188, which induce a huge conformational rearrangement that closes the substrate channel by pulling together the A helix and the β₁ sheet to the F/G loop. In turn, at the bottom of the substrate’s channel, residue Phe87 controls the regioselectivity by causing the substrate’s chain to curl up and juxtapose its CH₂ positions ω-1/ω-2/ω-3 to Compound I while preventing access to the endmost position, ω-CH₃. Phe87 also controls the stereoselectivity by the enantioselective steric blocking of the pro-<i>S</i> C–H bond, thus preferring <i>R</i> hydroxylation. Indeed, the MD simulations of the mutant Phe87Ala predict predominant ω hydroxylation. These findings, which go well beyond the X-ray structural data, demonstrate the predictive power of theory and its insight, which can potentially be used as a partner of experiment for eventual engineering of P450 BM3 with site-selective C–H functionalization capabilities

Computation Sheds Insight into Iron Porphyrin Carbenes’ Electronic Structure, Formation, and N–H Insertion Reactivity

Iron porphyrin carbenes constitute a new frontier of species with considerable synthetic potential. Exquisitely engineered myoglobin and cytochrome P450 enzymes can generate these complexes and facilitate the transformations they mediate. The current work harnesses density functional theoretical methods to provide insight into the electronic structure, formation, and N–H insertion reactivity of an iron porphyrin carbene, [Fe­(Por)­(SCH₃)­(CHCO₂Et)]⁻, a model of a complex believed to exist in an experimentally studied artificial metalloenzyme. The ground state electronic structure of the terminal form of this complex is an open-shell singlet, with two antiferromagnetically coupled electrons residing on the iron center and carbene ligand. <i>As we shall reveal, the bonding properties of [Fe­(Por)­(SCH</i>₃<i>)­(CHCO</i>₂<i>Et)]</i>^– <i>are remarkably analogous to those of ferric heme superoxide complexes.</i> The carbene forms by dinitrogen loss from ethyl diazoacetate. This reaction occurs preferentially through an open-shell singlet transition state: iron donates electron density to weaken the C–N bond undergoing cleavage. Once formed, the iron porphyrin carbene accomplishes N–H insertion via nucleophilic attack. The resulting ylide then rearranges, using an internal carbonyl base, to form an enol that leads to the product. The findings rationalize experimentally observed reactivity trends reported in artificial metalloenzymes employing iron porphyrin carbenes. Furthermore, these results suggest a possible expansion of enzymatic substrate scope, to include aliphatic amines. Thus, this work, among the first several computational explorations of these species, contributes insights and predictions to the surging interest in iron porphyrin carbenes and their synthetic potential

Enhancing Generalizability in Protein–Ligand Binding Affinity Prediction with Multimodal Contrastive Learning

Improving the generalization ability of scoring functions remains a major challenge in protein–ligand binding affinity prediction. Many machine learning methods are limited by their reliance on single-modal representations, hindering a comprehensive understanding of protein–ligand interactions. We introduce a graph-neural-network-based scoring function that utilizes a triplet contrastive learning loss to improve protein–ligand representations. In this model, three-dimensional complex representations and the fusion of two-dimensional ligand and coarse-grained pocket representations converge while distancing from decoy representations in latent space. After rigorous validation on multiple external data sets, our model exhibits commendable generalization capabilities compared to those of other deep learning-based scoring functions, marking it as a promising tool in the realm of drug discovery. In the future, our training framework can be extended to other biophysical- and biochemical-related problems such as protein–protein interaction and protein mutation prediction

Peroxo-Diiron(III/III) as the Reactive Intermediate for N‑Hydroxylation Reactions in the Multidomain Metalloenzyme SznF: Evidence from Molecular Dynamics and Quantum Mechanical/Molecular Mechanical Calculations

Upon oxygen activation, the non-heme diiron enzymes can generate various active species for oxidative transformations. In this work, the catalytic mechanism of the diiron active site (heme-oxygenase-like diiron oxidase (HDO) domain) in SznF has been comprehensively studied by molecular docking, classical molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) MD simulations, and hybrid QM/MM calculations. The HDO domain of SznF catalyzes the selective hydroxylation of Nω-methyl-l-arginine (l-NMA) to generate Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA) and Nδ,Nω-dihydroxy-Nω,-methyl-l-Arg (l-DHMA), which is a key step in the synthesis of the nitrosourea pharmacophore of the pancreatic cancer drug streptozotocin (SZN). Our study shows that the peroxo-diiron(III/III) intermediate in Sznf maintains a butterfly-like conformation, while the further protonation of the diiron(III/III) intermediate is found to be inaccessible and unfavorable thermodynamically. Among various mechanisms, we found that the most favorable mechanism involves the nucleophilic attack of the guanidium group onto the peroxo group of P1, which drives the heterolytic cleavage of the O–O bond. Moreover, the selectivity of N-hydroxylation by the peroxo-diiron(III/III) intermediate can be fully supported by MD simulations, suggesting that the peroxo-diiron(III/III) is the reactive intermediate for N-hydroxylation in SznF. The present study expands our understanding on the O2 activation and N-hydroxylation by the non-heme diiron enzymes

Preorganized Internal Electric Field Powers Catalysis in the Active Site of Uracil-DNA Glycosylase

Uracil-DNA glycosylase (UDG) is a monofunctional DNA glycosylase, which is involved in the base excision repair (BER) pathway and responsible for the excision of uracil from DNA. UDG is well known for its high catalytic efficiency and substrate autocatalysis character. Here, using quantum-mechanical/molecular-mechanical (QM/MM) and QM calculations as well as molecular dynamics (MD) simulations, we propose a revised catalytic mechanism of UDG and elucidate the nature of its strong catalytic efficiency. In the initial stage of the reaction, a heterolytic C–N bond cleavage is catalyzed by a strong internal electric field which stabilizes the charge distribution of the transition state more than that of the ground state, yielding an oxocarbenium-ion–uracil-anion species. The catalytic effect of substrate phosphate groups can be included in the effect of the internal electric field, which is counterbalanced by sodium ions, explaining the longstanding discrepancy between theory and experiment. Subsequently, Asp145 acts as the general base to activate the water nucleophile to attack the oxocarbenium ion, forming an abasic site. Interestingly, the proton further transfers from Asp145 to the neutral and more solvent-exposed residue His148 to facilitate its release, which is demonstrated to be driven by the internal electric field. This work deepens our understanding of the catalytic mechanism of UDG and more generally the catalytic effect of internal electric fields in enzymes

The Fe^III(H₂O₂) Complex as a Highly Efficient Oxidant in Sulfoxidation Reactions: Revival of an Underrated Oxidant in Cytochrome P450

This work demonstrates that the Fe^III(H₂O₂) complex, which has been considered as an unlikely oxidant in P450, is actually very efficient in sulfoxidation reactions. Thus, Fe^III(H₂O₂) undergoes a low-barrier nucleophilic attack by sulfur on the distal oxygen, <i>resulting in heterolytic O–O cleavage coupled to proton transfer</i>. We further show that Fe^III(H₂O₂) is an efficient sulfoxidation catalyst in synthetic iron porphyrin and iron corrolazine compounds. In all cases, Fe^III(H₂O₂) performs the oxidation <i>much faster than it converts to Cpd I</i> and will therefore bypass Cpd I in the presence of a thioether. Thus, this paper not only suggests a plausible resolution of a longstanding issue in P450 chemistry regarding the “second oxidant” but also highlights a new mechanistic pathway for sulfoxidation reactions in P450s and their multitude of synthetic analogues. These findings have far-reaching implications for transition metal compounds, where H₂O₂ is used as the terminal oxidant

Emergence of Function in P450-Proteins: A Combined Quantum Mechanical/Molecular Mechanical and Molecular Dynamics Study of the Reactive Species in the H₂O₂‑Dependent Cytochrome P450_SPα and Its Regio- and Enantioselective Hydroxylation of Fatty Acids

This work uses combined quantum mechanical/molecular mechanical and molecular dynamics simulations to investigate the mechanism and selectivity of H₂O₂-dependent hydroxylation of fatty acids by the P450_SPα class of enzymes. H₂O₂ is found to serve as the surrogate oxidant for generating the principal oxidant, Compound I (Cpd I), in a mechanism that involves homolytic O–O bond cleavage followed by H-abstraction from the Fe–OH moiety. Our results rule out a substrate-assisted heterolytic cleavage of H₂O₂ en route to Cpd I. We show, however, that substrate binding stabilizes the resultant Fe–H₂O₂ complex, which is crucial for the formation of Cpd I in the homolytic pathway. <i>A network of hydrogen bonds locks the HO· radical</i>, formed by the O–O homolysis, thus directing it to exclusively abstract the hydrogen atom from Fe–OH, thereby forming Cpd I, while preventing the autoxoidative reaction, with the porphyrin ligand, and the substrate oxidation. The so formed Cpd I subsequently hydroxylates fatty acids at their α-position with <i>S</i>-enantioselectivity. These selectivity patterns are controlled by the active site: substrate’s binding by Arg241 determines the α-regioselectivity, while the Pro242 residue locks the prochiral α-CH₂, thereby leading to hydroxylation of the <i>pro</i>-<i>S</i> C–H bond. Our study of the mutant Pro242Ala sheds light on potential modifications of the enzyme’s active site in order to modify reaction selectivity. Comparisons of P450_SPα to P450_BM3 and to P450_BSβ reveal that <i>function</i> has evolved <i>in these related metalloenzymes by strategically placing very few residues in the active site</i>

Proton-Shuttle-Assisted Heterolytic Carbon–Carbon Bond Cleavage and Formation

The conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) catalyzed by DXP reductoisomerase (DXR) is the committing step in the biosynthesis of terpenoids. This MEP pathway is essential for most pathogenic bacteria but absent in human, and thus, it is an attractive target for the development of novel antibiotics. To this end, it is critical to elucidate the conversion mechanism and identify the transition state, as many drugs are transition-state analogues. Here we performed extensive combined quantum mechanical (density functional theory B3LYP/6-31G*) and molecular mechanical molecular dynamics simulations to elucidate the catalytic mechanism. Computations confirmed the transient existence of two metastable fragments of DXP by the heterolytic C3–C4 bond cleavage, namely, 1-propene-1,2-diol and glycoaldehyde phosphate, in accord with the most recent kinetic isotope effect (KIE) experiments. Significantly, the heterolytic C3–C4 bond cleavage and C2–C4 bond formation are accompanied by proton shuttles, which significantly lower their reaction barriers to only 8.2–6.0 kcal/mol, compared with the normal single carbon–carbon bond energy 83 kcal/mol. This mechanism thus opens a novel way for the design of catalysts in the cleavage or formation of aliphatic carbon–carbon bonds