Isabella Daidone

Water molecules are key actors in enzymatic proton-coupled electron transfer (PCET), yet their mechanistic versatility remains underexplored. Using a multiscale computational approach, we show that a previously unidentified water molecule (Wat3) in fatty acid photodecarboxylase (FAP), a natural photoenzyme converting fatty acids into alkanes, mediates radical quenching via two PCET pathways: in the dominant one, which qualitatively agrees with the 100 ns experimental time scale, Wat3 stabilizes the transition state for direct proton transfer from Cys432 to the alkyl radical; in the alternative, the same molecule acts as a proton shuttle in a quasi-concerted two-proton transfer, with the flavin semiquinone providing the electron in both cases. The minor pathway, though less accessible in wild-type FAP, may become operative in engineered variants or related enzymes. Additionally, CO2 reacts with the thiolate to yield a thiocarbonate adduct, explaining unassigned crystallographic electron density. These results show how a single water molecule encodes mechanistic diversity in enzymatic PCET.

The dynamics of water in electrolyte solutions exhibits a striking, ion-specific anomaly: the diffusion coefficient of water is enhanced relative to the neat liquid in chaotropic CsI solutions, yet suppressed in kosmotropic NaCl solutions. This phenomenon, long challenging for classical force-field-based molecular dynamics, is studied here using classical molecular dynamics simulations with a many-body machine-learned force field trained within the MACE equivariant graph neural network framework. The force field is trained on energies, forces, and stresses computed at the density functional theory level with the revPBE-D3 exchange–correlation functional, which provides a reliable balance between accuracy and computational efficiency for aqueous systems. Simulations of NaCl and CsI aqueous solutions under ambient conditions over a concentration range of 0.89–3.56 mol/kg reproduce the experimentally observed anomalous diffusion and yield a quantitative improvement over previous results obtained with the DeePMD framework, which is trained on the same theory, particularly for NaCl solutions. This improvement is traced to a stronger Na+–water interaction in the first hydration shell and the non-negligible retarding contribution of the second hydration shell of Na+. For CsI solutions, the water acceleration is shown to be primarily driven by the anion I−, whose diffuse and weakly structured hydration shell facilitates rapid water exchange with the bulk. These results are rationalized through a shell-decomposition analysis of time-dependent water diffusivities and ion–oxygen potentials of mean force, providing a coherent microscopic picture of the acceleration–retardation mechanism in the studied aqueous electrolytes.

The interaction of a biologically active molecule, ethionamide (ETH), with a covalent porphyrinic cage in its open conformation, was studied by means of steady-state and time-resolved optical spectroscopies. The cage is composed of two free-base porphyrins, linked four times by ethylene glycol-based pillars through triazole units. The latter are able to bind to Ag(I) ions, resulting in the opening of the cage cavity. Both absorption and emission properties of the open cage are modified upon addition of ETH, indicating an interaction between the porphyrin array and the added organic molecule, which significantly affects the photophysical properties of the former. The nature of this interaction has been investigated by means of ultrafast transient absorption spectroscopy, NMR studies, quantum mechanical calculations and molecular dynamics simulations. The results indicate that two ETH molecules interact with two Ag(I) ions coordinated to the cage, resulting in their removal. Consequently, the cage retains only two Ag(I) ions, inducing a conformational change to a semi-closed V-shaped form, with a shorter distance between the porphyrins. Complexation of the cage with Na ⁺ is also performed to support the proposed rationale.

Membraneless compartmentalization via liquid–liquid phase separation (LLPS) has emerged as a powerful strategy to organize biochemical reactions. Recently, peptide‐based coacervates demonstrated the potential to function as microreactors by enhancing reaction kinetics through increased local concentrations and altered microenvironments. Here, we introduce an O‐methylated diphenylalanine‐based tripeptide ^LLL PFF‐OCH ₃ containing an N‐terminal proline, designed to undergo LLPS, and simultaneously function as an enantioselective organocatalyst. Comprehensive characterization via confocal microscopy, fluorescence recovery after photobleaching (FRAP), micro‐Raman and attenuated total reflection infrared (ATR‐IR) spectroscopy, diffusion‐surface plasmon resonance ( D ‐SPR), and molecular dynamics (MD) simulations revealed the formation of stable liquid droplets. In contrast, a racemic mixture of ^LLL PFF‐OCH ₃ and ^DDD PFF‐OCH ₃ failed to form liquid droplets and instead formed a solid precipitate, unveiling a critical role of enantiopurity in LLPS. Proof‐of‐concept catalytic studies proved enantioselective organocatalytic activity of the ^LLL PFF‐OCH ₃ liquid coacervates. Beyond catalysis these results may have broader implications in understanding prebiotic chemistry and neurodegeneration.

Cross-couplings are essential reactions in modern chemical synthesis, enabling the rapid construction of complex molecules from simple precursors. Transition metal catalysts are prized for these transformations because their reactivity and selectivity can be tuned via judicious selection of the metal and ligand. Although enzymes offer analogous opportunities for tuning via protein engineering, their application to cross-coupling remains limited, as nature relies on alternative paradigms for building molecular complexity. Here, we report the cross-coupling of alkyl halides and benzylic carboxylic acids using an engineered flavin-dependent lactate monooxygenase—a photoenzyme. The enzyme achieves this feat by exploiting the redox versatility of the flavin cofactor. Stoichiometric experiments, ultrafast spectroscopy, and computational studies support a mechanism in which photoexcited flavin quinone initiates the reaction via oxidative decarboxylation to generate a benzylic radical. The resulting flavin semiquinone can reduce the alkyl halide to form a second organic radical within the protein active site, which rapidly engages in C(sp ³ )–C(sp ³ ) bond formation. A variant was engineered to control the stereochemical outcome of this radical–radical coupling event, highlighting the ability of the protein to alter the energetic barrier for a mechanistic step that is traditionally understood to be near barrierless. This work demonstrates that the scope for nonnative reaction mechanisms in biocatalysis far exceeds previously established bounds and has potential to solve a variety of reactivity challenges in cross-coupling chemistry.

A graph-theory framework allows hydrogen-bond network mapping beyond the first solvation shell, capturing local connectivity and long-range organization.

Fatty acid photodecarboxylase (FAP) is one of the few known natural photoenzymes and has attracted considerable interest due to its ability to convert fatty acids into hydrocarbons upon photoexcitation of its oxidized flavin adenine dinucleotide (FAD) cofactor. Notably, FAD in FAP exhibits an absorption spectrum red-shifted by approximately 10-15 nm compared to many other flavoproteins. This shift might arise from the specific electrostatics of the binding pocket and/or the slightly bent conformation of the FAD, as suggested by the crystallographic data. During the photocycle, an even more red-shifted intermediate (FAD_RS) has been observed, which ultimately reverts to the original state. In this work, we simulate the absorption spectrum of FAD inside FAP using a hybrid computational approach that combines quantum mechanics (QM) and molecular dynamics (MD) simulations in the Perturbed Matrix Method (PMM) framework. The computed absorption spectrum matches and explains the experimental one, not only validating the effectiveness of the MD-PMM approach but also revealing that the observed red shift primarily originates from the electrostatic environment provided by the protein matrix, whereas the effect of bending is comparatively minor. Additionally, we show that the formation of FAD_RS is unrelated to changes in active-site residue protonation or FAD conformation, but instead is likely to arise from a stable interaction between the flavin ring and bicarbonate, one of the proposed reaction products.

Fatty acid photodecarboxylase catalyzes the light-driven decarboxylation of fatty acids into hydrocarbons via electron transfer (ET) from the substrate to the flavin adenine dinucleotide cofactor, proceeding through either proton-coupled ET or hydrogen-atom transfer. Through quantum mechanics/molecular mechanics calculations, we show that only the deprotonated fatty acid supports the charge-transfer states required to initiate catalysis. Molecular dynamics simulations combined with graph-theory-based analysis reveal that the crystallographic water site, Wat1, is consistently occupied by a stable, yet dynamically exchanging, population of water molecules. We also identify transient, solvent-accessible water channels connecting the active site to the bulk solvent, potentially facilitating proton transfer and water exchange. These findings support the notion that beyond preserving the structural integrity of the active site, this water population may also enable the flexible modulation of electron and proton transfer through an adaptive hydrogen-bonding network.