Luca Bertini

The enzymatic degradation of polyethylene (PE) by oxidative enzymes remains poorly understood at the molecular level, limiting the development of effective biotechnological strategies for plastic valorization. Here, we investigate the molecular basis of PE oxidation by the laccase-like multicopper oxidase LMCO2 from Rhodococcus opacus R7 with combined DFT and molecular docking calculations. LMCO2 is a particularly relevant model system because it has been experimentally shown to oxidatively modify low-density polyethylene without the need for mediators, despite its relatively low redox potential. DFT calculations indicate that direct oxidation of aliphatic C–H bonds in pristine PE is energetically prohibitive, and that hydroxylated defects do not significantly facilitate substrate activation. In contrast, oxidation at carbon atoms adjacent to carbonyl groups is associated with substantially lower barriers, owing to stabilization of the resulting radicals through conjugation and keto–enol tautomerism. Alternative pathways involving alkoxyl radicals and β-scission were also examined and found to be energetically unfavorable. Molecular docking calculations complement this picture by showing that both pristine and pre-oxidized PE segments can access the main cavity adjacent to the T1 copper site, although binding is weak and largely non-specific. Taken together, these results support a mechanism in which LMCO2 selectively acts on pre-oxidized, carbonyl-containing regions of polyethylene, promoting further radical chemistry that may ultimately lead to chain scission and the formation of low-molecular-weight oxygenated products. Graphical Abstract

[FeFe]-hydrogenases are highly efficient enzymes in the reversible catalysis of molecular hydrogen production and oxidation. Their active site, the H-cluster, consists of a [4Fe-4S]_H subcluster linked to a binuclear [2Fe]_H organometallic unit. Many [FeFe]-hydrogenases, such as the one from Desulfovibrio desulfuricans (DdHydAB), possess accessory Fe-S clusters (F and F') that mediate electron transfer. This study employs hybrid quantum mechanics/molecular mechanics (QM/MM) methods to characterize the electronic structure and thermodynamic landscape associated with redox and protonation events in the complete Fe-S cluster network of DdHydAB. Our calculations indicate that the F' cluster plays a key role in the initial reduction of the oxidized resting state, acting as the preferential site for the accumulation of the first electron. Analysis of protonated states, upon reduction events, reveals a strong correlation between protonation and electron transfer (PCET), with protonation at the H-cluster inducing electron transfer from the F' cluster to the H-cluster. Calculations indicate that the formation of a terminal hydride is energetically favored over ADT protonation, and subsequent isomerization to a bridging hydride (μ-H) is further stabilizing, albeit potentially kinetically limiting. The study highlights how accessory clusters influence the electronic distribution and redox properties of the H-cluster, underscoring the importance of considering the entire Fe-S cluster system for a complete understanding of the catalytic mechanism of [FeFe]-hydrogenases.

The performance of artificial molecular machines relies on the interplay between molecular design and environmental factors, yet how solvation shapes their energy landscapes and kinetics remains poorly understood. Here, we combine well-tempered and infrequent metadynamics to investigate equilibrium shuttling in a minimal [2]rotaxane inspired by Borsley's fuel-driven molecular motor. By systematically varying solvent polarity and hydrogen-bonding capacity, we uncover distinct thermodynamic and kinetic regimes that govern macrocycle motion. In highly polar, hydrogen-bond-accepting media, the macrocycle adopts a symmetric distribution between binding sites, with enthalpic and entropic forces in direct competition. Conversely, in low-polarity, hydrogen-bond-donating environments, the axle undergoes a conformational collapse that entropically biases occupancy toward a single station in the absence of chemical fuel. Despite comparable free-energy barriers across conditions (9-13 kcal/mol), the transition pathways exhibit pronounced solvent-dependent asymmetries and energetic ruggedness. These findings provide a molecular-level framework for understanding how solvation dictates passive ratchet behavior and offer strategic insights for designing high-performance molecular machines tailored to complex media.

The origin of recently reported anomalous fluorescence emissions from aqueous solutions of nonaromatic solutes remains elusive. To determine whether the solute nature influences the fluorescence characteristics and to identify a potential common mechanism, we measured the fluorescence spectra of 21 different solutions. We observed similar emission characteristics across all samples, suggesting that the solute nature plays a minimal role in the emission mechanism. Using time-dependent density functional theory on large water, NaCl/water, and glycerol/water clusters, we attributed the anomalous emission to the decay of charge-transfer-to-solvent excitations (CTTS) which populate a diradical zwitterionic excited state localized at hydrogen-bond network defects. The Arrhenius-like plots for NaCl and glycerol solutions revealed that the S₁ nonradiative decay pathway involves the diradical recombination via librational motion. We propose that the presence of solute molecules slows this process, thus increasing the lifetime of the CTTS excited states and facilitating emission.

Laccases that oxidize low‐density polyethylene (LDPE) represent a promising strategy for bioremediation purposes. To rationalize or optimize their PE‐oxidative activity, two fundamental factors must be considered: the enzyme‘s redox potential and its binding affinity/mode towards LDPE. Indeed, a stable laccase‐PE complex may facilitate a thermodynamically unfavorable electron transfer, even without redox mediators. In this study, we compared the redox potential and the LDPE‐binding properties of three different PE‐oxidizing laccases: a fungal high‐redox potential laccase from Trametes versicolor, a bacterial low‐redox potential laccase from Bacillus subtilis, and the recently characterized LMCO2 from Rhodococcus opacus R7. First we found that LMCO2 is a low‐potential laccase (E°=413 mV), as reported in other bacterial variants. Using computational tools, we simulated the interactions of these laccases with a large LDPE model and highlighted the key role of hydrophobic residues surrounding the T1 site. Notably, a methionine‐rich loop in LMCO2 appears to enhance the formation of a stable complex with LDPE, potentially facilitating electron transfer. This study underscores the necessity for comprehensive computational strategies to analyze enzyme‐polymer interactions beyond simplistic models, uncovering critical binding determinants and informing future mutagenesis experiments, in order to enhance laccase performance and rationalize variations in enzymatic activity.

The reactivity of laccases towards non-phenolic polycyclic aromatic hydrocarbons remains unclear. Using molecular modelling the oxidation of anthracene and benzo[a]pyrene by Trametes versicolor fungal laccase is investigated.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Oxidative stress and metal dyshomeostasis are considered crucial factors in the pathogenesis of Alzheimer’s disease (AD). Indeed, transition metal ions such as Cu(II) can generate reactive oxygen species (ROS) via O₂ Fenton-like reduction, catalyzed by Cu(II) coordinated to the amyloid-beta (Aβ) peptide. Despite intensive efforts, the mechanisms of ROS-induced molecular damage remain poorly understood. In the present paper, we investigate, on the basis of Density Functional Theory (DFT) computations, a possible mechanism of the OH radical propagation toward membrane phospholipid polar head and fatty acid chains starting from the end-product of the OH radical generation by Cu(II)-Aβ. Using phosphatidylcholine as a model of a single unit inside a membrane, we evaluated the thermochemistry of the OH propagation with the oxidation of a C-H bond and the formation of the radical moiety. The DFT results show that Cu(II)-Aβ-OH can oxidize only sn-2 C-H bonds of the polar head and can easily oxidize the C-H bond adjacent to the carbon–carbon double bond in a mono or bis unsaturated fatty acid chain. These results are discussed on the basis of the recent literature on in vitro Aβ metal-catalyzed oxidation and on the possible implications in the AD oxidative stress mechanism.