Metal-free catalysis for electrocatalytic hydrogen evolution from water is very demanding for the production of sustainable and clean fuel. Herein, we report the synthesis of a porphyrin-based metal-free covalent organic polymer (TpPAM) through a simple condensation between triformyl phloroglucinol (Tp) and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (PAM). The as-prepared porous TpPAM exhibited superior activity for the hydrogen evolution reaction (HER) current density of 10 mA cm −2 at a low overpotential of 250 mV and a small Tafel slope of 106 mV decade −1 , which are better than those of related metal-free electrocatalysts. The high HER activity of TpPAM was investigated in-depth via theoretical density functional theory (DFT) calculations. The theoretical findings were correlated with the experimental results, and these were in good agreement for high HER catalytic efficiency of the porous TpPAM polymer. The Faradaic efficiency of the TpPAMbased electrode was found to be 98%, which is very close to the ideal value of 100%, reflecting its potential for practical implementation. Moreover, the as-synthesized catalyst showed good stability by retaining 91% of the initial current density after 1000 cycles.

*Modern DFT functional as well as the continuum solvation model have been applied in order to theoretically predict carbon, chlorine and hydrogen kinetic isotope effects (KIEs) during aerobic degradation of four hexachlorocyclohexane isomers (α, β, δ and γ). A small model of the hexachlorocyclohexane dehydrochlorinase (LinA) active site comprising its catalytic dyad has been constructed based on the receptor-ligand complexes suggested by docking studies and compared to the respective reaction modeled in aqueous solution. In-depth analysis of chlorine and hydrogen KIEs patterns clearly indicates different transition states in aqueous solution and in the model mimicking the protein active site. Although all isomers seem to undergo a concerted E2 mechanism, the contribution of proton transfer and carbonchlorine leaving group bond stretch in the transition states differs for the different isomers giving rise to totally different magnitudes of predicted isotope effects.

The elucidation of the catalytic role of LinA dehydrohalogenase in the degradation processes of hexachlorocyclohexane (HCH) isomers is extremely important to further studies on the bioremediation of HCH polluted areas. Herein, QM/MM free energy simulations are employed to provide the details of the dehydrochlorination reaction of two HCH isomers (γ and β). In particular, the role of the protonation state of one of the catalytic residues-His73-is explored. Based on our calculations, two distinct minimum free energy pathways (concerted and stepwise) were found for γ-HCH and β-HCH. The choice of the reaction channel for the dehydrochlorination reactions of γ- and β-HCH was shown to depend on the initial mutual orientations of the reacting species in the active site and the protonation form of His73. The sequential pathway comprises the transfer of the proton (Hδ1) between His73 and Asp25 and subsequently the H1/Cl2 pair elimination from the substrate molecule. Within a concerted mechanism, the dehydrochlorination reaction of γ-/β-HCH is initiated with neutral His73 and the Hδ1 proton is transferred upon final product formation. We found that the concerted pathway for β-HCH results in significantly higher free energy of activation than the stepwise route and therefore can be disregarded as not a feasible mechanism. On the other hand, the reaction that occurs with much lower energetic barrier requires a stronger base (i.e., anionic His73) to abstract the proton (H1) from the substrate molecule. The presence of such transient form of His results in higher energy than the respective Michaelis complex and was observed only in the stepwise pathway for both isomers. Furthermore, we have concluded that both pathways (concerted and stepwise) are feasible for the dehydrochlorination reaction of γ-HCH. The activation free energies obtained from the M05-2X/6-31+G(d,p) corrected path coordinate PMF profiles for the dehydrochlorination reactions of the γ-/β-HCH are in good agreement with the experimental values.

2,4′-Dihydroxyacetophenone dioxygenase (DAD), a nonheme dioxygenase enzyme, shows exquisite selectivity in the aliphatic C–C bond cleavage of 2,4′-dihydroxyacetophenone (DHAP) in the presence of molecular oxygen (O2). Molecular dynamics simulations revealed the presence of a single water molecule at the active site of the enzyme. This lone water molecule is pivotal for facilitating the oxidative cleavage of the aliphatic C–C bond of 2,4′-DHAP catalyzed by DAD enzyme, as evident from the findings of our hybrid quantum mechanics/molecular mechanics (QM/MM) studies. 2,4′-DHAP is initially deprotonated through a relay proton transfer mechanism with the aid of the active site water molecule. This water molecule also actively participates in the O–O and C–C bond cleavage steps. The activated water molecule acts as catalytic acid–base species. The O–O cleavage step has been predicted to be the rate-determining step with an associated barrier of 20.3 kcal/mol calculated at the uB3LYP-D3/def2-TZVP/OPLS level of theory on the quintet spin surface. Multiple sequence alignment of the bacterial DAD enzyme has shown the evolutionary importance of the Tyr93 and Glu108 residues, in which Tyr93 acts as proton carrier and Glu108 acts as reservoir, during the relay proton transfer. Our study demonstrates the active role of water in the catalytic cycle of DAD enzyme and additionally unearthed important indirect roles of the two amino acids (Tyr93 and Glu108) in the enzymatic cycle.

Despite their routine use as protein denaturants, the comprehensive understanding of the molecular mechanisms by which urea and guanidinium chloride (GdmCl) disrupts proteins' structure is still lacking. Here, we use steered molecular dynamics simulations along with the umbrella sampling technique to elucidate the mechanism of unfolding of chicken villin headpiece (HP-36) in these two denaturants. We find that while urea denatures protein predominantly by forming hydrogen bonds with the protein backbone, GdmCl commences unfolding by weakening of the hydrophobic interactions present in the core. The potential of mean force calculation indicates the reduction of hydrophobic interactions between two benzene moieties in 6 M GdmCl as compared to 6 M urea. We observe a near parallel orientation between the guanidinium cation and aromatic side chains of the HP-36 suggesting π-cation type stacking interactions which play a crucial role in weakening of the hydrophobic interaction. We use QM/MM optimization calculations to estimate the energetics of this π-cation interaction. Additionally, the consistency of the unfolding paths between high temperature (400 K) unfolding simulations and steered molecular dynamics simulations strengthens the proposed molecular mechanism of unfolding further.

Large-scale conformational transition from open to closed state of adenylate kinase (ADK) is essential for its catalytic cycle. Apo-ADK undergoes conformational transition in a way that closely resembles an open-to-closed conformational transition. Here, equilibrium simulations, free-energy simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations in combination with several bioinformatics approaches have been used to explore the molecular origin of this conformational transition in apo-ADK. In addition to its conventional open state, Escherichia coli apo-ADK adopts conformations that resemble a closed-like intermediate, the “half-open-half-closed” (HOHC) state, and a π–cation interaction can account for the stability of this HOHC state. Energetics and the electronic properties of this π–cation interaction have been explored using QM/MM calculations. Upon rescinding the π–cation interaction, the conformational landscape of the apo-ADK changes completely. The apo-ADK population is shifted completely toward the open state. This π–cation interaction is highly conserved in bacterial ADK; the cationic guanidinium moiety of a conserved ARG interacts with the delocalized π-electron cloud of either PHE or TYR. Interestingly, this study demonstrates the modulation of a principal protein dynamics by a conserved specific π–cation interaction across different organisms.

Our findings suggest the definitive requirement of a proton relay process mediated by a water-chain and the Glu1742 residue in the ATP hydrolysis reaction of a dynein motor.

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