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A large number of catalysts for hydrogen evolution reaction (HER) that are related (structurally and/or functionally) to the active site of [FeFe]-hydrogenases (H-cluster) has been proposed in the past few decades. Very recently, a novel recipe for HER catalysts has been added to the list. This consists in the combination of a diiron core, a ligand that can act as an intramolecular weak base to support protonation (both features that are common to the H-cluster), and a peculiar carbyne ligand that bridges the two metal centers. For instance, complex [Fe2{μ-CNMe2}(μ-CO)(CO)(CN)Cp2] (4) proved to be catalytically active towards HER. The novelty related to this family of biomimicry is not only structural, but also mechanistic. Indeed, an unprecedented (in the context of hydrogenase mimicry) ligand-based mechanism of HER, which avoids protonation at metal, has been dissected by DFT. In order to increase catalytic activity of this “non dithiolate based” system, a systematic search of ligand modification is therefore desirable. In this regard, herein we present results of replacing CN− (the proton shuttle in 4) by other ligands such as CNCH2Ph (5), PPh3 (6) or azide (7). The redox behavior of these compounds has been tested, in the absence and presence of a proton source. CV experiments combined with DFT calculations have revealed and rationalized critical points entailed by modifications of stereo-electronic features of 4. However, understanding the key factors underlying the absence of catalytic activity of these new compounds can be beneficial for the design of future improved biomimicry related to HER. As an example, properly tailored hydrocarbyl complexes replacing dithiolates at the Fe2 core might allow recovering the fruitful electronic properties of cyanides, when coordinated to metal centers.

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 μm. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest “in vitro” activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Quantum chemical methods allow one to investigate chemical aspects that are often difficult to evaluate using only experimental approaches. In particular, the continuous increase in reliability and speed of quantum chemical methods has recently allowed the investigation of very complex molecular systems, such as biological macromolecules. In this contribution, we present applications of quantum chemical methods to the investigation of reaction paths of metalloenzymes and related biomimetic models, using hydrogenase models as a reference case. In particular, we discuss several examples from the literature, emphasizing the possibilities (and limitations) offered by present theoretical approaches to study structures, electronic properties and reactivity of metalloenzyme models. Some relevant aspects which have not yet been fully explored using theoretical methods, such as the role of antiferromagnetic coupling and photochemical reactions in [Fe] hydrogenases, are treated in more detail, with presentation and discussion of original data recently obtained in our laboratory.

It was recently discovered that some redox proteins can thermodynamically and spatially split two incoming electrons towards different pathways, resulting in the one-electron reduction of two different substrates, featuring reduction potential respectively higher and lower than the parent reductant. This energy conversion process, referred to as electron bifurcation, is relevant not only from a biochemical perspective, but also for the ground-breaking applications that electron-bifurcating molecular devices could have in the field of energy conversion. Natural electron-bifurcating systems contain a two-electron redox centre featuring potential inversion (PI), i. e. with second reduction easier than the first. With the aim of revealing key factors to tailor the span between first and second redox potentials, we performed a systematic density functional study of a 26-molecule set of models with the general formula Fe2 (μ-PR2 )2 (L)6 . It turned out that specific features such as i) a Fe-Fe antibonding character of the LUMO, ii) presence of electron-donor groups and iii) low steric congestion in the Fe's coordination sphere, are key ingredients for PI. In particular, the synergic effects of i)-iii) can lead to a span between first and second redox potentials larger than 700 mV. More generally, the "molecular recipes" herein described are expected to inspire the synthesis of Fe2 P2 systems with tailored PI, of primary relevance to the design of electron-bifurcating molecular devices.

This perspective aims at illustrating a computational viewpoint on some specific issues concerning structure–activity relationships related to [FeFe]-hydrogenase ([FeFe]-H2ase) biomimicry. Most of the research outlined herein has been addressed by means of density functional theory (DFT) based tools, in some cases in conjunction with experimental techniques. The number of computational, experimental and mixed approach studies on the “hydrogenase models” topic is extraordinarily high. Accordingly, several comprehensive reviews have been published over the last twenty years. Moreover, the number of iron compounds of increasing nuclearity that have been considered as “biomimetic models” of [FeFe]-H2ase has grown exponentially over time. Additionally, the issues regarding some mismatches existing between the natural system and related synthetic analogues, are quite variegated. As a consequence of the countless examples that could be considered as related to “hydrogenase mimicry”, the intent of the present contribution is to provide an account of a limited number of recent study cases, in which DFT has been employed to elucidate redox properties, reactivity issues of selected examples of diiron biomimicry. Herein the treated topics have been explicitly chosen to show how DFT has allowed us to rationalize and complement experimental observations, with a special focus on electrocatalysis aspects. Finally, the specific purpose of this perspective is to show how mutually intertwined are the various issues concerning a fascinating branch of biomimetic research.

Stability is a crucial feature for enzymes employed in biocatalytic processes where they might be exposed to non-natural or even harsh environments. We have studied the behaviour of the lipase from Burkholderia glumae (BGL) by exposing it to challenging experimental conditions and characterizing changes in conformation and activity by a large set of biochemical, biophysical (mainly ESI-mass spectrometry, CD, fluorimetry) and computational methods. We observed that deactivation is not strictly related to structural instability in the assay conditions (temperature, pH, solvents), in fact (i) thermal deactivation precedes denaturation; (ii) acid-induced deactivation arises at higher pH than partial or global protein unfolding; and (iii) activity in most organic solvents decreases at solvent concentrations where conformation is fully retained. In the native protein, calcium is not accessible unless specific flexible loops are displaced, for example, by a temperature increase. Such movements concern the whole calcium-binding pocket and particularly the environment of the coordinating aspartate residue 241. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. Our results are consistent with local unfolding phenomena causing deactivation and in a complex interplay between the mobility of loop structures and the ability of the protein to retain stabilizing Ca2+. This might suggest that a straightforward structural stabilization by site-directed mutagenesis aimed at increasing the general or local rigidity of the protein could be ineffective in relieving the subtle and elusive molecular effects that induce loss of activity in the “twilight” zone, i.e. under conditions that are mild enough to avoid protein unfolding but sub-optimal for activity. In this scenario, random approaches such as mutagenesis by directed evolution may prove to be more effective.

Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca2+ influx in basophils and mast cells, analogous to β-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of FcεRI+ cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1–86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46–60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded β-chemokine homologue that influences the directional migration of human FcεRI+ cells and the expression of surface receptor CCR3 on these cells.

Hydrogenases are enzymes that catalyze the oxidation and production of molecular hydrogen. For about fifteen years, there have been many reports about the successful connection of these enzymes to photosensitizers with the aim of designing H2 photoproduction systems, but relatively little attention has been paid to whether and why illumination may affect the catalytic properties of the enzyme. In all hydrogenases, hydrogen activation occurs at an inorganic active site that includes at least one Fe-carbonyl motif, which may make it sensitive to irradiation. Here we review the evidence that hydrogenases are indeed photosensitive. We focus mainly on the so-called FeFe hydrogenases; their active site, called the H-cluster, consists of a [4Fe4S] cluster that is bound by a cysteine sulfur to a diiron site. The iron atoms of the binuclear cluster are coordinated by carbonyl and cyanide ligands and an azadithiolate group. We describe the effects of UV-visible light irradiation on the enzyme under cryogenic or turnover conditions and the photoreactivity of model complexes that mimic the diiron site. We emphasize the dependence of the photochemical processes on wavelength, and warn about FeFe hydrogenase photoinhibition, which should probably be considered when attempts are made to use FeFe hydrogenases for the artificial photosynthesis of solar fuels. We also underline the relevance of studies of synthetic mimics of the H-cluster for understanding at atomistic level the photochemical processes observed in the enzyme.

The potentials of redox systems involving nitrogen, oxygen, and metal ions of the first-row transition series have been computed according to the general approach of the grand canonical ensemble, which leads to the equilibrium value of the reduction potential via a (complete) sampling of configuration space at a given temperature. The approach is a single configuration approach in the sense that identical molecular structures are sampled for both the oxidized and reduced species considered in water solution. In this study, the solute and a cluster of 11-12 water molecules are treated explicitly at the same level of theory and embedded in a continuum solvent. The molecular energies are computed in the framework of the density functional theory. Our approach is basically different from the approach based on the ThermoDynamic Cycle involving gas-phase calculations of the electron affinity of the oxidized species, corrected by the differential hydration energy (obtained from continuum solvent models only) between oxidized and reduced forms. The calculated redox potentials are in agreement with the available experimental data much closer than other results so far presented in the literature. Our results are very satisfactory also in the case of the 3+/2+ redox states of the first-row transition metals, i.e., systems with a high positive charge for which enhanced effects of the solvent are expected.

International audience; The synthesis, characterization, and protonation of [Fe2(CO)6{(μ-SCH2)2(Et)P═O}] (1) using the moderately strong acid CF3CO2H (pKaMeCN = 12.7) are reported. Digital simulations of the cyclic voltammetry of 1 in the presence of CF3CO2H and DFT calculations have allowed us to obtain a detailed mechanistic picture of the processes underlying the catalytic hydrogen evolution reaction (HER) that 1 can mediate. Moreover, DFT has shed light on the role of the P═O functionality in the whole catalytic cycle of proton reduction. The reductive behavior of 1 features a double electron transfer with potential inversion, which is associated with deep structural rearrangement of the catalyst. The double reduction appears also functional to the intramolecular proton transfer from the P═O group to the diiron core, a crucial process for the H+/H– heterocoupling yielding H2. The key intermediate for the H2 formation and release is predicted to be a 3H+/3e– species, in which P═O is perfectly poised to shuttle protons from solution to the Fe–H–Fe moiety. Therefore, the R-P═O bridgehead installed in a dithiolato linker of a diiron core proves a valid and versatile alternative to the natural nitrogen-based Fe2 strap.

Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4–previously identified as a water-soluble Ras inhibitor– targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.

FeIFeI Fe2(S2C3H6)(CO)6(µ-CO) (1a–CO) and its FeIFeII cationic species (2a+–CO) are the simplest model of the CO-inhibited [FeFe] hydrogenase active site, which is known to undergo CO photolysis within a temperature-dependent process whose products and mechanism are still a matter of debate. Using density functional theory (DFT) and time-dependent density functional theory (TDDFT) computations, the ground state and low-lying excited-state potential energy surfaces (PESs) of 1a–CO and 2a+–CO have been explored aimed at elucidating the dynamics of the CO photolysis yielding Fe2(S2C3H6)(CO)6 (1a) and [Fe2(S2C3H6)(CO)6]+ (2a+), two simple models of the catalytic site of the enzyme. Two main results came out from these investigations. First, a–CO and 2a+–CO are both bound with respect to any CO dissociation with the lowest free energy barriers around 10 kcal mol−1, suggesting that at least 2a+–CO may be synthesized. Second, focusing on the cationic form, we found at least two clear excited-state channels along the PESs of 2a+–CO that are unbound with respect to equatorial CO dissociation.

In view of the depletion of fossil fuel reserves and climatic effects of greenhouse gas emissions, Ni,Fe-containing carbon monoxide dehydrogenase (Ni-CODH) enzymes have attracted increasing interest in recent years for their capability to selectively catalyze the reversible reduction of CO2 to CO (CO2 + 2H+ + 2e- ⇌ CO + H2O). The possibility of converting the greenhouse gas CO2 into useful materials that can be used as synthetic building blocks or, remarkably, as carbon fuels makes Ni-CODH a very promising target for reverse-engineering studies. In this context, in order to provide insights into the chemical principles underlying the biological catalysis of CO2 activation and reduction, quantum mechanics calculations have been carried out in the framework of density functional theory (DFT) on different-sized models of the Ni-CODH active site. With the aim of uncovering which stereoelectronic properties of the active site (known as the C-cluster) are crucial for the efficient binding and release of CO2, different coordination modes of CO2 to different forms and redox states of the C-cluster have been investigated. The results obtained from this study highlight the key role of the protein environment in tuning the reactivity and the geometry of the C-cluster. In particular, the protonation state of His93 is found to be crucial for promoting the binding or the dissociation of CO2. The oxidation state of the C-cluster is also shown to be critical. CO2 binds to Cred2 according to a dissociative mechanism (i.e., CO2 binds to the C-cluster after the release of possible ligands from Feu) when His93 is doubly protonated. CO2 can also bind noncatalytically to Cred1 according to an associative mechanism (i.e., CO2 binding is preceded by the binding of H2O to Feu). Conversely, CO2 dissociates when His93 is singly protonated and the C-cluster is oxidized at least to the Cint redox state.

Oxidative stress and metal dyshomeostasis are considered as 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 effort, the mechanisms of ROS-induced molecular damage remain poorly understood. In the present paper, we investigate on the basis of molecular modelling computations the mechanism of OH radical propagation toward the Aβ peptide, starting from the end-product of OH radical generation by Cu(ii)·Aβ. We evaluate (i) the OH oxidative capacity, as well as the energetics of the possible Aβ oxidation target residues, by quantum chemistry Density Functional Theory (DFT) on coordination models of Cu(ii)/OH/Aβ and (ii) the motion of the OH˙ approaching the Aβ target residues by classical Molecular Dynamics (MD) on the full peptide Cu(ii)/OH/Aβ(1–16). The results show that the oxidative capacity of OH coordinated Cu(ii)Aβ is significantly lower than that of the free OH radical and that propagation toward Aβ Asp and His residues is favoured over Tyr residues. These results are discussed on the basis of the recent literature on in vitro Aβ metal-catalyzed oxidation and on the possible implications for the AD oxidative stress mechanism.

International audience; Electrochemical oxidation of the complex [Fe2(CO)4(κ2‐dmpe)(µ‐adtBn)] (adtBn = (SCH2)2NCH2C6H5, dmpe = Me2PCH2CH2PMe2) (1) has been studied by cyclic voltammetry (CV) in acetonitrile and in dichloromethane in presence of various substrates L (L =MeCN, trimethylphosphite, isocyanide). The oxidized species, [1‐MeCN](PF6)2, [1‐(P(OMe)3)2](PF6)2 and [1‐(RNC)4](PF6)2 (R = tButyl, Xylyl), have been prepared and characterized by IR, NMR spectroscopies and, excepted [1‐MeCN](PF6)2, by X‐ray diffraction analysis. The crystallographic structures of the new FeIIFeII complexes reveal in any case that the association of one additional ligand (P(OMe)3 or RNC) occurs and, according to the nature of the substrates, further substitutions of one or three carbonyl groups, by P(OMe)3 or RNC, respectively, arise. Density functional theory (DFT) calculations have been performed for elucidating and discriminating, in each case, the mechanisms leading to the corresponding oxidized species. Moreover, the different degree of ligand substitution in the diiron core has been theoretically rationalized.

International audience; FeFe hydrogenases catalyse H2 oxidation and production using a "H-cluster", where two Fe ions are bound by an aza-dithiolate (adt) ligand. Various hypotheses have been proposed (by us and others) to explain that the enzyme reversibly inactivates under oxidizing, anaerobic conditions: intramolecular binding of the N atom of adt, formation of the so-called Hox/inact state or non-productive binding of H2 to isomers of the H-cluster. Here we show that none of the above explains the new finding that the anaerobic, oxidative, H2-dependent reversible inactivation is strictly dependent on the presence of Cl- or Br-. We provide experimental evidence that chloride uncompetitively inhibits the enzyme: it reversibly binds to catalytic intermediates of H2 oxidation (but not to the resting "Hox" state), after which oxidation locks the active site into a stable, saturated, inactive form, the structure of which is proposed here based on DFT calculations. The halides interact with the amine group of the H-cluster but do not directly bind to iron. It should be possible to stabilize the inhibited state in amounts compatible with spectroscopic investigations to explore further this unexpected reactivity of the H-cluster of hydrogenase.

The apparently simple dihydrogen formation from protons and electrons (2H+ + 2e- ↔ H2) is one of the most challenging reactions in nature. It is catalyzed by metalloenzymes of amazing complexity, called hydrogenases. A better understanding of the chemistry of these enzymes, especially that of the [NiFe]-hydrogenases subgroup, has important implications for production of H2 as alternative sustainable fuel. In this work, reactivation mechanism of the oxidized and inactive Ni-B and Ni-A states of the [NiFe]-hydrogenases active site has been investigated using density functional theory. Results obtained from this study show that one-electron reduction and protonation of the active site promote the removal of the bridging hydroxide ligand contained in Ni-B and Ni-A. However, this process is sufficient to activate only the Ni-B state. H2 binding to the active site is required to convert Ni-A to the active Ni-SIa state. Here, we also propose a reasonable structure for the spectroscopically well-characterized Ni-SIr and Ni-SU species, formed respectively from the one-electron reduction of Ni-B and Ni-A. Ni-SIr, depending on the pH at which the reaction occurs, features a bridging hydroxide ligand or a water molecule terminally coordinated to the Ni atom, whereas in Ni-SU a water molecule is terminally coordinated to the Fe atom, and the Cys64 residue is oxidized to sulfenate. The sulfenate oxygen atom in the Ni-A state affects the stereoelectronic properties of the binuclear cluster by modifying the coordination geometry of Ni, and consequently, by switching the regiochemistry of H2O and H2 binding from the Ni to the Fe atom. This effect is predicted to be at the origin of the different reactivation kinetics of the oxidized and inactive Ni-B and Ni-A states

Catalytic H2 oxidation has been dissected by means of DFT into the key steps common to the Fe2 unit of both the [FeFe]-hydrogenase cofactor and selected biomimics. The aim was to elucidate the molecular details underlying the very different performances of the two systems. We found that the better enzyme performance is based on a single iron atom that is maintained electron-poor, favoring H2 binding, although embedded within a highly electron-rich cofactor, ensuring a facile oxidation of the Fe2 -H2 adduct. This is due to 1) CN- coordinating to both iron atoms, due to their amphipathic Lewis acid/base properties, and 2) the 4Fe4S subunit further withdrawing electrons from the Fe2 core. Preserving a moderate electron deficiency at a single iron also helps the cofactor preserve hydride affinity, which favors H2 cleavage. Such valuable characteristics allow the biocatalyst to turnover close to equilibrium conditions. All previous biomimicry has shown, in contrast, the impossibility to properly balance the two apparently contrasting aforementioned requisites, although evident progress has been made by the H2 -ase community. Disclosure of the differences identified could inspire the design of novel biomimics, for instance, reconsidering the use of CN- in the catalyst architecture. Indeed, in the presence of bases normally employed in oxidative catalysis, undesired stable protonation at coordinated CN- , which affects the opposite process (proton reduction), could be overcome.

The electrochemical behavior of complexes [FeMo(CO) 5 (κ 2 -dppe)(μ-pdt)] (1) and [FeMo(CO) 4 (MeCN)( κ 2 -dppe)(μ-pdt)] (2), in the absence and in the presence of acid, has been investigated. The reduction of 1 follows at slow scan rates, in CH 2 Cl 2 -[NBu 4 ][PF 6 ] and acid-free media, an EC rev E mechanism that is supported by cyclic voltammetry (CV) experiments and digital CV simulations. In MeCN-[NBu 4 ][PF 6 ], the electrochemical reduction of 1 is the same as in dichloromethane and follows an ECE mechanism at slow scan rates, but with a positive shift of the redox potentials. In contrast, the oxidation of 1 is strongly solvent-dependent. In dichloromethane, the oxidation of 1 is reversible and involves a single electron, while in acetonitrile, it is irreversible at moderate and slow scan rates (v ≤ ca. 1 V s -1 ), and some chemical reversibility is apparent at higher scan rates (v = 10 V s -1 ). Density functional theory calculations revealed that the chemical step in the EC rev E mechanism corresponds to the dissociation of one PPh 2 end of the diphosphine ligand and the transfer of the semibridging CO to the Fe atom, similarly to the mechanism observed in the FeFe analogue complex. However, in the case of 1, the subsequent coordination of the phosphine ligand to the other metal is an unfavorable process.

Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumor progression. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Here we report the preclinical evaluation of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N- and O-glycosylation level that cause also a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with the activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice, supporting the advantage of targeting HBP for therapeutic purpose and encouraging further investigation about the use of this small molecule as a promising compound for breast cancer therapy.

Alzheimer's disease (AD) involves a number of factors including an anomalous interaction of copper with the amyloid peptide (Aβ), inducing oxidative stress with radical oxygen species (ROS) production through a three-step cycle in which O2 is gradually reduced to superoxide, oxygen peroxide and finally OH radicals. The purpose of this work has been to investigate the reactivity of 14 different Cu(ii)-Aβ coordination models with the aim of identifying on an energy basis (Density Functional Theory (DFT) and classical Molecular Dynamics (MD)) the redox competent form(s). Accordingly, we have specifically focused on the first three steps of the cycle, i.e. ascorbate binding to Cu(ii), Cu(ii) → Cu(i) reduction and O2 reduction to O2-. Compared to the recent literature, our results broaden the set of possible redox competent metallopeptide forms responsible for ROS production. Indeed, in addition to the three-coordinated species containing one His ligand, a N-terminal amine group and the carboxylate side chain of the Asp1 residue of Aβ already proposed, we found two other Cu-Aβ coordination modes involving two histidines.

The extraordinary capability of [NiFe]-hydrogenases to catalyse the reversible interconversion of protons and electrons into dihydrogen (H2) has stimulated numerous experimental and theoretical studies addressing the direct utilization of these enzymes in H2 production processes. Unfortunately, the introduction of these natural H2-catalysts in biotechnological applications is limited by their inhibition under oxidising (aerobic and anaerobic) conditions. With the aim of contributing to overcome this limitation, we studied the oxidative inactivation mechanism of [NiFe]-hydrogenases by performing Density Functional Theory (DFT) calculations on a very large model of their active site in which all the amino acids forming the first and second coordination spheres of the NiFe cluster have been explicitly included. We identified an O2 molecule and two H2O molecules as sources of the two oxygen atoms that are inserted at the active site of the inactive forms of the enzyme (Ni-A and Ni-B) under aerobic and anaerobic conditions, respectively. Furthermore, our results support the experimental evidence that the Ni-A-to-Ni-B ratio strongly depends on the number of reducing equivalents available for the process and on the oxidizing conditions under which the reaction takes place.

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH(·) that is suspected to contribute to Alzheimer's disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)-Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH(·) radical production from H2O2 through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)-Aβ coordination modes proposed in the literature, in terms of OH(·) production. For each coordination model, we considered the corresponding H2O2 adduct and performed a potential energy surface scan along the reaction coordinate of O-O bond dissociation of the peroxide, resulting in the production of OH(·) radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)-Aβ two-histidine coordination is not able to perform efficiently H2O2 reduction, while a less populated three-coordinated form would be responsible for the OH(·) production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H2O2 reduction.

International audience; To learn from Nature how to create an efficient hydrogen-producing catalyst, much attention has been paid to the investigation of structural and functional biomimics of the active site of [FeFe]-hydrogenase. To understand their catalytic activities, the μ-S atoms of the dithiolate bridge have been considered as possible basic sites during the catalytic processes. For this reason, a series of [FeFe]-H2ase mimics have been synthesized and characterized. Different [FeFe]-hydrogenase model complexes containing bulky Si–heteroaromatic systems or fluorene directly attached to the dithiolate moiety as well as their mono-PPh3-substituted derivatives have been prepared and investigated in detail by spectroscopic, electrochemical, X-ray diffraction, and computational methods. The assembly of the herein reported series of complexes shows that the μ-S atoms can be a favored basic site in the catalytic process. Small changes in the (hetero)-aromatic system of the dithiolate moiety are responsible for large differences in their structures. This was elucidated in detail by DFT calculations, which were consistent with the experimental results.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenum‑iron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Iron‑molybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70 A crystal structure of a nitrogenase MoFe protein α-96Arg ➔ Gln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), “native” mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+complex, yielding a specific signature. The interaction of Tb3+with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+or Fe3+binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of ∼3) of analytical sensitivity. [Figure not available: see fulltext.]

International audience; Electrochemical studies of [Fe2(CO)4(κ2-dmpe)(μ-dithiolate)] (dithiolate=adtBn, pdt) and density functional theory (DFT) calculations reveal the striking influence of an amine functionality in the dithiolate bridge on their oxidative properties. [Fe2(CO)4(κ2-dmpe)(μ-adtBn)] (1) undergoes two one-electron oxidation steps, with the first being partially reversible and the second irreversible. When the adtBn bridge is replaced with pdt, a shift of 60 mV towards more positive potentials is observed for the first oxidation whereas 290 mV separate the oxidation potentials of the two cations. Under CO, oxidation of azadithiolate compound 1 occurs according to an ECE process whereas an EC mechanism takes place for the propanedithiolate species 2. The dication species [1-CO]2+ resulting from the two-electron oxidation of 1 has been spectroscopically and structurally characterized. The molecular details underlying the reactivity of oxidized species have been explored by DFT calculations. The differences in the behaviors of 1 and 2 are mainly due to the presence, or not, of favored interactions between the dithiolate bridge and the diiron site depending on the redox states, FeIFeII or FeIIFeII, of the complexes.

The Mo/Cu-dependent CO dehydrogenase from Oligotropha carboxidovorans is an enzyme that is able to catalyze CO oxidation to CO2; moreover, it can also oxidize H2, thus eliciting a characteristic EPR signal. Interestingly, the Ag-substituted enzyme form proved unable to catalyze H2 oxidation. In the present contribution, we characterized the reactivity of the enzyme with H2 by quantum-chemical calculations. It was found that dihydrogen binding to the wild-type enzyme requires significant structural rearrangements of the active site Theoretical EPR spectra for plausible H2-bound models of the partially reduced, paramagnetic active site are also presented and compared with the experimental counterpart. Finally, density functional theory modeling shows that Ag substitution impairs H2 binding at the active site.

DFT has been used to investigate viable mechanisms of the hydrogen evolution reaction (HER) electrocatalyzed by [Fe2(CN){μ-CN(Me)2}(μ-CO)(CO)(Cp)2] (1) in AcOH. Molecular details underlying the proposed ECEC electrochemical sequence have been studied, and the key functionalities of CN- and amino-carbyne ligands have been elucidated. After the first reduction, CN- works as a relay for the first proton from AcOH to the carbyne, with this ligand serving as the main electron acceptor for both reduction steps. After the second reduction, a second protonation occurs at CN- that forms a Fe(CNH) moiety: i.e., the acidic source for the H2 generation. The hydride (formally 2e/H+), necessary to the heterocoupling with H+ is thus provided by the μ-CN(Me)2 ligand and not by Fe centers, as occurs in typical L6Fe2S2 derivatives modeling the hydrogenase active site. It is remarkable, in this regard, that CN- plays a role more subtle than that previously expected (increasing electron density at Fe atoms). In addition, the role of AcOH in shuttling protons from CN- to CN(Me)2 is highlighted. The incompetence for the HER of the related species [Fe2{μ-CN(Me)2}(μ-CO)(CO)2(Cp)2]+ (2+) has been investigated and attributed to the loss of proton responsiveness caused by CN- replacement with CO. In the context of hydrogenase mimicry, an implication of this study is that the dithiolate strap, normally present in all synthetic models, can be removed from the Fe2 core without loss of HER, but the redox and acid-base processes underlying turnover switch from a metal-based to a ligand-based chemistry. The versatile nature of the carbyne, once incorporated in the Fe2 scaffold, could be exploited to develop more active and robust catalysts for the HER.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

Graphical abstract, Highlights • Cdc34 is an E2 enzyme involved in protein ubiquitination and associated with some cancers. • Cdc34 activity is modulated by phosphorylation-induced conformational changes of the acidic loop. • We used computational approaches to identify potential inhibitory compounds for Cdc34. • These inhibitors as molecular hinges stabilizing the acid loop in its inactive, closed conformation., Among the different classes of enzymes involved in the ubiquitin pathway, E2 ubiquitin-conjugating enzymes occupy a central role in the ubiquitination cascade. Cdc34-like E2 enzymes are characterized by a 12–14 residue insertion in the proximity of the catalytic site, known as the acidic loop. Cdc34 ubiquitin-charging activity is regulated by CK2-dependent phosphorylation and the regulatory mechanism involves the acidic loop. Indeed, the phosphorylation stabilizes the loop in an open conformation that is competent for ubiquitin charging. Cdc34 is associated with a variety of diseases, such as hepatocellular carcinomas and prostatic adenocarcinomas. In light of its role, the discovery of potential inhibitory compounds would provide the mean to effectively modulate its activity. Here, we carried out a computational study based on molecular dynamics, virtual screening and docking to identify potential inhibitory compounds of Cdc34, modulating the acidic loop conformation. The molecules identified in this study have been designed to act as molecular hinges that can bind the acidic loop in its closed conformation, thus inhibiting the Cdc34-mediated ubiquitination cascade at the ubiquitin-charging step. In particular, we proposed a pharmacophore model featuring two amino groups in the central part of the model and two lateral aromatic chains, which respectively establish electrostatic interactions with the acidic loop (Asp 108 and Glu 109) and a hydrogen bond with Ser 139, which is one of the key residues for Cdc34 activity.

International audience; [FeFe] hydrogenases are H2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O2. The [2Fe]H cluster can span various redox states. The active-ready form (Hox) attains the Fe(II)Fe(I) state. States more oxidized than Hox were shown to be inactive and/or resistant to O2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an Fe(II)Fe(II) active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding Fe(II)Fe(II) synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H ox (inact) state.

International audience; FeFe hydrogenases catalyze H-2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe-2(CO)(3)(CN)(2)(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the Use of hydrogenase for the photoproduction of H-2. Here, we explore in great detail the reactivity of the excited states of the H-duster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This could be used for probing other, aspects of the photoreactivity of the H-cluster.

[NiFe]-hydrogenases catalyse the relevant H2 → 2H+ + 2e- reaction. Aerobic oxidation or anaerobic oxidation of this enzyme yields two inactive states called Ni-A and Ni-B. These states differ for the reactivation kinetics which are slower for Ni-A than Ni-B. While there is a general consensus on the structure of Ni-B, the nature of Ni-A is still controversial. Indeed, several crystallographic structures assigned to the Ni-A state have been proposed, which, however, differ for the nature of the bridging ligand and for the presence of modified cysteine residues. The spectroscopic characterization of Ni-A has been of little help due to small differences of calculated spectroscopic parameters, which does not allow to discriminate among the various forms proposed for Ni-A. Here, we report a DFT investigation on the nature of the Ni-A state, based on systematic explorations of conformational and configurational space relying on accurate energy calculations, and on comparisons of theoretical geometries with the X-ray structures currently available. The results presented in this work show that, among all plausible isomers featuring various protonation patterns and oxygenic ligands, the one corresponding to the crystallographic structure recently reported by Volbeda et al. (J Biol Inorg Chem 20:11-22, 19)-featuring a bridging hydroxide ligand and the sulphur atom of Cys64 oxidized to bridging sulfenate-is the most stable. However, isomers with cysteine residues oxidized to terminal sulfenate are very close in energy, and modifications in the network of H-bond with neighbouring residues may alter the stability order of such species.

It is successfully shown that photocatalytic proton reduction to dihydrogen in the presence of a sacrificial electron donor, such as trimethylamine (TEA) and ascorbate, can be driven by compact sensitizer–catalyst dyads, that is, dithiolate-bridged [FeFe] hydrogenase models tethered to organic sensitizers, such as fluorenes and silafluorenes (1 a–4 a). The sensitizer–catalyst dyads 1 a–4 a show remarkable and promising catalytic activities as well as enhanced stabilities during photocatalysis performed under UV-light irradiation. The photocatalysis was carried out both in non-aqueous and aqueous media. The latter experiments were performed by solubilizing the photocatalysts within micelles formed by either sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB). In this study a turnover number of 539 (7 h) is achieved under optimized conditions, which corresponds to an exceptionally high turnover frequency of 77 h−1. Theoretical investigations as well as emission decay experiments were performed to understand the observed phenomena together with the mechanisms of photocatalytic H2 generation.

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling.

We synthesized a small library of N-spirofused bicyclic derivatives of 1-deoxynojirimycin (DNJ), as quaternary ammonium salts, through a double SN2 annulation process. The spirofused rings are of different size and structural characteristics. Preliminary biological evaluation showed no antibacterial activity towards both Gram+ and Gram- bacteria. The DNJ derivative bearing a 6 member spirofused cycle revealed a promising inhibitor activity towards amyloglucosidase. Binding energies calculated through docking studies resembled the in vitro capability of such compound and of DNJ to inhibit amyloglucosidase activity, while showing significant difference in the binding poses.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

This paper reports on the protonation of phosphine-substituted diiron diphosphido carbonyls, analogues of diiron dithiolato centers at the active sites of hydrogenase enzymes. Reaction of the diphosphines (CH2)(n)(PPhH)(2) (n = 2 (edpH(2)) and n = 3 (pdpH(2))) with Fe-3(CO)(12) gave excellent yields of Fe-2(edp)(CO)(6) (1) and Fe-2(pdp)(CO)(6) (2). Substitution of Fe-2(edp)(CO)(6) with PMe3 afforded Fe-2(edp)(CO)(2)(PMe3)(4) (3; nu(CO) 1855 and 1836 cm(-1)). Crystallographic analysis showed that 3 adopts an idealized C-2 symmetry, with pairs of phosphine ligands occupying apical-basal sites on each Fe center. Relative to that in the dithiolato complex, the Fe-Fe bond (2.7786(8) angstrom) is elongated by 0.15 angstrom. Treatment of 3 with H(OEt2)(2)BAr4F (Ar-F = C6H3-3,5-(CF3)(2)) gave exclusively the C-2-symmetric mu-hydride complex [HFe2(edp)(CO)(2)(PMe3)(4)](+). This result contrasts with the behavior of the analogous ethanedithiolate Fe-2(edt)(CO)(2)(PMe3)(4) (edt = 1,2-C2H4S2), protonation of which gives both the bridging and terminal hydride complexes. This difference points to the participation of the sulfur centers in the formation of terminal hydrides. The absence of terminal hydride intermediates was also revealed in the protonation of the diphosphine diphosphido complexes Fe-2(pdp)(CO)(4)(dppv) (4; dppv = cis-1,2-C2H2(PPh2)(2)) and Fe-2(edp)(CO)(4)(dppbz) (5; dppbz = 1,2-C6H4(PPh2)(2)). Protonation of these cliphosphine complexes afforded mu-hydrido cations with apical-basal diphosphine ligands, which convert to the isomer where the diphosphine is dibasal. In contrast, protonation of the dithiolato complex Fe-2(pdt)(CO)(4)(dppv) gave terminal hydrides, which isomerize to mu-hydrides. In a competition experiment, 4 was shown to protonate faster than Fe-2(pdt)(CO)(4)(dppv)

The DFTB and DFT methods are applied to the study of different forms of the [Cu(HGGG)(Py)] complex in water, with the aim of identifying the most stable isomer. The DFTB calculations were possible thanks to a careful parametrization of the atom-atom repulsive energy terms for Cu-H, Cu-C, Cu-N, and Cu-O. The speciation process is carried out by computing different DFTB-steered molecular dynamics (SMD) trajectories, each of which ends in a well-defined different form. The last frame of each trajectory is subjected to geometry optimization at both DFTB and DFT levels, leading to a different isomer. From the corresponding energy values, a rank of relative stability of the isomers can be established. The computational protocol developed here is of general applicability to other metal-peptide systems and represents a new powerful tool for the study of speciation of metal-containing systems in water solution, particularly useful when the full characterization of the compound cannot be carried out on the basis of experimental results only.

In the present contribution, we report a theoretical investigation of the magnetic properties of the dihydrogen-evolving enzyme [FeFe]-hydrogenase, based on both DFT models of the active site (the H-cluster, a Fe6S 6 assembly including a binuclear portion directly involved in substrates binding), and QM/MM models of the whole enzyme. Antiferromagnetic coupling within the H-cluster has been treated using the broken-symmetry approach, along with the use of different density functionals. Results of g value calculations turned out to vary as a function of the level of theory and of the extension of the model. The choice of the broken-symmetry coupling scheme also had a significant influence on the calculated g values, for both the active-ready (Hox) and the CO-inhibited (Hox-CO) enzyme forms. However, hyperfine coupling-constant calculations were found to provide more consistent results. This allowed us to show that the experimentally detected delocalization of an unpaired electron at the binuclear subcluster in Desulfovibrio desulfuricans Hox is compatible with a weak interaction between the catalytic centre and a low-weight exogenous ligand like a water molecule. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

The photochemistry originated by the Fe2(CO)6S 2 low-lying excited states is investigated using density functional theory (DFT), time-dependent density functional theory (TDDFT), and the quantum theory of atoms in molecules (QTAIM) methods.The 11A1 excitation is the most intense among the low-lying excited states computed at the TDDFT level, which is assigned to the 449 nm metal-to-ligand charge transfer (MLCT) band observed experimentally. We then investigated the nine excited states in a range of ±35 nm centered on the 11A1 excitation energy, which reproduces the range of wavelengths covered by a recent 450 ± 35 nm low-energy laser photolysis experiment. The results presented in this paper suggest that the 450 nm photochemistry recently investigated proceeds mainly through the 11B2 lowest energy singlet excited state. The comparison between tetrahedrane ground state and 11B2 vertical excited state QTAIM atomic net charges evidenced the Fe→S MLCT character but also a significant CO→S ligand-to-ligand charge transfer (LLCT). Geometry relaxation of the nine excited state structures shows a S-S bond distance elongation that reaches the highest value for the 11B2 state. Moreover, during geometry optimization of the 11B2 state, the HOMO/LUMO crossing occurs, favoring the formation of the Fe-Fe butterfly isomer upon decay to the ground state, in agreement with experimental findings. Delocalization indexes allow us to describe the shift of the bonding electron density along the 1 1B2 photochemical reaction path that connects the tetrahedrane ground state to the Fe-Fe butterfly intermediate. Along this path, the S-S bond is progressively weakened until its breaking in the Fe-Fe butterfly isomer, while the Fe-Fe bond is only partially weakened. The S atom is progressively reduced with a total increasing of its negative charge by 0.211 electron, leading to the Fe-Fe butterfly intermediate suitable for oxidative addition. In light of the results obtained, a mechanism of the photochemical ethylene oxidative addition to Fe2(CO)6S2 is proposed. © 2011 American Chemical Society.

Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2-ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a π-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols. © 2010 Elsevier Inc. All rights reserved.

In this work a density functional theory study of the CO addition reaction to FeIFeI and FeIFeII models of the active site of [FeFe] hydrogenases is presented. A series of model complexes, ranging from simple diiron model complexes of the binuclear [2Fe]H subcluster to the full H-cluster, have been investigated. For each system, the thermodynamic parameters for the CO adduct formation, a reaction that mimics the enzyme CO inhibition, were computed. Parallel to the investigation of the CO addition reaction, the structural features of the various FeIFeI and FeIFeII species have been evaluated, with particular attention to the issue of the ligand arrangement as a function of the redox state. CO affinity depends on the redox state of the model and the chemical nature of its ligands. FeIFeII species are more favorable to form the CO adducts than the reduced FeIFeI species. According to the computed free energies and enthalpies for the CO adduct formation from Fe2(pdt)(CO)5L models, the CO affinity follows the ligand sequence L = SCH3- > CN- > PPh3 > CO (FeIFeI) and L = CO > CN- > PPh3 > SCH3- (FeIFeII). As the models become more similar to the H-cluster, the CO affinity increases, although the FeIFeI CO -inhibited H-cluster is not stable. The bonding properties of the models considered have been investigated by means of the quantum theory of atoms in molecules approach. Upon CO addiction, the new Fe-C bond is formed to the detriment of the Fe-Fe bonds and, to a lesser extent, the Fe-S bonds. Regarding the FeIFeII systems investigated, the spin density is initially localized on the rotated Fe atom, and the formation of the CO adducts results in a delocalization of the spin density. Consequently, the FeIFeII CO-inhibited forms are better described as (Fe+1.5)2.

This paper reports the synthesis of a panel of small molecules with arylamides and arylsulfonamides groups and their biological activity in inhibiting nucleotide exchange on human Ras. The design of these molecules was guided by experimental and molecular modelling data previously collected on similar compounds. Aim of this work is the validation of the hypothesis that a phenyl hydroxylamine group linked to a second aromatic moiety generates a pharmacophore capable to interact with Ras and to inhibit its activation. In vitro experiments on purified human Ras clearly show that the presence of an aromatic hydroxylamine and a sulfonamide group in the same molecule is a necessary condition for Ras binding and nucleotide exchange inhibition. The inhibitor potency is lower in molecules in which either the hydroxylamine has been replaced by other functional groups or the sulfonamide has been replaced by an amide. In the case both these moieties, the hydroxylamine and sulfonamide are absent, inactive compounds are obtained.

The cold-active, chloride-dependent a-amylase from Pseudoalteromonas haloplanktis (AHA) is one of the best characterized psychrophilic enzymes, and shares high sequence and structural similarity with its mesophilic porcine counterpart (PPA). An atomic detail comparative analysis was carried out by performing more than 60 ns of multiple-replica explicit-solvent molecular dynamics simulations on the two enzymes in order to characterize the differences in ensemble properties and dynamics in solution between the two homologues. We find in both enzymes high flexibility clusters in the surroundings of the substrate-binding groove, primarily involving the long loops that protrude from the main domain's barrel structure. These loops are longer in PPA and extend further away from the core of the barrel, where the active site is located: essential fluctuations in PPA mainly affect the highly solvent-accessible portions of these loops, whereas AHA is characterized by greater flexibility in the immediate surroundings of the active site. Furthermore, detailed analysis of active- site dynamics has revealed that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis. © 2009 American Chemical Society.

By combining in the same molecule Ras-interacting aromatic moieties and a sugar, we prepared a water-soluble Ras ligand that binds Ras and inhibits guanine nucleotide exchange. With this compound it was possible to determine experimentally by a 15 N-edited HSQC NMR experiment the ligand-Ras binding interface.

Nature's recipe: A theoretical study analyzes how the environment of the [FeFe] hydrogenase's catalytic cofactor affects its chemical properties, particularly the relative stability of complexes with bridging and terminal hydride ligands (see picture; Fe teal, S yellow, C green, N blue, O red, H gray). The results help to elucidate key rules for the design of bioinspired synthetic catalysts for H2 production. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.