Search

T cell activation plays a central role in immune response and in the maintenance of self-tolerance. We analyzed the evolutionary history of Tcell regulatory molecules. Nine genes involved in triggering Tcell activation or in regulating the ensuing response evolved adaptively in mammals. Several positively selected sites overlap with positions interacting with the binding partner or with cellular components. Population genetic analysis in humans revealed acomplex scenario of local (FASLG, CD40LG, HAVCR2) and worldwide (FAS, ICOSLG) adaptation and H.sapiens-to-Neandertal gene flow (gene transfer between populations). Disease variants in these genes are preferential targets of pathogen-driven selection, and a Crohn's disease risk polymorphism targeted by bacterial. -driven selection modulates the expression of ICOSLG in response to a bacterial superantigen. Therefore, we used evolutionary information to generate experimentally testable hypotheses concerning the function of specific genetic variants and indicate that adaptation to infection underlies the maintenance of autoimmune risk alleles. © 2013 Elsevier Inc.

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)

A lucky break: A combination of steric pressure and electron asymmetry has provided the first example of a diiron dithiolate that is both rotated and basic. The present work establishes the feasibility of a hydride free rotated structure for the Hred state of the enzyme (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rotated geometry: The first example of a dinuclear iron(I)-iron(I) complex featuring a fully rotated geometry related to the active site of [Fe-Fe] hydrogenase is reported (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The transport of small ligands to active sites of proteins is the basis of vital processes in biology such as enzymatic catalysis and cell signaling, but also of more destructive ones including enzyme inhibition and oxidative damage. Here, we show how a diffusion-reaction model solved by means of molecular dynamics and density functional theory calculations provides novel insight into the transport of small ligands in proteins. In particular, we unravel the existence of an elusive, dynamically formed gas channel, which CO2 takes to diffuse from the solvent to the active site (C-cluster) of the bifunctional multisubunit enzyme complex carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). Two cavities forming this channel are temporarily created by protein fluctuations and are not apparent in the X-ray structures. The ligand transport is controlled by two residues at the end of this tunnel, His113 and His 116, and occurs on the same time scale on which chemical binding to the active site takes place (0.1-1 ms), resulting in an overall binding rate on the second time scale. We find that upon reduction of CO2 to CO, the newly formed Fe-hydroxy ligand greatly strengthens the hydrogen-bond network, preventing CO from exiting the protein through the same way that CO2 takes to enter the protein. This is the basis for directional transport of CO from the production site (C-cluster of CODH subunit) to the utilization site (A-cluster of ACS subunit). In view of these results, a general picture emerges of how large proteins guide small ligands toward their active sites

[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 H 2, 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 FeIIFeI 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 FeIIFeII active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding FeIIFeII 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 oxinact state. © 2013 SBIC.

The multi-domain protein hSos1 plays a major role in cell growth and differentiation through its Ras-specific guanine nucleotide exchange domain whose complex regulation involves intra-molecular, inter-domain rearrangements. We present a stochastic mathematical model describing intra-molecular regulation of hSos1 activity. The population macroscopic effect is reproduced through a Monte-Carlo approach. Key model parameters have been experimentally determined by BIAcore analysis. Complementation experiments of a Saccharomyces cerevisiae cdc25(ts) strain with Sos deletion mutants provided a comprehensive data set for estimation of unknown parameters and model validation. The model is robust against parameter alteration and describes both the behavior of Sos deletion mutants and modulation of activity of the full length molecule under physiological conditions. By incorporating the calculated effect of aminoacid changes at an inter-domain interface, the behavior of a mutant correlating with a developmental syndrome could be simulated, further validating the model. The activation state of Ras-specific guanine nucleotide exchange domain of hSos1 arises as an "emergent property" of its multi-domain structure that allows multi-level integration of a complex network of intra- and inter-molecular signals.

The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.

Molecular dynamics (MD) simulations have the ability to help reveal the relationship between protein structure, dynamics and function. Here, we describe MD simulations of the representative members of family 3 of E2 enzymes that we performed and analyzed with the aim of providing a quantitative description of the functional dynamics in this biologically important set of proteins. In particular, we combined a description of the protein as a network of interacting residues with the dynamical cross-correlation method to characterize the correlated motions observed in the simulations. This approach enabled us to detect communication between distal residues in these enzymes, and thus to reliably define all the likely intramolecular pathways of communication. We observed functionally relevant differences between the closed and open conformations of the enzyme, and identified the critical residues involved in the long-range communication paths. Our results highlight how molecular simulations can be used to aid in providing atomic-level details to communication paths within proteins. This journal is © the Owner Societies 2012.

The toxicity of heavy metals, which is associated with the high affinity of the metals for thiolate rich proteins, constitutes a problem worldwide. However, despite this tremendous toxicity concern, the binding mode of AsIII and PbII to proteins is poorly understood. To clarify the requirements for toxic metal binding to metalloregulatory sensor proteins such as AsIII in ArsR/ArsD and PbII in PbrR or replacing ZnII in d-aminolevulinc acid dehydratase (ALAD), we have employed computational and experimental methods examining the binding of these heavy metals to designed peptide models. The computational results show that the mode of coordination of AsIII and PbII is greatly influenced by the steric bulk within the second coordination environment of the metal. The proposed basis of this selectivity is the large size of the ion and, most important, the influence of the stereochemically active lone pair in hemidirected complexes of the metal ion as being crucial. The experimental data show that switching a bulky leucine layer above the metal binding site by a smaller alanine residue enhances the PbII binding affinity by a factor of five, thus supporting experimentally the hypothesis of lone pair steric hindrance. These complementary approaches demonstrate the potential importance of a stereochemically active lone pair as a metal recognition mode in proteins and, specifically, how the second coordination sphere environment affects the affinity and selectivity of protein targets by certain toxic ions

Cyclin-dependent kinase inhibitors (CKIs) are key regulatory proteins of the eukaryotic cell cycle, which modulate cyclin-dependent kinase (Cdk) activity. CKIs perform their inhibitory effect by the formation of ternary complexes with a target kinase and its cognate cyclin. These regulators generally belong to the class of intrinsically disordered proteins (IDPs), which lack a well-defined and organized three-dimensional (3D) structure in their free state, undergoing folding upon binding to specific partners. Unbound IDPs are not merely random-coil structures, but can present intrinsically folded structural units (IFSUs) and collapsed conformations. These structural features can be relevant to protein function in vivo. The yeast CKI Sic1 is a 284-amino acid IDP that binds to Cdk1 in complex with the Clb5,6 cyclins, preventing phosphorylation of G1 substrates and, therefore, entrance to the S phase. Sic1 degradation, triggered by multiple phosphorylation events, promotes cell-cycle progression. Previous experimental studies pointed out a propensity of Sic1 and its isolated domains to populate both extended and compact conformations. The present contribution provides models for compact conformations of the Sic1 kinase-inhibitory domain (KID) by all-atom molecular dynamics (MD) simulations in explicit solvent and in the absence of interactors. The results are integrated by spectroscopic and spectrometric data. Helical IFSUs are identified, along with networks of intramolecular interactions. The results identify a group of putative hub residues and networks of electrostatic interactions, which are likely to be involved in the stabilization of the globular states

Reaction of Fe-3(CO)(12) with pyridinyl thioester ligand PyCH2SCOCH3 (L-1, Py = pyridin-2-yl) produced complex, [Fe-2(kappa-COCH3)(mu-SCH2Py)(CO)(5)] (1) (PyCH2S = pyridin-2-ylmethanethiolate). When complex 1 reacted with PPh3, a monosubstituted complex, [Fe-2(kappa-COCH3)(mu-SCH2Py)(CO)(4)PPh3] (2), was derived. Reaction of the same precursor with analogous thioester ligand PyCH2SCOPy (L-2) generated three novel diiron complexes, [Fe-2(kappa-Py)(mu-SCH2Py)(CO)(5)] (3), [Fe-2(kappa-Py)'(mu-SCH2Py)(CO)(5)] (4), and [Fe-2(kappa-Py)(mu-SCH2Py)(CO)(6)] (5). Complexes 3 and 4 are structural isomers. Complex 5 could be converted into complex 4 but the conversion from complex 5 to the isomer 3 was not observed. All the five complexes were fully characterised using FTIR, NMR, and other techniques. Their structures were determined using X-ray single crystal diffraction analysis. The oxidative formation of complexes 1, 3, 4, and 5 involved C-S and/ or C-C bonds cleavages. To probe possible mechanisms for these cleavages, DFT calculations were performed. From the calculations, viable reaction pathways leading to the formation of all the isolated products were delineated. The results of the theoretic calculations also allowed rationalisation of the experimental observations.

The behavior of [Fe2(CO)4(?2-PNPR)(mu-pdt)] (PNPR=(Ph2PCH2)2NR, R=Me (1), Ph (2); pdt=S(CH2)3S) in the presence of acids is investigated experimentally and theoretically (using density functional theory) in order to determine the mechanisms of the proton reduction steps supported by these complexes, and to assess the role of the PNPR appended base in these processes for different redox states of the metal centers. The nature of the R substituent of the nitrogen base does not substantially affect the course of the protonation of the neutral complex by CF3SO3H or CH3SO3H; the cation with a bridging hydride ligand, 1?mu H+ (R=Me) or 2?mu H+ (R=Ph) is obtained rapidly. Only 1?mu H+ can be protonated at the nitrogen atom of the PNP chelate by HBF4 center dot Et2O or CF3SO3H, which results in a positive shift of the proton reduction by approximately 0.15 V. The theoretical study demonstrates that in this process, dihydrogen can be released from a ?2-H2 species in the FeIFeII state. When R=Ph, the bridging hydride cation 2?mu H+ cannot be protonated at the amine function by HBF4 center dot Et2O or CF3SO3H, and protonation at the N atom of the one-electron reduced analogue is also less favored than that of a S atom of the partially de-coordinated dithiolate bridge. In this situation, proton reduction occurs at the potential of the bridging hydride cation, 2?mu H+. The rate constants of the overall proton reduction processes are small for both complexes 1 and 2 (kobs approximate to 47 s-1) because of the slow intramolecular proton migration and H2 release steps identified by the theoretical study.

The reactivity of [Fe-2(CO)(4)(kappa(2)-LL)(mu-pdt)](+) complexes (pdt = S(CH2)(3)S, propanedithiolate) with different substrates L' (L' = CO, MeCN, P(OMe)(3)) was investigated electrochemically in order to assess the influence of the chelating ligand kappa(2)-LL (LL = I-Me-CH2-I-Me (1(+)), dppe (2(+)); I-Me = 1-methylimidazol-2-ylidene). This latter ligand is effectively shown to affect the reactivity of the cations in different ways: when L' = CO, the adduct [Fe-2(CO)(4)(mu-CO)(kappa(2)-dppe)(mu-pdt)](+) (2-CO+) was clearly observed by cyclic voltammetry, whereas [Fe-2(CO)(4)(mu-CO)(kappa(2)-I-Me-CH2-I-Me)(mu-pdt)](+) (1-CO+) was not detected, although DFT calculations show that the energies of the products and the activation barriers to their formation are similar. When L' = MeCN, the adducts X-MeCN+ with X = 1, 2 are both observed, but the formation is easier when LL = dppe. Finally, the reaction of [Fe-2(CO)(4)(kappa(2)-I-Me-CH2-I-Me)(mu-pdt)](+) (1(+)) with P(OMe)(3) produces the disubstituted dication [Fe-2(CO)(2){P(OMe)3}(2)(kappa(2)-I-Me-CH2-I-Me)(mu-CO)(mu-pdt)](2+) (4(2+)) via a disproportionation reaction, while previous studies demonstrated that monocationic derivatives were obtained when LL = dppe. Complex 4[PF6](2) was fully characterized, and its X-ray crystal structure confirms the presence of a carbonyl ligand in a bridging position, which did not exist in the related P(OMe)(3)-substituted kappa(2)-dppe cations.

DFT/BP86/TZVP and DFT/B3LYP/TZVP have been used to investigate systematically the reaction pathways associated with the H-transfer step, which is the rate-determining step of the reaction HCOO- reversible arrow CO2 + H+ + 2e(-), as catalyzed by metalloenzyme formate dehydrogenase (FDH). Actually, the energetics associated with the transfer from formate to all H (proton or hydride) acceptors that are present within the FDH active site have been sampled. This study points to a viable intimate mechanism in which the metal center mediates H transfer from formate to the final acceptor, i.e. a selenocysteine residue. The Mo-based reaction pathway, consisting of a beta-H elimination to metal with concerted decarboxylation, turned out to be favored over previously proposed routes in which proton transfer occurs directly from HCOO- to selenocysteine. The proposed reaction pathway is reminiscent of the key step of metal-based catalysis of the water-gas shift reaction.

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

We present a DFT study of the structural and spectroscopic properties of the complex formed by Cu2? with the peptide fragment Ac-PHREN-NH2, which encompasses the putative cell binding domain of angiogenin, as well as with its Ac-PHRQN-NH2 variant. Analysis of structures, energies and spectroscopic parameters has allowed to conclude that the metal coordination environment at pH 8 is formed by a nitrogen atom of His, two deprotonated amide groups, and an oxygen atom from the COO- side chain of Glu, in nice agreement with recent experimental results (La Mendola et al. in Dalton Trans, 39:10678, 2010). Moreover, DFT results allowed to reveal that the Glu side chain of the Ac-PHREN-NH2 peptide is coordinated in equatorial position, in a tetrahedrically distorted square planar arrangement, fully disclosing the effects of Cu2? binding on the structural properties of this key angiogenin portion. In the Ac-PHRQN-NH2 variant, the carboxylate group is replaced by a H2O molecule in a coordination arrangement similar to that of the wild-type system.

Recent advances aimed at modeling the chemistry of the active site of [FeFe]-hydrogenases (the H-cluster, composed by a catalytic Fe 2S 2 subcluster and an Fe 4S 4 portion) have led to the synthesis of binuclear coordination compounds containing a noninnocent organophosphine ligand [2,3-bis(diphenylphosphino)maleic anhydride, bma] that is able to undergo monoelectron reduction, analogously to the tetranuclear Fe 4S 4 subcluster portion of the H-cluster. However, such a synthetic model was shown to feature negligible electronic communication between the noninnocent ligand and the remaining portion of the cluster, at variance with the enzyme active site. Here, we report a theoretical investigation that shows why the electron transfer observed in the enzyme upon protonation of the catalytic Fe 2S 2 subsite cannot take place in the bma-containing cluster. In addition, we show that targeted modifications of the bma ligand are sufficient to restore the electronic communication within the model, such that electron density can be more easily withdrawn from the noninnocent ligand, as a result of protonation of the iron centers. Similar results were also obtained with a ligand derived from cobaltocene. The relevance of our findings is discussed from the perspective of biomimetic reproduction of proton reduction to yield molecular hydrogen. © 2011 American Chemical Society.

A density functional theory study is presented regarding the energetics and the Mulliken population analyses of a quantum mechanical/molecular mechanical (QM/MM) system including multiple iron-sulfur clusters in the QM region. The [FeFe]-hydrogenase from Desulfovibrio desulfuricans was studied, and both the active site (an Fe6S6 assembly generally referred to as the H-cluster) and an ancillary Fe4S4 site were treated at the BP86-RI/TZVP level. The antiferromagnetic coupling that characterizes both sites was modeled using the broken-symmetry (BS) approach. For such a QM system, 36 different BS couplings can be defined, depending on the localization of spin excess on the various spin centers. All the BS states were obtained by means of an effective and simple method for spin localization, that is here described and compared with more sophisticated approaches already available in literature. The variation of the QM/MM energy among the various geometry-optimized protein models was found to be less than 25 kJ mol-1. This energy variation almost doubles if no geometry optimization is performed. A detailed analysis of the additive nature of these variations in QM/MM energy is reported. The Mulliken charges show very small variations among the 36 BS states, whereas the Mulliken spin populations were found to be somewhat more variable. The relevance of such variations is discussed in light of the available Mössbauer and Electron Paramagnetic Resonance (EPR) spectroscopic data for the enzyme. Finally, the influence of the basis set on the spin populations, charges, and structural parameters of the models was investigated, by means of QM/MM computations on the same system at the BP86-RI/SVP level. © 2010 Wiley Periodicals, Inc.

The one-electron oxidation of the diiron complex [Fe-2(CO)(4)(kappa(2)-dppe)(mu-pdt)] (1) (dppe = Ph2PCH2CH2PPh2; pdt = S(CH2)(3)S) has been investigated in the absence and in the presence of P(OMe)(3), by both electrochemical and theoretical methods, to shed light on the mechanism and the location of the oxidatively induced structure change. While cyclic voltammetric experiments did not allow to discriminate between a two-step (EC) and a concerted, quasi-reversible (QR) process, density functional theory (DFT) calculations favor the first option. When P(OMe)(3) is present, the one-electron oxidation produces singly and doubly substituted cations, [Fe-2(CO)(4-n){P(OMe)(3)}(n)(kappa(2)-dppe)(mu-pdt)](+) (n = 1: 2(+); n = 2: 3(+)) following mechanisms that were investigated in detail by DFT. Although the most stable isomer 1(+) and 2(+) (and 3(+)) show a rotated Fe(dppe) center, binding of P(OMe)(3) occurs at the neighboring iron center of both 1(+) and 2(+). The neutral compound 3 was obtained by controlled-potential reduction of the corresponding cation, while 2 was quantitatively produced by reaction of 3 with CO. The CO dependent conversion of 3 into 2 as well as the 2(+) <-> 3(+) interconversion were examined by DFT.

Experimental and computational experiments show that the electrophile MeS+ attacks a single Fe center in Fe-2(propanedithiolate)(CO)(4)(PMe3)(2) followed by isomerization of this terminal thiolato complex to the corresponding mu-SMe derivative

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.

A QM/MM investigation of the active-ready (Hox) form of [FeFe]-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H2 formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in H ox is unlikely, and in any case it would not trigger electron transfer from the accessory Fe4S4 clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Key stereoelectronic properties of Desulfovibrio desulfuricans [FeFe]-hydrogenase (DdH) were investigated by quantum mechanical description of its complete inorganic core, which includes a Fe6S6 active site (the H-cluster), as well as two ancillary Fe4S4 assemblies (the F and F' clusters). The partially oxidized, active-ready form of DdH is able to efficiently bind dihydrogen, thus starting H2 oxidation catalysis. The calculations allow us to unambiguously assign a mixed Fe(II)Fe(I) state to the catalytic core of the activeready enzyme and show that H2 uptake exerts subtle, yet crucial influences on the redox properties of DdH. In fact, H2 binding can promote electron transfer from the H-cluster to the solvent-exposed F'-cluster, thanks to a 50% decrease of the energy gap between the HOMO (that is localized on the H-cluster) and the LUMO (which is centered on the F'-cluster). Our results also indicate that the binding of the redox partners of DdH in proximity of its F'-cluster can trigger one-electron oxidation of the H2-bound enzyme, a process that is expected to have an important role in H2 activation. Our findings are analyzed not only from a mechanistic perspective, but also in consideration of the physiological role of DdH. In fact, this enzyme is known to be able to catalyze both the oxidation and the evolution of H2, depending on the cellular metabolic requirements. Hints for the design of targeted mutations that could lead to the enhancement of the oxidizing properties of DdH are proposed and discussed. © 2011 American Chemical Society.

The presence of Fe-bound cyanide ligands in the active site of the proton-reducing enzymes [FeFe]-hydrogenases has led to the hypothesis that such Brønsted-Lowry bases could be protonated during the catalytic cycle, thus implying that hydrogen isocyanide (HNC) might have a relevant role in such crucial microbial metabolic paths. We present a hybrid quantum mechanical/molecular mechanical (QM/MM) study of the energetics of CN - protonation in the enzyme, and of the effects that cyanide protonation can have on [FeFe]-hydrogenase active sites. A detailed analysis of the electronic properties of the models and of the energy profile associated with H2 evolution clearly shows that such protonation is dysfunctional for the catalytic process. However, the inclusion of the protein matrix surrounding the active site in our QM/MM models allowed us to demonstrate that the amino acid environment was finely selected through evolution, specifically to lower the Brønsted-Lowry basicity of the cyanide ligands. In fact, the conserved hydrogen-bonding network formed by these ligands and the neighboring amino acid residues is able to impede CN- protonation, as shown by the fact that the isocyanide forms of [FeFe]-hydrogenases do not correspond to stationary points on the enzyme QM/MM potential-energy surface. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A small virtual library of iminosugar derivatives was evaluated by docking experiments carried out by sampling a protein region corresponding to the phosphoinositide binding site of the PH domain of Akt. Four compounds were selected and efficiently synthesised from a common precursor. All compounds were subjected to preliminary biological evaluation on purified enzyme, and - among them - compound 9 exhibited the best inhibitory activity. A small virtual library of iminosugar-based Akt inhibitors have been designed and evaluated by using docking calculations. Selected compounds have been conveniently synthesised, and preliminary biological evaluation identified compound 9 as a possible lead compound for further development of iminosugar-based Akt inhibitors.

Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H2 binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe2 subsite.

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 p

Reaction of 2-(1-(pyridin-2-yl)ethyl)propane-1,3-dithiol with tri-iron dodecacarbonyl afforded a diiron pentacarbonyl complex, [Fe(2)L(CO)(5)] (A and H(2)L = 2-methyl-2-(1,2,5,6-tetrahydropyridin-2-yl)propane-1,3-dithiol). In the reaction, the pyridinyl ring of the original ligand was partially hydrogenated during the reaction. This complex was fully characterised by using crystallographic, infrared, and NMR spectroscopic techniques. Formation reaction of its bridging hydride and subsequent conversion into its protonated diiron hexacarbonyl complex, [Fe(2)L(CO)(6)] (ACOH(+) in which the N atom of L is decoordinated and protonated), were experimentally and theoretically investigated. Results for this complex alongside with theoretic investigations into other diiron pentacarbonyl analogues revealed positive correlation of basicity of the internal bases of these investigated complexes to bridging hydrides formation. But subsequent conversion of these bridging hydrides into protonated diiron hexacarbonyl complexes was not solely dictated by the basicity. Protophilicity of the internal base and lability of its coordination with the diiron centre play also an important role as revealed by experimental and theoretic investigations. (C) 2009 Elsevier B.V. All rights reserved.

Reacting a bidentate ligand H2L, 2-(2-methoxybenzyl)-2-methylpropane-1,3-dithiol, with Fe-3(CO)(12) formed a diiron hexacarbonyl complex (1Me) from which a diiron hexacarbonyl complex (1H) pendant with a phenolic group was derived via in-situ demethylation. Further deprotonation of complex 1H gave a diiron pentacarbonyl species (1) in which a rare bond between the soft metal Fe-I and the relatively hard base phenolate formed, Fe-I-OR (R = phenolic moiety). This bonding may be a suitable mimic of the bonding feature, ((FeFeI)-Fe-I)R-OH/OH2 found in the FeFel-hydrogenase. (C) 2010 Elsevier B.V. All rights reserved.

A DFT study of protonation thermodynamics in H-2-evolving biomimetic catalysts related to [FeFe]-hydrogenases active site is presented here. Taking as a reference system the electrocatalytic dihydrogen evolution mechanism recently proposed for the synthetic assembly [Fe-2(CO)(4)(kappa(2)-Ph2PCH2CH2PPh2)(mu-S(CH2)(3)S)] (a, which is able to release H-2 after having undergone monoelectron reduction steps and three sequential protonation reactions), we show how the reduction of model complexes to oxidation states lower than those observed in [FeFe]-hydrogenases cofactor leads to a protonation regiochemistry that has no counterpart in the enzymatic mechanism of H-2 production. In particular, double protonation of the metal centers turned out to be disfavored in a by up to 12.5 kcal mol(-1) with respect to alternative protonation paths; as for the regiochemistry of triple protonation, the formation of eta(2)-H-2 adducts is disfavored by at least similar to 25 kcal mol(-1). Structural analysis of the theoretical models also revealed that over-reduction of synthetic complexes, though necessary for observing H-2 evolution from the currently available biomimetic electrocatalysts, can generally impair their structural integrity. Possible approaches for the modulation of protonation regiochemistry are then proposed; in particular, it turned out that a targeted use of sigma-donating ligands showing low basicity can favor double protonation of iron centers

The active site of the [FeFe] hydrogenases contains two Fe ions bound to one Cys ligand, three CO molecules, two CN- ions, and a dithiolate ligand. The nature of the last of these has been much discussed, and it has been suggested that it contains C, N, or O as the bridgehead atom. Most experimental studies indicate a N atom, whereas a recent density functional theory (DFT) study of a crystal structure indicated an O atom. Here, we performed quantum refinement on the same crystal structure with five different models of the dithiolate ligand X(CH2S-)2, with X = CH 2, NH2+, NH (two conformations), or O; we found that structures with a N bridgehead atom actually provide the best fit to the raw crystallographic data. Quantum refinement is standard crystallographic refinement in which the molecular mechanics force field normally used to supplement the experimental raw data to give a more chemical structure is replaced by more accurate DFT calculations for the active site. Thereby, we obtain structures that are an ideal compromise between DFT and crystallography.

The mechanism of terminal- to mu-hydride isomerisation in models of synthetic complexes resembling the [FeFe]-hydrogenase active site has been elucidated by DFT calculations, revealing that Ray-Dutt reaction pathways are generally favoured, and providing some clues for the rational design of novel synthetic catalysts to produce H-2.

Natriuretic peptides (NP) are a family of structurally related but genetically distinct hormones/paracrine factors that regulate blood volume, blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth. In this work we present computational structural analysis of the three human NP in solution, the synthesis and preliminary biological assays of a short fragment of CNP, I(14)GSM(17), together with one small mimetic, GGSM. Synthetic peptides IGSM, GGSM, and full length CNP were preliminary tested for their ability to influence cell growth of three human osteosarcoma cell lines. Synthetic peptides were shown to successfully mimic the biological activity of the full length natural peptide: their effects, although different upon the cell types used, are in accordance with the current literature, designating a possible role for CNP, and its derivatives, in skeletogenesis.

Natriuretic peptides (NPs) are a family of structurally related hormone/paracrine factors (ANP, BNP and CNP), which mediate a broad array of physiological effects by interacting with specific guanylyl cyclase receptors (NPR) and have promising therapeutic and clinical applications. NPs are specific for different NPRs and share a common ring structure in which a disulfide bond between two conserved cysteine residues is formed. Residues within the cyclic loop are largely responsible for receptor selectivity. Structural features of free NPs in solution have not been investigated in details even if their characterization would be very useful in order to identify important aspects related to NPs function and receptor selectivity. In light of the above scenario, we carried out a 0.1 mu s molecular dynamics investigation of NPs with the aim of providing a high-resolution atomistic view of specific of their conformational ensemble in solution. Our results clearly indicate that NP receptor-bound conformations are not stable solution structure and that induced-fit mechanisms are involved in the formation of NP-NPR complexes. Moreover, in agreement with the current view on strictly relationship between protein dynamics and protein function and activity, it turns out that differences in activity and NPR specificity of CNP and ANP/BNP might be correlated to different amino acid composition of the cyclic loop, propensity to form beta-sheet structures, flexibility patterns, dynamics properties and free conformations explored during the simulations. (C) 2010 Elsevier Inc. All rights reserved.

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.

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.

[FeFe]-hydrogenases are highly efficient H-2-evolving metalloenzymes that include cyanides and carbonyls in the active site. The latter is an Fe6S6 cluster (the so-called H-cluster) that can be subdivided into a binuclear portion carrying the CO and CN- groups and a tetranuclear subcluster. The fundamental role of cyanide ligands in increasing the basicity of the H-cluster has been highlighted previously. Here a more subtle but crucial role played by the two CN- ligands in the active site of [FeFe]-hydrogenases is disclosed. In fact, QM/MM calculations on all-atom models of the enzyme from Desulfovibrio desulfuricans show that the cyanide groups fine-tune the electronic and redox properties of the active site, affecting both the protonation regiochemistry and electron transfer between the two subclusters of the H-cluster. Despite the crucial role of cyanides in the protein active site, the currently available bioinspired electrocatalysts generally lack CN- groups in order to avoid competition between the latter and the catalytic metal centers for proton binding. In this respect, we show that a targeted inclusion of phosphine ligands in hexanuclear biomimetic clusters may restore the electronic and redox features of the wild-type H-cluster.