Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond

BACKGROUND: The microscopic events of ribonuclease (RNase) catalyzed phosphoryl transfer reactions are still a matter of debate in which the contenders adhere to either the classical concerted acid-base mechanism or a more sequential triester-like mechanism. In the case of RNase A, small thio-effects of the nonbridging oxygens have been invoked in favor of the classical mechanism. However, the RNase T1 catalyzed transphosphorylation of phosphorothioate RNA is highly stereoselective. R(P) thio-substituted RNA is depolymerized 60000 times faster than S(P) thio-substituted RNA by this enzyme, whereas the uncatalyzed cleavage of both substrates occurs at comparable rates. We combined site-directed mutagenesis in the RNase active site and stereospecific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S(P) oxygen that bring about this stereoselectivity of RNase T1. RESULTS: Thio-substitution of the nonbridging pro-S(P) oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-R(P) oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme's ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves R(P) and S(P) phosphorothioate RNA with similar rates. The dependence of the pro-S(P) thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-S(P) oxygen and the catalytic base Glu58 in the wild type enzyme. CONCLUSIONS: Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.     

Implications of protonation and substituent effects for C-O and O-P bond cleavage in phosphate monoesters

A recent study of phosphate monoesters that broke down exclusively through C-O bond cleavage and whose reactivity was unaffected by protonation of the nonbridging oxygens (Byczynski et al. J. Am. Chem. Soc. 2003, 125, 12541) raised several questions about the reactivity of phosphate monoesters, R-O-P(i). Potential catalytic strategies, particularly with regard to selectively promoting C-O or O-P bond cleavage, were investigated computationally through simple alkyl and aryl phosphate monoesters. Both C-O and O-P bonds lengthened upon protonating the bridging oxygen, R-O(H(+))-P(i), and heterolytic bond dissociation energies, DeltaH(C)(-)(O) and DeltaH(O)(-)(P), decreased. Which bond will break depends on the protonation state of the phosphoryl moiety, P(i), and the identity of the organosubstituent, R. Protonating the bridging oxygen when the nonbridging oxygens were already protonated favored C-O cleavage, while protonating the bridging oxygen of the dianion form, R-O-PO(3)(2)(-), favored O-P cleavage. Alkyl R groups capable of forming stable cations were more prone to C-O bond cleavage, with tBu > iPr > F(2)iPr > Me. The lack of effect on the C-O cleavage rate from protonating nonbridging oxygens could arise from two precisely offsetting effects: Protonating nonbridging oxygens lengthens the C-O bond, making it more reactive, but also decreases the bridging oxygen proton affinity, making it less likely to be protonated and, therefore, less reactive. The lack of effect could also arise without bridging oxygen protonation if the ratio of rate constants with different protonation states precisely matched the ratio of acidity constants, K(a). Calculations used hybrid density functional theory (B3PW91/6-31++G) methods with a conductor-like polarizable continuum model (CPCM) of solvation. Calculations on Me-phosphate using MP2/aug-cc-pVDZ and PBE0/aug-cc-pVDZ levels of theory, and variations on the solvation model, confirmed the reproducibility with different computational models.     

紫杉醇类似物C13侧链手性构型与活性关系的从头算研究

应用从头算RHF/STO-3G方法优化了四个紫杉醚异构体,在RHF/6-31G*水平进行单点计算,并计算了四个分子的静电势。分子立体构型表明:C13侧链上2 R构型的2-OH和3 S构型的3-NH均处于分子外侧,有利于与受体极性基团结合;2 S构型的2-OH和3 R构型的3-NH在分子的内侧,不利于与受体极性基团结合。静电势的结果表明,R构型的2-OH和S构型的3-NH可以产生相应的负电势区域。当C13侧链为天然构型(2 R,3 S)时,分子可能以双基结合的方式通过负静电势区域与受体作用,活性较高。     

Probing the origin of the compromised catalysis of E. coli alkaline phosphatase in its promiscuous sulfatase reaction

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.     

Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.     

Synthesis, Conformational Studies, X-ray Structures, and Complexation Properties of Semirigid Macrocyclic Phosphonamides

The semirigid phosphonamide ligands 1-5 have been synthesized from the macrocyclic precursors 6-9 by reaction with 1,3-propanediol ditosylate or 1,2-dichloroethane. For the thiophosphoryl compounds 1 and 2, and the phosphoryl derivative 5, the reactions were carried out in biphasic aqueous NaOH solutions. The phosphoryl derivatives 3 and 4 were better obtained from NaH in anhydrous tetrahydrofuran. The conformations of the hosts in solution were deduced from low-temperature NMR and NOE difference experiments. Conformational equilibria between exo and endo forms are observed for the 18-membered macrocycles 1 and 3. The exo conformer predominates in solution for the 21-membered macrocycle 2, whereas 4 exists as rapidly exchanging conformers. The X-ray crystal structures of macrocycles 1, 2, and 5 have been determined as well as the complexes 1.Hg(SCN)(2) and 5.LiNO(3). In the Hg(2+) complex the metal ion is located out of the macrocyclic cavity and is coordinated to the thiophosphoryl unit. In 5.LiNO(3)()()the Li(+) cation is located inside the macrocyclic cavity and is coordinated to a tetrahedral array of oxygen donors. Free energies of complexation (DeltaG degrees ) of the phosphorylated ligands 3-5 with alkali metal and ammonium cations were determined in CHCl(3) saturated with H(2)O by picrate extraction experiments. The -DeltaG degrees values are greatest for 4 complexing K(+) and NH(4)(+) (7.3 and 8.0 kcal/mol, respectively). The relationships between structure and binding are discussed.     

Alkaline phosphatase catalysis is ultrasensitive to charge sequestered between the active site zinc ions

Escherichia coli alkaline phosphatase (AP) is a prototypical bimetalloenzyme, facilitating catalysis of phosphate monoester hydrolysis with two Zn2+ metal ions that are only 4 A apart. In the reaction's transition state, one of the nonbridging oxygen atoms of the transferred group appears to interact directly with the Zn2+ ion metallocluster. To determine the importance and the energetic properties of this interaction, we systematically varied the charge on this oxygen atom, exploiting the ability of AP to catalyze reactions of different classes of substrates. We observed that the AP catalytic proficiency correlates very well (R2 = 0.98) with the charge on this oxygen atom, over 8 orders of magnitude of catalytic proficiency. The slope of this linear correlation (31 +/- 2 kcal/mol per unit charge) is extraordinarily steep, indicating that AP greatly discriminates between differentially charged substrates. We suggest that this discrimination arises via an electrostatic interaction with the bimetallocluster. The dependence of the AP catalytic proficiency on the nonbridging oxygen charge is much larger than charge perturbation effects observed previously for other proteins. We propose that AP uses folding energy to position the two Zn2+ metal ions in close proximity, thereby creating an active site with a high electrostatic potential that is extraordinarily sensitive to the charge that "solvates" the metallocluster. The sensitivity of enzyme energetics to systematic variation in electrostatic properties provides a powerful measure of the active site environment. Future work comparing the sensitivity of related enzymes that have been optimized to catalyze different reactions will help reveal how natural selection has tuned related active sites to favor different reactions.     

Enhancement of ammonia dehydrogenation by introduction of oxygen onto cobalt and iron cluster cations

Reactions of oxygen-chemisorbed cobalt and iron cluster cations (Co(n)O(m)(+) and Fe(n)O(m)(+); n = 3-6, m = 1-3) with an NH(3) molecule have been investigated in comparison with their bare metal cluster cations at a collision energy of 0.2 eV by use of a guided ion beam tandem mass spectrometer. We have observed three kinds of reaction products, which come from NH(3) chemisorption with and without release of a metal atom from the cluster and dehydrogenation of the chemisorbed NH(3). Reaction cross sections and branching fractions are strongly influenced by the number of oxygen atoms introduced onto the metal clusters. Oxygen-chemisorbed metal clusters with particular compositions such as Co(4)O(+), Co(5)O(2)(+), and Fe(5)O(2)(+) are extremely reactive for NH(3) dehydrogenation, whereas Co(4)O(2)(+) and Fe(4)O(2)(+) exhibit high reactivity for NH(3) chemisorption with metal release. The enhancement of dehydrogenation for specific compositions can be interpreted in terms of competition between O-H and neighboring Co-H (or Fe-H) formation.     

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

Diastereoselectivity in the Mukaiyama-Michael Reaction Employing alpha-Acyl beta,gamma-Unsaturated Phosphonates

The unique electronic and structural nature of the alpha-acylphosphonate functional group affords both dimeric and chelated complexes of diethyl crotonyl phosphonate (1; DECP) with stannic chloride (SnCl(4)). The dimeric complex, SnCl(4).(DECP)(2) (5) results from the coordination of two DECP molecules, ligated via the phosphoryl oxygens to the tin atom. The chelated complex, SnCl(4).(DECP) (6), is best represented with both phosphoryl and carbonyl oxygens coordinated to the metal center. Both metal ligated and chelated complexes have unique (13)C (31)P, and (119)Sn NMR spectra. In complex 5, the (13)C NMR resonances attributed to the carbonyl carbons were shifted upfield of free DECP. A monocoordinating Lewis acid, BF(3).OEt(2), produced a similar chemical shift trend in both the (13)C and (31)P NMR spectra of the BF(3).DECP complex. Essentially quantitative yields and moderate diastereomeric excesses favoring anti (or trans) diethyl 6-phenyl-4,5-dimethyl-6-(trimethylsilyloxy)-2-dihydropyranphosphonate (3) and diethyl 5-phenyl-3,4-dimethyl-1,5-dioxopentanephosphonate (4) were obtained from both chelated and dimeric SnCl(4).(DECP)(n) (n = 1, 2) when treated with either diastereomeric (Z)- or (E)-1-phenyl-1-(trimethylsilyloxy)-1-propene 2. Diethyl crotonylphosphonate (1), 3, and 4 were fully characterized.     

Rate of enolate formation is not very sensitive to the hydrogen bonding ability of donors to carboxyl oxygen lone pair acceptors; a ramification of the principle of non-perfect synchronization for general-base-catalyzed enolate formation

Two series of structures (1 and 2) possessing intramolecular hydrogen bonds to the lone-pair electrons of carbonyl oxygens have been examined to reveal the influence of the pK(a) of the hydrogen-bond donor on the rate of general-base-catalyzed enolate formation. The geometry of the hydrogen bonds is well accepted to be appropriate for intramolecular hydrogen-bond formation. Yet, as revealed by Br?nsted plots, both series show very little dependence of the rate of enolate formation on the hydrogen-bond donor ability. The intramolecular hydrogen bonds give rate enhancements only on the order of 10-100-fold, and corrected Br?nsted alpha-values are slightly below 0.1. The results can be understood by interpreting them in light of the Principle of Non-Perfect Synchronization. The results are consistent with the proton transfer occurring through an asynchronous transition state with the developing negative charge localized on carbon. We postulate that catalysts of enolate formation will be most effective if the binding groups are focused on stabilizing negative charge that is forming on the enolate carbon rather than on the enolate oxygen.     

Electronic structure and normal vibrations of CH3(OCH2CH2)nOCH3-M+-CF3SO3- (n = 2-4, M = Li, Na, and K)

Electronic structure and the vibrational frequencies of CH(3)(OCH(2)CH(2))(n)OCH(3)-M(+)-CF(3)SO(3)(-) (n = 2-4, M = Li, Na, and K) complexes have been derived from ab initio Hartree-Fock calculations. The metal ion shows varying coordination from 5 to 7 in these complexes. In tetraglyme-lithium triflate, Li(+) binds to one of the oxygens of CF(3)SO(3)(-) (triflate or Tf(-)) unlike for potassium or sodium ions, which possess bidentate coordination. Structures of glyme-MTf complexes thus derived agree well with those determined from X-ray diffraction experiments. The metal ion binds more strongly to ether oxygens of tetraglyme than its di- or triglyme analogues and engenders contraction of SO (for oxygens binding to metal ion) bonds with consequent frequency upshift for the corresponding vibration in the complex relative to those in the free MTf ion pairs. Complexation of the diglyme with LiTf engenders the largest downshift (91 cm(-1)) for the SO(2) stretching vibration of the free anion, which suggests stronger binding of lithium to the diglyme than the tri- (79 cm(-1)) or tetraglyme (70 cm(-1)). A frequency shift in the opposite direction for the SO (where oxygens do not coordinate to the metal) and CF(3) stretchings, which stems from the ion-polymer and anion-ion interactions, has been noticed. These frequency shifts have been analyzed using natural bond orbital analysis and difference electron density maps coupled with molecular electron density topography.     

Synthesis and structural and spectroscopic characterization of a complex between Co(II) and imino-bis(methylphosphonic acid): gaining insight into biologically relevant metal-ion phosphonate interactions or looking at a new Co(II)-organophosphonate material?

Cobalt is an essential metal ion involved in key biomolecules, regulating processes in human physiology. As a metal ion, Co(II) assumes forms, which are dictated by the nature of organic binders in biological fluids, and the conditions under which metal ion ligand interactions arise. Among the various low molecular mass metal ion binders in biological fluids are amino acids, organic acids, as well as their variably phosphorylated forms. As a representative metal ion binder, the organophosphonate ligand H(2)O(3)P-CH(2)-NH(2)(+)-CH(2)-PO(3)H(-) was employed in aqueous reactions with Co(II), ultimately leading to the isolation of complex [Co(C(2)H(8)O(6)NP(2))(2)(H(2)O)(2)] (1) at pH 2. The complex was characterized analytically, spectroscopically (FTIR, UV-vis, EPR), and magnetically. Compound 1 crystallizes in the monoclinic space group P2(1)/n, with a = 7.361(3) A, b = 8.133(3) A, c = 14.078(5) A, beta = 104.40(1) degrees, V = 816.3(5) A(3), and Z = 2. X-ray crystallography reveals that 1 is a compound with a molecular type of lattice. In it, there exist mononuclear octahedral sites of Co(II) surrounded by oxygens, belonging to terminal phosphonates and bound water molecules. Both ends of the ligand zwitterionic form are involved in binding to adjacent Co(II) ions, thus creating tetranuclear 32-membered rings, with cavities extending in two dimensions and holes in the third dimension throughout the lattice of 1. Similar structural features were observed in other metal organophosphonate lattices of potential catalytic and chemical reactivity. The magnetic and EPR data on 1 support the presence of a high-spin octahedral Co(II) in an oxygen environment, having a ground state with an effective spin S = (1)/(2). The solution UV-vis and EPR data suggest retention of the high-spin octahedral Co(II) ion, consistent with the magnetization measurements on 1. Collectively, the data reflect the existence of a soluble Co(II)-iminodiphosphonate species not unlike those expected in biological fluids containing the specific ligand or ligands similar to that. Both biologically relevant perspectives and a synthetic outlook into Co(II)-organophosphonate materials are discussed.     

An Isotope (<Superscript>18</Superscript>O, <Superscript>15</Superscript>N,and <Superscript>2</Superscript>H) Technique to Investigate the Metal Ion Interactions Between the Phosphoryl Group and Amino Acid Side Chains by Electrospray Ionization Mass Spectrometry

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS ⁿ ). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope (¹⁸O, ¹⁵N, and ²H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.     

Oxygen sites and network coordination in sodium germanate glasses and crystals: high-resolution oxygen-17 and sodium-23 NMR

Sodium germanate glasses are well-studied materials in which, unlike silicates but analogous to borates, the major structural consequence of alkali addition is generally thought to involve a coordination number increase of the network-forming Ge cations. However, the nature of this change, in particular quantifying fractions of nonbridging oxygens and of five- and/or six-coordinated Ge, has remained unresolved. We present here high-resolution 17O results, including triple-quantum MAS NMR (3QMAS), on a series of crystalline model compounds that allow the definition of ranges of chemical shifts corresponding to oxygens bonded to various coordinations of Ge. These include quartz- and rutile-structured GeO2, Na4Ge9O20, Na2Ge4O9, and Na2GeO3 (germanium dioxide, sodium enneagermanate, sodium tetragermanate, and sodium metagermanate). 3QMAS spectra of Na-germanate glasses ranging from 0% to 27% Na2O clearly show the development of partially resolved peaks as alkali is added, corresponding to signals from nonbridging oxygens (in the highest Na glasses) and to oxygen bridging between one four-coordinated and one higher coordinated Ge. As in conventional models of this system, nonbridging oxygen contents are much lower than in corresponding silicates. Although we do not directly distinguish between five- and six-coordinated Ge, modeling of bridging oxygen populations and comparison with measured speciation suggest that substantial proportions of both species are likely to be present. High-field 23Na MAS NMR shows systematic decreases in mean Na-O bond distance and/or coordination number with increasing alkali content that can be compared with published results for high-temperature liquids. These results, as well as comparison of molar volumes of glasses and high-temperature liquids, suggest the possibility of significant temperature effects on liquid structure.     

Characteristics of CO3(2-)-water hydrogen bonds in aqueous solution: insights from HF/MM and B3LYP/MM MD simulations

Two combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations, namely HF/MM and B3LYP/MM, have been performed to investigate the local hydration structure and dynamics of carbonate (CO(3)(2-)) in dilute aqueous solution. With respect to the QM/MM scheme, the QM region, which contains the CO(3)(2-) and its surrounding water molecules, was treated at HF and B3LYP levels of accuracy, respectively, using the DZV+ basis set, while the rest of the system is described by classical MM potentials. For both the HF/MM and B3LYP/MM simulations, it is observed that the hydrogen bonds between CO(3)(2-) oxygens and their nearest-neighbor waters are relatively strong, i.e., compared to water-water hydrogen bonds in the bulk, and that the first shell of each CO(3)(2-) oxygen atom somewhat overlaps with the others, which allows migration of water molecules among the coordinating sites to exist. In addition, it is observed that first-shell waters are either "loosely" or "tightly" bound to the respective CO(3)(2-) oxygen atoms, leading to large fluctuations in the number of first-shell waters, ranging from 1 to 6 (HF/MM) and 2 to 7 (B3LYP/MM), with the prevalent value of 3. Upon comparing the HF and B3LYP methods in describing this hydrated ion, the latter is found to overestimate the hydrogen-bond strength in the CO(3)(2-)-water complexes, resulting in a slightly more compact hydration structure at each of the CO(3)(2-) oxygens.     

Communication: CO oxidation by silver and gold cluster cations: identification of different active oxygen species

The oxidation of carbon monoxide with nitrous oxide on mass-selected Au(3)(+) and Ag(3)(+) clusters has been investigated under multicollision conditions in an octopole ion trap experiment. The comparative study reveals that for both gold and silver cations carbon dioxide is formed on the clusters. However, whereas in the case of Au(3)(+) the cluster itself acts as reactive species that facilitates the formation of CO(2) from N(2)O and CO, for silver the oxidized clusters Ag(3)O(x)(+) (n=1-3) are identified as active in the CO oxidation reaction. Thus, in the case of the silver cluster cations N(2)O is dissociated and one oxygen atom is suggested to directly react with CO, whereas a second kind of oxygen strongly bound to silver is acting as a substrate for the reaction.     

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2',3'-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10(5)-fold in their k(cat)/K(m) values for catalysis, but <40-fold in their K(i) values for inhibition of catalysis by U>v. Plots of logK(i) vs log(K(m)/k(cat)) for three distinct substrates [poly(cytidylic acid), uridine 3'-(p-nitrophenyl phosphate), and cytidine 2',3'-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K(i) for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.     

Interaction mechanisms between uranium(VI) and rutile titanium dioxide: from single crystal to powder

This paper is devoted to the study of the mechanisms of interaction between uranyl ion and rutile TiO2. Among the radionuclides of interest, U(VI) can be considered as a model of the radionuclides oxo-cations. The substrate under study here is the rutile titanium dioxide (TiO2) which is an interesting candidate as a methodological solid since it can be easily found as powder and as manufactured single crystals. This material presents also a wide domain of stability as a function of pH. Then, it allows the study of the retention processes on well-defined crystallographic planes, which can lead to a better understanding of the surface reaction mechanisms. Moreover, it is well-established that the (110) crystallographic orientation is dominating the surface chemistry of the rutile powder. Therefore, the spectroscopic results obtained for the U(VI)/rutile (110) system and other relevant crystallographic orientations were used to have some insight on the nature of the uranium surface complexes formed on rutile powder. This goal was achieved by using time-resolved laser-induced fluorescence spectroscopy (TRLFS) which allows the investigation, at a molecular scale, of the nature of the reactive surface sites as well as the surface species. For rutile surfaces, oxygen atoms can be 3-fold, 2-fold (bridging oxygens), or single-fold (top oxygens) coordinated to titanium atoms. However, among these three types of surface oxygen atoms, the 3-fold coordinated ones are not reactive toward water molecules or aqueous metallic cations. This study led to conclude on the presence of two uranium(VI) surface complexes: the first one corresponds to the sorption of aquo UO22+ ion sorbed on two bridging oxygen atoms, while the second one, which is favored at higher surface coverages, corresponds to the retention of UO22+ by one bridging and one top oxygen atom. Thus, the approach presented in this paper allows the establishment of experimental constraints that have to be taken into account in the modeling of the sorption mechanisms.     

The hydrogen bonding and hydration of 2'-OH in adenosine and adenosine 3'-ethyl phosphate

The 2'-OH group has major structural implications in the recognition, processing, and catalytic properties of RNA. We report here intra- and intermolecular H-bonding of 2'-OH in adenosine 3'-ethyl phosphate (1), 3'-deoxyadenosine (2), and adenosine (3) by both temperature- and concentration-dependent NMR studies, as well as by detailed endo ((3)J(H,H)) and exocyclic ((3)J(H,OH)) coupling constant analyses. We have also examined the nature of hydration and exchange processes of 2'-OH with water by a combination of NOESY and ROESY experiments in DMSO-d(6) containing 2 mol % HOD. The NMR-constrained molecular modeling (by molecular mechanics as well as by ab initio methods both in the gas and solution phase) has been used to characterize the energy minima among the four alternative dihedrals possible from the solution of the Karplus equation for (3)J(H2',OH) and (3)J(H3',OH) to delineate the preferred orientation of 2'-O-H proton in 1 and 2 as well as for 2'/3'-O-H protons in 3. The NMR line shape analysis of 2'-OH gave the DeltaG(H-bond)(298K) of 7.5 kJ mol(-1) for 1 and 8.4 kJ mol(-1) for 3; similar analyses of the methylene protons of 3'-ethyl phosphate moiety in 1 also gave comparable DeltaG(H-bond)(298K) of 7.3 kJ mol(-1). The donor nature of the 2'-OH in the intramolecular H-bonding in 3 is evident from its relatively reduced flexibility [-TDeltaS++](2'-OH) = -17.9(+/-0.5) kJ mol(-1)] because of the loss of conformational freedom owing to the intramolecular 2'O-H...O3' H-bonding, compared to the acceptor 3'-OH in 3 [-TDeltaS++](3'-OH) = -19.8 (+/- 0.6) kJ mol(-1)] at 298 K. The presence of intramolecular 2'-OH...O3' H-bonding in 3 is also corroborated by the existence of weak long-range (4)J(H2',OH3') in 3 (i.e., W conformation of H2'-C2'-C3'-O3'-H) as well as by (3)J(H,OH) dependent orientation of the 2'- and 3'-OH groups. The ROESY spectra for 1 and 3 at 308 K, in DMSO-d(6), show a clear positive ROE contact of both 2'- and 3'-OH with water. The presence of a hydrophilic 3'-phosphate group in 1 causes a much higher water activity in the vicinity of its 2'-OH, which in turn causes the 2'-OH to exchange faster, culminating in a shorter exchange lifetime (tau) for 2'-OH proton with HOD in 1 (tau2'-OH: 489 ms) compared to that in 3 (tau2'-OH: 6897 ms). The activation energy (E(a)) of the exchange with the bound-water for 2'- and 3'-OH in 3 (48.3 and 45.0 kJ mol(-1), respectively) is higher compared to that of 2'-OH in 1 (31.9 kJ mol(-1)), thereby showing that the kinetic availability of hydrated 2'-OH in 1 for any inter- and intramolecular interactions, in general, is owing to the vicinal 3'-phosphate residue. It also suggests that 2'-OH in native RNA can mediate other inter- or intramolecular interactions only in competition with the bound-water, depending upon the specific chemical nature and spatial orientation of other functions with potential for hydrogen bonding in the neighborhood. This availability of the bound water around 2'-OH in RNA would, however, be dictated by whether the vicinal phosphate is exposed to the bulk water or not. This implies that relatively poor hydration around a specific 2'-OH across a polyribonucleotide chain, owing to some hydrophobic microenvironmental pocket around that hydroxyl, may make it more accessible to interact with other donor or acceptor functions for H-bonding interactions, which might then cause the RNA to fold in a specific manner generating a new motif leading to specific recognition and function. Alternatively, a differential hydration of a specific 2'-OH may modulate its nucleophilicity to undergo stereospecific transesterification reaction as encountered in ubiquitous splicing of pre-mRNA to processed RNA or RNA catalysis, in general.