Theoretical examination of two opposite mechanisms proposed for hepatitis delta virus ribozyme

Two mechanisms were previously proposed for the hepatitis delta virus (HDV) ribozyme where an active-site cytosine residue (C75) either functioned as a general base to deprotonate the 2'-OH at the rupture site or as a general acid to protonate the O5' leaving group. Here, we reported the first theoretical examination of the two mechanisms using a combination of the quantum mechanics (QM)/molecular mechanics (MM), molecular dynamics (MD), and near-attack-conformation (NAC) techniques. Our theoretical results supported the C75-acid mechanism, which was demonstrated to have an unfavorable starting geometry (in agreement with the crystallographic data) but a significantly lower energy barrier as compared to the C75-base mechanism. Therefore, the chemical details of the transition state in the HDV ribozyme may dramatically differ from those inferred from the structural studies.     

Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation

Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2'OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2'OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.     

Dinuclear Zn2+ complexes in the hydrolysis of the phosphodiester linkage in a diribonucleoside monophosphate diester

Dizinc complexes that were formed from 2:1 mixtures of Zn(NO3)2 and dinucleating ligands TPHP (1), TPmX (2) or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence of (Zn2+)(2)-1 was sigmoidal, whereas those of (Zn2+)(2)-2 and (Zn2+)(2)-3 were bell-shaped. The pH titration study indicated that (Zn2+)(2)-1 dissociates only one aquo proton (up to pH 12), whereas (Zn2+)(2)-2 dissociates three aquo protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn2+)(2)-1, the NpN cleavage using (Zn2+)(2)-2 showed a greater rate constant, with a higher product ratio of 3'-NMP/2'-NMP. The saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis-Menten type kinetics. Although the binding of (Zn2+)(2)-2 to ApA was weaker than that of (Zn2+)(2)-1, (Zn2+)(2)-2 showed a greater kcat value than (Zn2+)(2)-1, resulting in higher ApA cleavage activity of the former.     

Detection of a proton-transfer process by kinetic solvent isotope effects in NH(4)(+)-mediated reactions catalyzed by a hammerhead ribozyme

Hammerhead ribozymes have been considered to be metalloenzymes. However, this proposal was recently questioned by the finding that the reaction proceeds in the presence of high concentrations of monovalent ions such as NH(4)(+) ions and in the absence of any divalent metal ions. Our present analysis based on solvent isotope effects indicates that (1) a proton transfer(s) occurs only in the NH(4)(+)-mediated reaction but not in metal-ion-mediated reactions such as Mg(2+)- and Li(+)-mediated reactions, (2) the catalyst that stabilizes the 5' leaving group in the NH(4)(+)-mediated reaction is different from that in the metal-ion-mediated HH ribozyme reactions, (3) an NH(4)(+) ion seems to act as a general acid catalyst, and (4) a nucleobase alone should not be the catalyst.     

Determination of the intrinsic affinities of multiple site-specific Mg(2+) ions coordinated to domain 6 of a group II intron ribozyme

Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 (D6) contains a conserved adenosine whose 2'-OH group acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long D6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5gamma, also identifying five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di- and triphosphate groups is strongest (e.g., log KA,TP = 4.55 +/- 0.10) and is evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 +/- 0.06) and thus compete for the free Mg2+ ions in solution, having a distinct influence on the individual affinities of the various sites. For the first time, we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA molecule undergoing tertiary contacts to a second RNA molecule first needs to be loaded evenly and specifically with metal ions to compensate for the repulsion between the negatively charged RNA molecules.     

Magnesium-tetrazaporphyrin incorporated PVC matrix as a new material for fabrication of Mg(2+) selective potentiometric sensor

Membrane incorporating [Mg{(TAP)(SBn)₈}] complex, (I), as ionophore with composition I:NaTPB:DOP:PVC in the ratio 10:2:133:200 (w/w) exhibits the best result for potentiometric sensing of Mg²⁺ ions. This gives linear potential response in the concentration range of 9.4×10⁻⁶ to 1.0×10⁻¹ M with a slope of 29.2±0.4 mV per decade of activity of Mg²⁺. Standard deviation in observed values of potentials in this concentration range, from the least square fit line, found to be 2.91 mV with 90% confidence limit lying within ±0.4 mV per decade of activity. The electrode works satisfactorily in the pH range 3.5-7.8 and shows a fast response time of 13±2 s. It shows good selectivity for Mg²⁺ over other mono-, bi- and tri-valent cations. Only K⁺ and Zn²⁺ cause slight interference if present at concentrations ≥1.0×10⁻⁵ M. The electrode is durable and can be used over a period of 5 months with good reproducibility (∼1% error). It has been successfully used as an indicator electrode in potentiometric titration of Mg²⁺ against EDTA as well as for the determination of Mg²⁺ in simulated mixtures.     

Differential binding of Mg2+, Zn2+, and Cd2+ at two sites in a hammerhead ribozyme motif, determined by 15N NMR

A decamer duplex model of Domain II of the hammerhead ribozyme was synthesized with [8-13C-1,7,NH2-15N3]-guanosine at the known metal binding site, G10.1 and, for comparison, [2-13C-1,7,NH2-15N3]-guanosine at G16.2. The 15N NMR chemical shifts of the labeled N7s monitored during addition of Mg2+, Cd2+, and Zn2+ showed the same preference for binding at G10.1 over G16.2 for each metal. These results demonstrate that 15N labeling can be used to evaluate the binding of different metals, including Mg2+, to a given nitrogen, as well as to compare the binding potential of different sites.     

Unimolecular and hydrolysis channels for the detachment of water from microsolvated alkaline earth dication (Mg2+, Ca2+, Sr2+, Ba2+) clusters

We examine theoretically the three channels that are associated with the detachment of a single water molecule from the aqueous clusters of the alkaline earth dications, [M(H₂O) _n ]²⁺, M = Mg, Ca, Sr, Ba, n ≤ 6. These are the unimolecular water loss (M²⁺(H₂O) _n?1 + H₂O) and the two hydrolysis channels resulting the loss of hydronium ([MOH(H₂O) _n?2]⁺ + H₃O⁺) and Zundel ([MOH(H₂O) _n?3]⁺ + H₃O⁺(H₂O)) cations. Minimum energy paths (MEPs) corresponding to those three channels were constructed at the Møller–Plesset second order perturbation (MP2) level of theory with basis sets of double- and triple-ζ quality. We furthermore investigated the water and hydronium loss channels from the mono-hydroxide water clusters with up to four water molecules, [MOH(H₂O) _n ]⁺, 1 ≤ n ≤ 4. Our results indicate the preference of the hydronium loss and possibly the Zundel-cation loss channels for the smallest size clusters, whereas the unimolecular water loss channel is preferred for the larger ones as well as the mono-hydroxide clusters. Although the charge separation (hydronium and Zundel-cation loss) channels produce more stable products when compared to the ones for the unimolecular water loss, they also require the surmounting of high-energy barriers, a fact that makes the experimental observation of fragments related to these hydrolysis channels difficult.     

Complexation in the System Paramagnetic Ion (Tm3 +)-Diamagnetic Ion (Mg2 +)-Carboxylic Acid

The dependence of the NMR chemical shift in the system containing complexes of a paramagnetic cation PL and PL₂ on the concentration of a diamagnetic salt was simulated. The complexation in the system paramagnetic ion (Tm^{3 +})-diamagnetic ion (Mg^{2 +})-carboxylic acid (acetic, propionic, n-butyric) was studied experimentally. The effect of the second cation on the calculated complexation constants was detected.     

Theoretical study on the Co~(2+)OH_2/Co~(3+)OH_2 electron transfer reactivity

This paper presents a contact distance dependence analysis scheme and an ab initio calculation application for the electron transfer (ET) reactivity of Co2+OH2/Co3+OH2 reacting pair. The applicability of these schemes and the corresponding models has been discussed. The contact distance (Rcoco) dependence of the relevant quantities has been analyzed. The results indicate that the activation energy from the accurate PES method agrees well with that from the anharmonic potential method, and they are obviously better than that from the harmonic potential method. The pair distribution function varies from 10~(-2) to 10~(-5) along with Rcoco changing from 1.20 to 0.35 nm. The coupling matrix element exponentially decays along with the increase of Rcoco, and the effective electronic coupling requires Rcoco smaller than 0.75 nm. In the range from 0.50 to 0.75 nm for Rcoco, the corresponding electronic transmission coefficient falls within 1.0-10~(-6). The local ET rate also exponentially decays along with the incre     

Coordination environment of a site-bound metal ion in the hammerhead ribozyme determined by 15N and 2H ESEEM spectroscopy

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a K(d,app) < 10 microM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin-echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands.     

Carboxylic acid functionalized cobalt(III) cyclen complexes for catalytic hydrolysis of phosphodiester bonds

4-(1,4,7,10-Tetraazacyclotetradec-1-yl)methylbenzoic acid (cycmba, 1) has been synthesized, as a step towards the eventual development of sequence-specific hydrolytic complexes. A cobalt(III) complex of 1, [Co(cycmba)Cl2]Cl.1.5H2O (.1.5H2O) was found to be active against both an activated phosphodiester compound, bis(nitrophenyl)phosphate (BNPP), and supercoiled DNA. The presence of the benzoate group depresses the rate of hydrolysis of the ligand-Co(III) system at neutral pH, as confirmed by the kinetics results of a methyl ester analog. The ability of (2.1.5H2O) to bind to solid substrates and remain active was also demonstrated by attachment of the molecule to agarose beads.     

Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy

BACKGROUND: Divalent metal ions serve as structural as well as catalytic cofactors in the hammerhead ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the hammerhead ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces. RESULTS: Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the hammerhead ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z(2)/r of the aqua ion (where Z is the charge and r is the radius of the ion). CONCLUSIONS: This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins.     

Molecular dynamics analysis of Mg2+-dependent cleavage of a pistol ribozyme reveals a fail-safe secondary ion for catalysis

Pistol ribozymes comprise a class of small, self-cleaving RNAs discovered via comparative genomic analysis. Prior work in the field has probed the kinetics of the cleavage reaction, as well as the influence of various metal ion cofactors that accelerate the process. In the current study, we performed unbiased and unconstrained molecular dynamics simulations from two current high-resolution pistol crystal structures, and we analyzed trajectory data within the context of the currently accepted ribozyme mechanistic framework. Root-mean-squared deviations, radial distribution functions, and distributions of nucleophilic angle-of-attack reveal insights into the potential roles of three magnesium ions with respect to catalysis and overall conformational stability of the molecule. A series of simulation trajectories containing in silico mutations reveal the relatively flexible and partially interchangeable roles of two particular magnesium ions within solvated hydrogen-bonding distances from the catalytic center.     

Theoretical Researches on the Charge Transport Properties of Humic Acid Coordinating with Fe~(3+)Cu~(2+)Al~(3+) Metal Ions

Humic acid is a key component of extracellular electron acceptor. Experimental study elucidates that humic acid molecular ligand with different metal elements has different abilities to accept electrons. By using density functional theory, this article selected the leonardite humic acid(LHA) organic macromolecule as ligand to study interactions between the ligand and different metals. At the same time, the calculation of binding energy, the analysis of characteristics for the complex structure and the distribution of frontier molecular orbital were also completed. On the basis of Marcus theory, the reorganization energy, matrix element and charge transport rate constant were calculated. The results show that the order of the charge transfer rates is Fe~(3+)Cu~(2+)Al~(3+) for different metal complexes, and are in good agreement with the experimental ones.     

Characterization of the glycosidic linkage of underivatized disaccharides by interaction with Pb(2+) ions

Electrospray ionization in combination with tandem mass spectrometry and lead cationization is used to characterize the linkage position of underivatized disaccharides. Lead(II) ions react mainly with disaccharides by proton abstraction to generate [Pb(disaccharide)(m)-H](+) ions (m = 1-2). At low cone voltages, an intense series of doubly charged ions of general formula [Pb(disaccharide)(n)](2+) are also observed. Our study shows that MS/MS experiments have to be performed to differentiate Pb(2+)-coordinated disaccharides. Upon collision, [Pb(disaccharide)-H](+) species mainly dissociate according to glycosidic bond cleavage and cross-ring cleavages, leading to the elimination of C(n)H(2n)O(n) neutrals (n = 2-4). The various fragmentation processes allow the position of the glycosidic bond to be unambiguously located. Distinction between glc-glc and glc-fru disaccharides also appears straightforward. Furthermore, for homodimers of D-glucose our data demonstrate that the anomericity of the glycosidic bond can be characterized for the 1 --> n linkages (n = 2, 4, 6). Consequently, Pb(2+) cationization combined with tandem mass spectrometry appears particularly useful to identify underivatized disaccharides.