The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions

Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

Electrochemistry of P450cin: new insights into P450 electron transfer

Electrochemistry of bacterial cytochrome P450cin (CYP176A) reveals that, unusually, substrate binding does not affect the heme redox potential, although a marked pH dependence is consistent with a coupled single electron/single proton transfer reaction in the range 6 < pH < 10.

The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding

DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Angstrom, and present a proposal for its interaction with peptide. The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified-the typo IV beta-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains. Three unique features on the active site surface of the DsbA molecule-a groove, hydrophobic pocket, and hydrophobic patch-form an extensive uncharged surface surrounding the active-sits disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins. A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Leading coordinate analysis of reaction pathways in proton chain transfer: Application to a two-proton transfer model for the green fluorescent protein

The ‘leading coordinate’ approach to computing an approximate reaction pathway, with subsequent determination of the true minimum energy profile, is applied to a two-proton chain transfer model based on the chromophore and its surrounding moieties within the green fluorescent protein (GFP). Using an ab initio quantum chemical method, a number of different relaxed energy profiles are found for several plausible guesses at leading coordinates. The results obtained for different trial leading coordinates are rationalized through the calculation of a two-dimensional relaxed potential energy surface (PES) for the system. Analysis of the 2-D relaxed PES reveals that two of the trial pathways are entirely spurious, while two others contain useful information and can be used to furnish starting points for successful saddle-point searches. Implications for selection of trial leading coordinates in this class of proton chain transfer reactions are discussed, and a simple diagnostic function is proposed for revealing whether or not a relaxed pathway based on a trial leading coordinate is likely to furnish useful information.

Numerical modelling of double diffusion driven reactive flow transport in deformable fluid-saturated porous media with particular consideration of temperature-dependent chemical reaction rates

Numerical methods ave used to solve double diffusion driven reactive flow transport problems in deformable fluid-saturated porous media. in particular, thp temperature dependent reaction rate in the non-equilibrium chemical reactions is considered. A general numerical solution method, which is a combination of the finite difference method in FLAG and the finite element method in FIDAP, to solve the fully coupled problem involving material deformation, pore-fluid flow, heat transfer and species transport/chemical reactions in deformable fluid-saturated porous media has been developed The coupled problem is divided into two subproblems which are solved interactively until the convergence requirement is met. Owing to the approximate nature of the numerical method, if is essential to justify the numerical solutions through some kind of theoretical analysis. This has been highlighted in this paper The related numerical results, which are justified by the theoretical analysis, have demonstrated that the proposed solution method is useful for and applicable to a wide range of fully coupled problems in the field of science and engineering.

Copolymerization of methyl methacrylate and allyl acetate Part I. Rate of reaction

Free radical bulk copolymerization of methyl methacrylate (MMA) and allyl acetate (AAc) has been investigated using electron spin resonance (ESR) and FT-near infrared (FTNIR) spectroscopy. Data are used to evaluate the rate constants. The mole fraction of AAc plays an important role in the copolymerization of these two monomers. AAc not only delays the Trommsdorff effect but also increases the onset of percentage total conversion at which the Trommsdorff region begins. With AAc fraction 0.5 and higher, no Trommsdorff effect was observed. Inclusion of AAc into copolymer structure mainly occurs in the Trommsdorf region or when the AAc fraction in the comonomer feed is dominant. This is associated with a drop in the concentration of propagating radicals. However, ESR spectra indicate that the MMA propagating radical is predominant during the reaction. In the comonomer mixtures where a Trommsdorff region can be observed, the addition of AAc does not produce any significant change in k(p) and k(t) in the steady state region. Major changes in k(p) and k(t) are observed after the gel point and glassy state, respectively. (C) 2001 Society of Chemical Industry.

Time-dependent master equation simulation of complex elementary reactions in combustion: Application to the reaction of (CH2)-C-1 with C2H2 from 300-2000 K

Computational simulations of the title reaction are presented, covering a temperature range from 300 to 2000 K. At lower temperatures we find that initial formation of the cyclopropene complex by addition of methylene to acetylene is irreversible, as is the stabilisation process via collisional energy transfer. Product branching between propargyl and the stable isomers is predicted at 300 K as a function of pressure for the first time. At intermediate temperatures (1200 K), complex temporal evolution involving multiple steady states begins to emerge. At high temperatures (2000 K) the timescale for subsequent unimolecular decay of thermalized intermediates begins to impinge on the timescale for reaction of methylene, such that the rate of formation of propargyl product does not admit a simple analysis in terms of a single time-independent rate constant until the methylene supply becomes depleted. Likewise, at the elevated temperatures the thermalized intermediates cannot be regarded as irreversible product channels. Our solution algorithm involves spectral propagation of a symmetrised version of the discretized master equation matrix, and is implemented in a high precision environment which makes hitherto unachievable low-temperature modelling a reality.

Prediction of absolute rate coefficients and product branching ratios for the C(P-3) plus allene reaction system

Complex chemical reactions in the gas phase can be decomposed into a network of elementary (e.g., unimolecular and bimolecular) steps which may involve multiple reactant channels, multiple intermediates, and multiple products. The modeling of such reactions involves describing the molecular species and their transformation by reaction at a detailed level. Here we focus on a detailed modeling of the C(P-3)+allene (C3H4) reaction, for which molecular beam experiments and theoretical calculations have previously been performed. In our previous calculations, product branching ratios for a nonrotating isomerizing unimolecular system were predicted. We extend the previous calculations to predict absolute unimolecular rate coefficients and branching ratios using microcanonical variational transition state theory (mu-VTST) with full energy and angular momentum resolution. Our calculation of the initial capture rate is facilitated by systematic ab initio potential energy surface calculations that describe the interaction potential between carbon and allene as a function of the angle of attack. Furthermore, the chemical kinetic scheme is enhanced to explicitly treat the entrance channels in terms of a predicted overall input flux and also to allow for the possibility of redissociation via the entrance channels. Thus, the computation of total bimolecular reaction rates and partial capture rates is now possible. (C) 2002 American Institute of Physics.

Determination of external and internal mass transfer limitation in nitrifying microbial aggregates

In this article we present a study of the effects of external and internal mass transfer limitation of oxygen in a nitrifying system. The oxygen uptake rates (OUR) were measured on both a macro-scale with a respirometric reactor using off-gas analysis (Titrimetric and Off-Gas Analysis (TOGA) sensor) and on a micro-scale with microsensors. These two methods provide independent, accurate measurements of the reaction rates and concentration profiles around and in the granules. The TOGA sensor and micro-sensor measurements showed a significant external mass transfer effect at low dissolved oxygen (DO) concentrations in the bulk liquid while it was insignificant at higher DO concentrations. The oxygen distribution with anaerobic or anoxic conditions in the center clearly shows major mass transfer limitation in the aggregate interior. The large drop in DO concentration of 22 - 80% between the bulk liquid and aggregate surface demonstrates that the external mass transfer resistance is also highly important. The maximum OUR even for floccular biomass was only attained at much higher DO concentrations ( approximate to 8 mg/L) than typically used in such systems. For granules, the DO required for maximal activity was estimated to be > 20mg/L, clearly indicating the effects of the major external and internal mass transfer limitations on the overall biomass activity. Smaller aggregates had a larger volumetric OUR indicating that the granules may have a lower activity in the interior part of the aggregate. (C) 2004 Wiley Periodicals, Inc.

Modeling the molecular weight distribution of block copolymer formation in a reversible addition-fragmentation chain transfer mediated living radical polymerization

Block copolymers have become an integral part of the preparation of complex architectures through self-assembly. The use of reversible addition-fragmentation chain transfer (RAFT) allows blocks ranging from functional to nonfunctional polymers to be made with predictable molecular weight distributions. This article models block formation by varying many of the kinetic parameters. The simulations provide insight into the overall polydispersities (PDIs) that will be obtained when the chain-transfer constants in the main equilibrium steps are varied from 100 to 0.5. When the first dormant block [polymer-S-C(Z)=S] has a PDI of 1 and the second propagating radical has a low reactivity to the RAFT moiety, the overall PDI will be greater than 1 and dependent on the weight fraction of each block. When the first block has a PDI of 2 and the second propagating radical has a low reactivity to the RAFT moiety, the PDI will decrease to around 1.5 because of random coupling of two broad distributions. It is also shown how we can in principle use only one RAFT agent to obtain block copolymers with any desired molecular weight distribution. We can accomplish this by maintaining the monomer concentration at a constant level in the reactor over the course of the reaction. (c) 2005 Wiley Periodicals, Inc.

Controlled Radical Polymerization of Styrene and Methyl Acrylate in the Presence of Reversible Addition-Fragmentation Chain Transfer Agents, Phenylethyl Phenyl Dithioacetate and Phenyldithioacetic Acid

The use of phenyldithioacetic acid (PDA) in homopolymerizations of styrene or methyl acrylate produced only a small fraction of chains with dithioester end groups. The polymerizations using 1-phenylentyl phenyldithioacetate (PEPDTA) and PDA in the same reaction showed that PDA had little or no influence on the rate or molecular weight distribution even when a 1:1 ratio is used. The mechanistic pathway for the polymerizations in the presence of PDA seemed to be different for each monomer. Styrene favors addition of styrene to PDA via a Markovnikov type addition to form a reactive RAFT agent. The polymer was shown by double detection SEC to contain dithioester end groups over the whole distribution. This polymer was then used in a chain extension experiment and the M-n was close to theory. A unique feature of this work was that PDA could be used to form a RAFT agent in situ by heating a mixture of styrene and PDA for 24 h at 70 degrees C and then polymerizing in the presence of AIBN to give a linear increase in Mn and low values of PDI (< 1.14). In the case of the polymerization of MA with PDA, the mechanism was proposed to be via degradative chain transfer. (c) 2005 Wiley Periodicals, Inc.

Pressure and temperature effects on metal-to-metal charge transfer in cyano-bridged Co-III-Fe-II complexes

The effects of pressure and temperature on the energy (E-op) of the metal-to-metal charge transfer (MMCT, Fe-II --> Co-III) transition of the cyano-bridged complexes trans - [(LCoNCFe)-Co-14(CN)(5)](-) and cis-[(LCoNCFe)-Co-14(CN)(5)](-) (where L-14 = 6-methyl-1,4,8,11-tetraazacyclotetradecan-6-amine) were examined. The changes in the redox potentials of the cobalt and iron metal centres with pressure and temperature were also examined and the results interpreted with Marcus Hush theory. The observed redox reaction volumes can mainly be accounted for in terms of localised electrostriction effects. The shifts in E-op due to both pressure and temperature were found to be less than the shifts in the energy difference (E degrees) between the Co-III-Fe-II and Co-II-Fe-III redox isomers. The pressure and temperature dependence of the reorganisational energy, as well as contributions arising from the different spin states of Co-II, are discussed in order to account for this trend. To study the effect of pressure on Co-III electronic absorption bands, a new cyano-bridged complex, trans - [(LCoNCCo)-Co-14(CN)(5)], was prepared and characterised spectroscopically and structurally. X-Ray crystallography revealed this complex to be isostructural with trans -[(LCoNCFe)-Co-14(CN)(5)] center dot 5H(2)O.

Intra- vs intermolecular photoinduced electron transfer reactions of a macrocyclic donor-acceptor dyad

The synthesis, structural characterization, and photophysical behavior of a 14-membered tetraazamacrocycle with pendant 4-dimethylaminobenzyl (DMAB) and 9-anthracenylmethyl groups is reported (L-3, 6-((9-anthracenylmethyl)amino)-trans-6,13-dimethyl-13-((4-dimethylaminobenzyl)amino)-1,4,8,11-tetraaza-cyclotetradecane). In its free base form, this compound displays rapid intramolecular photoinduced electron transfer (PET) quenching of the anthracene emission, with both the secondary amines and the DMAB group capable of acting as electron donors. When complexed with Zn(II), the characteristic fluorescence of the anthracene chromophore is restored as the former of these pathways is deactivated by coordination. Importantly, it is shown that the DMAB group, which remains uncoordinated and PET active, acts only very weakly to quench emission, by comparison to the behavior of a model Zn complex lacking the pendant DMAB group, [ZnL2](2+) (Chart 1). By contrast, Stern-Volmer analysis of intermolecular quenching of [ZnL2](2+) by N,N-dimethylaniline (DMA) has shown that this reaction is diffusion limited. Hence, the pivotal role of the bridge in influencing intramolecular PET is highlighted.