Facilitate Proton Transfer Research Articles

1.55 Å-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis

BackgroundThe diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained. MethodUse of crystallization additives yielded CPS crystals diffracting to 1.55Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis. ResultsThis work clarifies structure–function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1, as well as extensive solvent structure, are observed. ConclusionsTwo channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded “proton wires” that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1, is proposed to be a ligand to Mg2+ required for optimal catalytic activity. General significanceThis work establishes structure–function relationships for class II terpenoid cyclases.

Mechanisms of Horseradish Peroxidase and α-Chymotrypsin

The pH range for Compound I formation of horseradish peroxidase (2.5 to 11) is the largest for any known enzyme reaction. A key part of the reaction is proton transfer from hydrogen peroxide to distal His42. This proton is retained to complete formation of a water leaving group as the ferryl porphyrin π-cation radical is formed. How can the imidazolium side chain of His42 retain a proton at very high pH? And how can it give up the proton when required at very low pH? The answer is rearrangement of electronic charge through Electron Density Circuits (EDCs) in the protein matrix. An increase of at least 9 p Ka units, which occurs on the imidazole side chain of His42 as the Compound I reactive intermediate is formed, is facilitated by an EDC. A reverse EDC facilitates proton transfer to form the water leaving group. The pathway of the EDC involves the heme, its propionate side chains, Arg41, and His42. The occurrence of EDCs in chymotrypsin reactions reinforces the nucleophilic attack, and the subsequent electron and proton transfers. They also can shift p Ka values. The catalytic triad of chymotrypsin is Ser195, His57, and Asp102. The currently accepted one-proton transfer mechanism involves Ser195 and His57, with Asp102 being regarded as too acidic to accept a proton. A comparatively small shift in the p Ka value of Asp102 is all that is required to make it a proton acceptor from His57 so a two-proton mechanism may occur.

Independent control of water retention and acid–base pairing through double-shelled microcapsules to confer membranes with enhanced proton conduction under low humidity

Proton exchange membranes (PEM) with affordable and controllable proton conductivity under low humidity are crucial to the commercial application of PEM fuel cells. In this study, double-shelled polymer microcapsules bearing a carboxylic acid inner shell and imidazole outer shell (PMC-Ns) are synthesized via distillation–precipitation polymerization and then incorporated into a sulfonated poly(ether ether ketone) matrix to fabricate composite membranes. The inner shell renders the absorbed water of lower chemical potential and higher bound water content, yielding the composite membrane with enhanced water retention properties. The higher water uptake ensures composite membrane facilitated proton transfer via a vehicle mechanism and the lower water loss confers a reduced proton conductivity decline. The outer shell generates sulfonic acid–imidazole pairs within the membranes, which construct low-energy-barrier pathways to facilitate proton transfer via the Grotthuss mechanism. Under identical conditions, PMC-Ns endow much higher proton conductivity to composite membranes than the microcapsules with both carboxylic acid inner shell and outer shell, or both imidazole inner shell and outer shell. Particularly, incorporating 20 wt% PMC-Ns affords the composite membrane a 1.7 times increase in proton conductivity under 100% relative humidity (RH) and a 41.8 times increase in proton conductivity under 20% RH. Moreover, the methanol barrier property of the composite membranes is explored.

H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters form a family of molecular motor proteins that couple ATP hydrolysis to substrate translocation across cell membranes. Each nucleotide binding domain of ABC-transporters contains a highly conserved H-loop histidine residue, whose precise mechanistic role in motor functions has remained elusive. By using combined quantum mechanical and molecular mechanical (QM/MM) calculations, we showed that the conserved H-loop residue H662 in E. coli HlyB, a bacterial ABC-transporter, can act first as a general acid and then as a general base to facilitate proton transfer in ATP hydrolysis. Without the assistance of H662, direct proton transfer from the lytic water to ATP results in a substantially higher barrier height. Our findings suggest that the essential function of the H-loop residue H662 is to provide a "chemical linchpin" that shuttles protons between reactants through a relay mechanism, thereby catalyzing ATP hydrolysis in HlyB.

Embedding of Hollow Polymer Microspheres with Hydrophilic Shell in Nafion Matrix as Proton and Water Micro-Reservoir

Assimilating hydrophilic hollow polymer spheres (HPS) into Nafion matrix by a loading of 0.5 wt % led to a restructured hydrophilic channel, composed of the pendant sulfonic acid groups (–SO3H) and the imbedded hydrophilic hollow spheres. The tiny hydrophilic hollow chamber was critical to retaining moisture and facilitating proton transfer in the composite membranes. To obtain such a tiny cavity structure, the synthesis included selective generation of a hydrophilic polymer shell on silica microsphere template and the subsequent removal of the template by etching. The hydrophilic HPS (100–200 nm) possessed two different spherical shells, the styrenic network with pendant sulfonic acid groups and with methacrylic acid groups, respectively. By behaving as microreservoirs of water, the hydrophilic HPS promoted the Grotthus mechanism and, hence, enhanced proton transport efficiency through the inter-sphere path. In addition, the HPS with the –SO3H borne shell played a more effective role than those with the –CO2H borne shell in augmenting proton transport, in particular under low humidity or at medium temperatures. Single H2-PEMFC test at 70 °C using dry H2/O2 further verified the impactful role of hydrophilic HPS in sustaining higher proton flux as compared to pristine Nafion membrane.

Oxygen and hydrogen peroxide reduction by 1,2-diferrocenylethane at a liquid/liquid interface

Molecular oxygen and hydrogen peroxide reduction by 1,2-diferrocenylethane (DFcE) was investigated at a polarized water/1,2-dichloroethane (W/DCE) interface. The overall reaction points to a proton-coupled electron transfer (PCET) mechanism, where the first step consists of the protonation of DFcE to form the DFcE–H+ in DCE phase, either by DFcE facilitated proton transfer across the liquid–liquid interface or by the homogeneous protonation of DFcE in the presence of protons extracted in the oil phase by tetrakis(pentafluorophenyl)borate. The formation of DFcE–H+ is followed up by the O2 reduction to hydrogen peroxide and further reduction to water. The final products of DFcE oxidation, namely DFcE+ or DFcE2+, were investigated by ion transfer voltammetry, ultramicroelectrode voltammetry and UV/visible spectroscopy. These results show that mostly DFcE+ is produced, although DFcE+ can also reduce oxygen at longer time scales. Hydrogen peroxide reduction is actually faster than oxygen reduction, but both reactions are slow due to relatively low thermodynamic driving force.

Effect of increasing anodic NaCl concentration on microbial fuel cell performance

High salinity effluents represent an estimated 5% of the wastewater generated worldwide. In microbial fuel cells, high salinity is usually considered beneficial to power production because increased conductivity facilitates proton transfer and therefore decreases the internal resistance of the system. However, high salt concentrations are known to adversely affect the physiology of anaerobic microbial consortia. In this study, the effect of increasing NaCl concentration in the anode chamber of a microbial fuel cell fed with sodium acetate was tested. Adding up to 20gL−1 of NaCl enhanced the overall performance of the system, reducing the internal resistance by 33% and increasing the maximum power production by 30%. Higher NaCl concentration proved detrimental to the system. However, the Coulombic efficiency started to be affected at a much lower NaCl concentration of 10gL−1, showing that the anodophilic bacteria are sensitive to NaCl at relatively low concentrations.

Effects of solvation shells and cluster size on the reaction of aluminum clusters with water

Reaction of aluminum clusters, Aln (n = 16, 17 and 18), with liquid water is investigated using quantum molecular dynamics simulations, which show rapid production of hydrogen molecules assisted by proton transfer along a chain of hydrogen bonds (H-bonds) between water molecules, i.e. Grotthuss mechanism. The simulation results provide answers to two unsolved questions: (1) What is the role of a solvation shell formed by non-reacting H-bonds surrounding the H-bond chain; and (2) whether the high size-selectivity observed in gas-phase Aln-water reaction persists in liquid phase? First, the solvation shell is found to play a crucial role in facilitating proton transfer and hence H2 production. Namely, it greatly modifies the energy barrier, generally to much lower values (< 0.1 eV). Second, we find that H2 production by Aln in liquid water does not depend strongly on the cluster size, in contrast to the existence of magic numbers in gas-phase reaction. This paper elucidates atomistic mechanisms underlying these observations.

Proton transport in a binary biomimetic solution revealed by molecular dynamics simulation

We report the simulation results of the proton transport in a binary mixture of amphiphilic tetramethylurea (TMU) molecules and water. We identify different mechanisms that either facilitate or retard the proton transport. The efficiency of these mechanisms depends on the TMU concentration. The overall picture is more complicated than a recent suggestion that the presence of amphiphilic molecules suppresses the proton mobility by slowing down the reorientation of the surrounding water molecules. It has also been suggested that the hydronium ion induces local water orientational order, which results in an ordered region that has to move along with the proton potentially slowing down the proton transport as suggested by experiment. We find that water-wire like structures formed at low amphiphile concentrations facilitate proton transfer, and reduction of the hydrogen bond connectivity induced at high concentrations retards it.

Proton Delivery to Ferryl Heme in a Heme Peroxidase: Enzymatic Use of the Grotthuss Mechanism

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.

Immobilization of imidazole in polymer electrolyte membranes for elevated temperature anhydrous applications

AbstractPolymer electrolyte composite membranes were cast from the mixture of Nafion® ionomer and 2‐substituted imidazole. The existing flexible hydrocarbon chain on 2‐position of imidazole facilitates proton transfer in the membrane at elevated temperature. The formed composite membrane showed an increased glass transition temperature and improved mechanical property compared with plain Nafion® membrane. At temperature above 100°C, the ionic conductivity of the composite membrane increases with the increase in temperature, reaching 5 × 10−3 S cm−1 at 160°C under anhydrous condition. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Proton transfer via a transient linear water-molecule chain in a membrane protein

High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109-112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

Facilitated Proton Transfer by a Novel 2-Aminothiazole Derivative Across the Water/1,2-Dichloroethane Interface

The behavior of proton transfer facilitated by a novel thiazole derivative, N-methyl-4-(4-phenoxyphenyl)thiazol-2-amine (MPPT), across the water/1,2-dichloroethane (1,2-DCE) interface was investigated electrochemically. The ionic partition diagram for MPPT was obtained from interpretation of the cyclic voltammograms. The apparent partition coefficient of MPPT was evaluated by the shaking-flask method under experimental conditions, while that for the protonated form of MPPT was calculated from its transfer potential obtained from the ionic partition diagram. It was suggested that the mechanism for transfer of MPPT across the water/1,2-DCE) interface depends on the pH of the aqueous phase. The parameters of the facilitated proton transfer across the water/1,2-DCE interface were evaluated as a quantitative measure of its lipophilicity.

Chemical response of aldehydes to compression between (0001) surfaces of α-alumina

First-principles molecular dynamics simulations are used to investigate the chemical response of acetaldehyde molecules (MeCHO) to compression and decompression between (0001) surfaces of α-alumina (Al(2)O(3)), with pressures reaching approximately 40 GPa. The results demonstrate that the MeCHO molecules are transformed into other chemical species through a range of chemical processes involving the formation of C-O and C-C bonds between MeCHO monomers as well as proton transfer. The mechanistic details of a representative set of the observed reactions are elucidated through analysis of maximally localized Wannier functions. Analysis of the changes in structure demonstrates that the main role of compression is to reduce the distances between MeCHO molecules to facilitate the formation of C-O bonds. Additional examination of the electronic structure demonstrates that the surface plays a role in facilitating proton transfer by both rendering hydrogen atoms in adsorbed MeCHO molecules more acidic and by acting as a proton acceptor. In addition, adsorption of the MeCHO molecules on the surface renders the sp(2) carbon atoms in these molecules more electrophilic, which promotes the formation of C-C and C-O bonds. It is suggested that the reaction products may be beneficial in the context of wear inhibition. Comparison of the surface structure before compression and after decompression demonstrates that the aldehydes and reaction products are capable of inhibiting irreversible changes in the structure as long as there is at least a monolayer coverage of these species. As a whole, the study sheds light on the chemical behavior of the aldehydes in response to uniaxial compression in nanoscopic contacts that likely applies to other molecules containing carbonyl groups and other metal oxide surfaces.

Tuning the activity of glutathione peroxidase mimics through intramolecular Se⋯N,O interactions: A DFT study incorporating solvent-assisted proton exchange (SAPE)

Diaryl diselenide mimics of the antioxidant selenoprotein glutathione peroxidase (GPx) often incorporate intramolecular Se···N,O interactions to enhance their GPx-like activity. Although the strength of the interaction is defined by the Lewis basicity of the donating group and the strength of the Se-X bond, there is not a clear relationship between the interaction and the GPx-like activity. Density-functional theory and natural bond orbital (NBO) calculations are used to show the range of Se···N,O interactions for various functional groups. The strongest interactions are found for groups which stabilize the donor-acceptor interaction through aromatic stabilization. The activation barriers for the GPx-like mechanism of activity of several substituted areneselenols are calculated using DFT and solvent-assisted proton exchange (SAPE), a technique that incorporates networks of solvent molecules into the theoretical model to facilitate proton transfer between sites in the reactant and product. DFT-SAPE models show that, in addition to decreasing the barrier to oxidation of the selenol, Se···N,O interactions generally increase the barriers for selenenic acid reduction and selenol regeneration because the Se···N,O interaction must be broken for the reaction to proceed. Calculated activation barriers for the rate-determining step are consistent with the relative experimental GPx-like activities of a series of diaryl diselenides.

Reactions of simple and peptidic alpha-carboxylate radical anions with dioxygen in the gas phase

α-Carboxylate radical anions are potential reactive intermediates in the free radical oxidation of biological molecules (e.g., fatty acids, peptides and proteins). We have synthesised well-defined α-carboxylate radical anions in the gas phase by UV laser photolysis of halogenated precursors in an ion-trap mass spectrometer. Reactions of isolated acetate (˙CH(2)CO(2)(-)) and 1-carboxylatobutyl (CH(3)CH(2)CH(2)˙CHCO(2)(-)) radical anions with dioxygen yield carbonate (CO(3)˙(-)) radical anions and this chemistry is shown to be a hallmark of oxidation in simple and alkyl-substituted cross-conjugated species. Previous solution phase studies have shown that C(α)-radicals in peptides, formed from free radical damage, combine with dioxygen to form peroxyl radicals that subsequently decompose into imine and keto acid products. Here, we demonstrate that a novel alternative pathway exists for two α-carboxylate C(α)-radical anions: the acetylglycinate radical anion (CH(3)C(O)NH˙CHCO(2)(-)) and the model peptide radical anion, YGGFG˙(-). Reaction of these radical anions with dioxygen results in concerted loss of carbon dioxide and hydroxyl radical. The reaction of the acetylglycinate radical anion with dioxygen reveals a two-stage process involving a slow, followed by a fast kinetic regime. Computational modelling suggests the reversible formation of the C(α) peroxyl radical facilitates proton transfer from the amide to the carboxylate group, a process reminiscent of, but distinctive from, classical proton-transfer catalysis. Interestingly, inclusion of this isomerization step in the RRKM/ME modelling of a G3SX level potential energy surface enables recapitulation of the experimentally observed two-stage kinetics.

Proton transfer reactions and dynamics of sulfonic acid group in Nafion®

Proton transfer reactions and dynamics of the hydrophilic group (-SO(3)H) in Nafion® were studied at low hydration levels using the complexes formed from CF(3)SO(3)H, H(3)O(+) and nH(2)O, 1 ≤n≤ 3, as model systems. The equilibrium structures obtained from DFT calculations suggested at least two structural diffusion pathways at the -SO(3)H group namely, the "pass-through" and "pass-by" mechanisms. The former involves the protonation and deprotonation at the -SO(3)H group, whereas the latter the proton transfer in the adjacent Zundel complex. Analyses of the asymmetric O-H stretching frequencies (ν(OH)) of the hydrogen bond (H-bond) protons showed the threshold frequencies (ν(OH*)) of proton transfer in the range of 1700 to 2200 cm(-1). Born-Oppenheimer Molecular Dynamics (BOMD) simulations at 350 K anticipated slightly lower threshold frequencies (ν(A)(OH*,MD)), with two characteristic asymmetric O-H stretching frequencies being the spectral signatures of proton transfer in the H-bond complexes. The lower frequency (ν(A)(OH,MD))) is associated with the oscillatory shuttling motion and the higher frequency (ν(B)(OH,MD))) the structural diffusion motion. Comparison of the present results with BOMD simulations on protonated water clusters indicated that the -SO(3)H group facilitates proton transfer by reducing the vibrational energy for the interconversion between the two dynamic states (Δν), resulting in a higher population of the H-bonds with the structural diffusion motion. One could therefore conclude that the -SO(3)H groups in Nafion® act as active binding sites which provide appropriate structural, energetic and dynamic conditions for effective structural diffusion processes in a proton exchange membrane fuel cell (PEMFC). The present results suggested for the first time a possibility to discuss the tendency of proton transfer in H-bond using Δν(BA)(OH,MD)) and provided theoretical bases and guidelines for the investigations of proton transfer reactions in theory and experiment.

Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/o-Xylene Monooxygenase Hydroxylase

Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201(peroxo), were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O(2) activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation, and possible structures of oxygenated intermediates are discussed.

Kinetic and structural studies of Haemophilus influenzae carbonic anhydrase with altered proton transfer capacity

The rate‐determining step for CO2 hydration by β‐carbonic anhydrase (β‐CA) is believed to be the loss of a hydrogen ion from the zinc‐bound water to regenerate the zinc‐bound hydroxide ion. Intramolecular acceptors, including a Tyr residue in the active site cleft of Haemophilus influenzae β‐CA (HICA) and a His residue near the exterior of the active site cleft of plant β‐CAs, are hypothesized to facilitate proton transfer from the zinc‐bound water. We report that in HICA the replacement of Tyr83 with Phe decreased kcat ≈30% of wild type, and displays substrate inhibition similar to the analogous Y212F variant of Arabidopsis thaliana β‐CA. Conversely, the replacement of Val87 with His increased the kcat to ≈250% of wild‐type, similar to the kcat of plant β‐CAs. X‐ray crystallographic structures of the V87H variant reveal a unique orientation of Tyr181 to hydrogen bond to the “unique” water molecule in HICA, and alternate “in” and “out” conformations of His87 that could facilitate intermolecular proton transfer to bulk solution; V87H crystals soaked in bicarbonate ion demonstrate that the enzyme still retains the ability to bind bicarbonate in the allosteric site. These results support the hypothesis of proton transfer as the rate‐determining step of β‐CA, and that Tyr and His residues can play important roles in this proton transfer step. This work is supported by NSF grants CHE‐0819686 and MCB‐0741396.

The use of nylon and glass fiber filter separators with different pore sizes in air-cathode single-chamber microbial fuel cells

Separators are needed in microbial fuel cells (MFCs) to reduce electrode spacing and preventing electrode short circuiting. The use of nylon and glass fiber filter separators in single-chamber, air-cathode MFCs was examined for their effect on performance. Larger pore nylon mesh were used that had regular mesh weaves with pores ranging from 10 to 160 μm, while smaller pore-size nylon filters (0.2–0.45 μm) and glass fiber filters (0.7–2.0 μm) had a more random structure. The pore size of both types of nylon filters had a direct and predictable effect on power production, with power increasing from 443 ± 27 to 650 ± 7 mW m−2 for pore sizes of 0.2 and 0.45 μm, and from 769 ± 65 to 941 ± 47 mW m−2 for 10 to 160 μm. In contrast, changes in pore sizes of the glass fiber filters resulted in a relatively narrow change in power (732 ± 48 to 779 ± 43 mW m−2) for pore sizes of 0.7 to 2 μm. An ideal separator should increase both power density and Coulombic efficiency (CE). However, CEs measured for the different separators were inversely correlated with power production, demonstrating that materials which reduced the oxygen diffusion into the reactor also hindered proton transport to the cathode, reducing power production through increased internal resistance. Our results highlight the need to develop separators that control oxygen transfer and facilitate proton transfer to the cathode.