Proton Relay Mechanism Research Articles

Gliding on Ice in Search of Accurate and Cost-Effective Computational Methods for Astrochemistry on Grains: The Puzzling Case of the HCN Isomerization.

The isomerization of hydrogen cyanide to hydrogen isocyanide on icy grain surfaces is investigated by an accurate composite method (jun-Cheap) rooted in the coupled cluster ansatz and by density functional approaches. After benchmarking density functional predictions of both geometries and reaction energies against jun-Cheap results for the relatively small model system HCN···(H2O)2, the best performing DFT methods are selected. A large cluster containing 20 water molecules is then employed within a QM/QM′ approach to include a realistic environment mimicking the surface of icy grains. Our results indicate that four water molecules are directly involved in a proton relay mechanism, which strongly reduces the activation energy with respect to the direct hydrogen transfer occurring in the isolated molecule. Further extension of the size of the cluster up to 192 water molecules in the framework of a three-layer QM/QM′/MM model has a negligible effect on the energy barrier ruling the isomerization. Computation of reaction rates by the transition state theory indicates that on icy surfaces, the isomerization of HNC to HCN could occur quite easily even at low temperatures thanks to the reduced activation energy that can be effectively overcome by tunneling.

Role of Water in Proton-Coupled Electron Transfer between Tyrosine and Cysteine in Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and is critical for DNA synthesis and repair in all organisms. Its mechanism requires radical transfer along a ∼32 Å pathway through a series of proton-coupled electron transfer (PCET) steps. Previous simulations suggested that a glutamate residue (E623) mediates the PCET reaction between two stacked tyrosine residues (Y730 and Y731) through a proton relay mechanism. This work focuses on the adjacent PCET reaction between Y730 and a cysteine residue (C439). Quantum mechanical/molecular mechanical free energy simulations illustrate that when Y730 and Y731 are stacked, E623 stabilizes the radical on C439 through hydrogen bonding with the Y730 hydroxyl group. When Y731 is flipped away from Y730, a water molecule stabilizes the radical on C439 through hydrogen bonding with Y730 and lowers the free energy barrier for radical transfer from Y730 to C439 through electrostatic interactions with the transferring hydrogen but does not directly accept the proton. These simulations indicate that the conformational motions and electrostatic interactions of the tyrosines, cysteine, glutamate, and water strongly impact the thermodynamics and kinetics of these two coupled PCET reactions. Such insights are important for protein engineering efforts aimed at altering radical transfer in RNR.

Enzyme‐Inspired Design of CoII‐Based H2 Generation Catalysts: A Toolbox with Guiding Principles Revealed by a Systematic DFT Study

AbstractInspired by the proton relay pathway, as found in the hydrogenase enzymes, herein we report a series of highly promising pentadentate CoII‐[N2Nring3]‐based H2 generation molecular catalysts using experimentally easily accessible, redox‐innocent oxamide and pyridine‐/N‐methyl imidazole‐rich motifs. These newly designed catalysts can display proton relay using the donor nitrogen atom of the pendant arm in the catalytically active CoI state. The ligand framework contains three tether chains connecting the oxamide to pyridine/N‐methyl imidazole rings. The length of the tethers and position of attachment of the third tether are found to have a significant impact on the stability of the CoII catalyst. The electronic property of the pendant ligand can be modulated by judicious choice of the functional group such that the proton relay mechanism can be triggered at acidic, basic, and neutral pH of the medium. Moreover, it has been found that electron donating functional groups can avoid formation of an unproductive ligand‐reduced intermediate which can contribute to catalyst deactivation. Detailed DFT analysis revealed a set of much‐needed guiding principles that can be used by an experimental chemist as a toolbox for synthesis of novel H2 generation catalysts that can successfully demonstrate the biomimetic proton relay pathway while minimizing the decomposition of the metal catalysts.

Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis for all living organisms. It reduces ribonucleotides to the corresponding deoxyribonucleotides by a reversible radical transfer mechanism. The active form of E. coli Ia RNR is composed of two subunits, α and β, which form an active asymmetric α2β2 complex. The radical transfer pathway involves a series of proton-coupled electron transfer (PCET) reactions spanning α and β over ∼32 Å. Herein, quantum mechanical/molecular mechanical free energy simulations of PCET between tyrosine residues Y730 and Y731 are performed on the recently solved cryo-EM structure of the active α2β2 complex, which includes a pre-turnover α/β pair with an ordered PCET pathway and a post-turnover α'/β' pair. The free energy surfaces in both the pre- and post-turnover states are computed. According to the simulations, forward radical transfer from Y731 to Y730 is thermodynamically favored in the pre-turnover state, and backward radical transfer is favored in the post-turnover state, consistent with the reversible mechanism. E623, a glutamate residue that is near these tyrosines only in the pre-turnover state, is discovered to play a key role in facilitating forward radical transfer by thermodynamically stabilizing the radical on Y730 through hydrogen-bonding and electrostatic interactions and lowering the free energy barrier via a proton relay mechanism. Introduction of fluorinated Y731 exhibits expected thermodynamic trends without altering the basic mechanism. These simulations suggest that E623 influences the directionality of PCET between Y731 and Y730 and predict that mutation of E623 will impact catalysis.

A Proposed Proton Relay Mechanism in Methylenetetrahydrofolate Reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) by NAD(P)H. This reaction can be divided into two half‐reactions: the NAD(P)H reductive halfreaction and the folate oxidative half‐reaction. The X‐ray crystal structure of the E. coli enzyme has revealed a hydrogen‐bonded network of three active site amino acids, Glu28, Ser26 and His273. Previous studies with a Glu28Gln mutant indicated that Glu28, located near N10 of CH3‐H4folate, is an essential amino acid. Since Ser26 and His273 form hydrogen bonds to Glu28, we propose that Ser26, His273 and Glu28 are involved in a proton relay mechanism which donates a proton to CH2‐H4folate in the folate oxidative half‐reaction. In order to elucidate the role of Ser26 in this mechanism, we have characterized Ser26Thr and Ser26Ala mutants using the steady‐state CH3‐H4folate‐menadione oxidoreductase assay, in which the folate oxidative half‐reaction is rate limiting. We have hypothesized that the Ser26Thr mutant will have activity comparable to the wild‐type enzyme and the Ser26Ala mutant will have reduced activity due to its inability to hydrogen bond. Contrary to our hypothesis, we show that both mutants catalyze the folate reaction similar to the wild‐type enzyme. We also demonstrate that the Ser26Ala mutant shows competitive substrate inhibition in the CH3‐H4folate‐menadione assay, which is not observed for the wild‐type enzyme. Examination of the mutants in the NADH‐menadione assay shows kinetic parameters similar to those of the wild‐type enzyme. Taken together, our results suggest that Ser26 may not play an important role in either folate or NADH catalysis of MTHFR. However, we propose that in the case of the folate reaction, a water molecule inserted between the alanine and His273 might function like a serine in the proton relay mechanism, explaining the lack of a defect in the Ser26Ala mutant. To investigate the proton relay mechanism and the role of Ser26 further, we are studying the pH‐dependency of MTHFR in the CH3‐H4folate‐menadione assay.

Kinetic Characterization of Mutant Methylenetetrahydrofolate Reductase Enzymes His273Ala and His273Gln

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism that binds flavin adenine dinucleotide (FAD) and uses NAD(P)H to catalyze the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate by a ping‐pong Bi‐Bi mechanism. The x‐ray crystal structure of the 5‐methyltetrahydrofolate and FAD ‐bound E. coli enzyme has revealed a hydrogen bonding network that involves amino acid residues Ser26, His273, Glu28, a structural water molecule, and the N10 of the folate. Based on previous studies that confirmed the importance of Glu28 in the catalysis of the folate half‐reaction, we hypothesized a proton relay mechanism that transports a proton from the aqueous solvent to the N10 of the folate through this hydrogen bonding network and catalyzes the formation of the putative 5‐iminium cation folate intermediate. In our study, we employed steady‐state oxidoreductase assays using menadione as an artificial electron receptor and measured kinetic parameters of the wild‐type E. coli enzyme and two mutants, His273Gln and His273Ala MTHFRs. We found that in the methyltetrahydrofolate‐menadione assay, where the folate half‐reaction is rate‐limiting, the kcat of the His273Ala mutant (0.047 ± 0.002 s−1) is more than 10‐fold lower than the wild‐type enzyme (kcat = 0.54 ± 0.01 s−1). His273Gln mutant showed similar activity to the wild‐type enzyme, but strong competitive substrate inhibition was observed. In the NADH‐menadione assay, both His273Ala and His273Gln showed insignificant change in kcat (1.2 – 1.3‐fold) and in Km (1.7 – 2‐fold). Together, these results suggest that the ability of the His273 residue to form hydrogen bonds is important in the catalysis of the folate half‐reaction and support the hypothetical proton relay mechanism. In addition, we report our use of Liquid Chromatography‐Mass Spectrometry (LC‐MS) to quantify the product in the folate‐menadione assay more accurately.Support or Funding InformationGrinnell College Department of ChemistryHydrogen‐bonding network in the X‐ray structure of E28Q MTHFRFigure 1v/E vs. [substrate] plots obtained from methyltetrahydrofolate‐menadione oxidoreductase assays at 4C.Figure 2

Self‐catalyzed keto‐enol tautomerization of malonic acid

AbstractWe demonstrate through quantum chemical calculations that the keto‐enol tautomerization of malonic acid can be catalyzed by the two tautomers of malonic acid itself. This self‐catalyzed process proceeds with a relatively low barrier (Gibbs energy ca. 13 kcal/mol in gas phase, 20 kcal/mol in aqueous phase), and involves the concerted transfer of two protons between the substrate and the carboxylic acid functionality of the malonic acid catalyst. This mechanism is expected to compete with the proton relay mechanism currently favored to explain the tautomerization of malonic acid in aqueous media. Malonic acid is an important constituent of secondary organic aerosol where the present chemistry may play a role in determining chemical composition.

Modulating the mechanism of electrocatalytic CO2 reduction by cobalt phthalocyanine through polymer coordination and encapsulation

The selective and efficient electrochemical reduction of CO2 to single products is crucial for solar fuels development. Encapsulating molecular catalysts such as cobalt phthalocyanine within coordination polymers such as poly-4-vinylpyridine leads to dramatically increased activity and selectivity for CO2 reduction. In this study, we use a combination of kinetic isotope effect and proton inventory studies to explain the observed increase in activity and selectivity upon polymer encapsulation. We provide evidence that axial-coordination from the pyridyl moieties in poly-4-vinylpyridine to the cobalt phthalocyanine complex changes the rate-determining step in the CO2 reduction mechanism accounting for the increased activity in the catalyst-polymer composite. Moreover, we show that proton delivery to cobalt centers within the polymer is controlled by a proton relay mechanism that inhibits competitive hydrogen evolution. These mechanistic findings provide design strategies for selective CO2 reduction electrocatalysts and serve as a model for understanding the catalytic mechanism of related heterogeneous systems.

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Microbial formate-nitrite transporter-type proteins (FNT) exhibit dual transport functionality. At neutral pH, electrogenic anion currents are detectable, whereas upon acidification transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of the energy metabolism, such as l-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. Two opposing hypotheses on the site of substrate protonation are plausible. The proton relay mechanism postulates proton transfer from a highly conserved histidine centrally positioned in the transport path. The dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Here, we defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also show that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including l-lactate, a low substrate affinity as is typical for FNTs, and, strikingly, proton motive force-dependent transport as observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules. Furthermore, EhFNT is the sole annotated FNT in the Entamoeba genome suggesting that it could be a putative new drug target with similar utility as that of the malarial PfFNT.

Proton-Enhanced Dielectric Properties of Polyoxometalates in Water under Radio-Frequency Electromagnetic Waves.

Electromagnetic waves, such as microwaves, have been used to enhance various chemical reactions over polyoxometalates. The dielectric properties of catalysts are among the relevant parameters facilitating catalytic reactions under electromagnetic radiation. This study describes the dielectric properties of polyoxometalate catalysts in aqueous and organic solutions to understand the mechanism of interactions between polyoxometalates and electromagnetic waves. Specific loss factors of polyoxometalates were observed at lower frequencies (<1 GHz) by the ionic conduction of the polyoxometalate solution. The evolution of ionic conduction depended strongly on cations rather than anions. Proton-type polyoxometalates exhibited significantly higher loss factors than other cations did. The activation energy for ionic conduction in protonated silicotungstic acid (H4SiW12O40) was significantly low in water (7.6–14.1 kJ/mol); therefore, the high loss factor of protonated polyoxometalates in water was attributed to the proton relay mechanism (i.e., Grotthuss mechanism). The results suggested that the proton relay mechanism at the radio-frequency band is critical for generating selective interactions of polyoxometalates with applied electromagnetic fields.

Nitrogen-Mediated Graphene Oxide Enables Highly Efficient Proton Transfer

Two-dimensional (2D) graphene and graphene oxide (GO) offer great potential as a new type of cost-efficient proton-exchange membranes (PEM) for electrochemical devices. However, fundamental issues of proton transfer mechanism via 2D membranes are unclear and the transfer barrier for perfect graphene are too high for practical application. Using ab initio molecular dynamic simulations, we screened the proton transfer barrier for different un-doped and nitrogen doped GO membranes, and clarified the corresponding transfer mechanisms. More significantly, we further identify that N-mediated GO can be built into a highly efficient PEM with a proton transfer rate of seven orders of magnitude higher than an un-doped case via. a proton relay mechanism between a ketone-like oxygen and a pyridine-like nitrogen across the vacancy site. The N-doped 2D GO is also impermeable to small molecules, and hence a highly efficient PEM for practical applications.

The structure and function of Mycobacterium tuberculosis MazF-mt6 toxin provide insights into conserved features of MazF endonucleases

Toxin-antitoxin systems are ubiquitous in prokaryotic and archaeal genomes and regulate growth in response to stress. Escherichia coli contains at least 36 putative toxin-antitoxin gene pairs, and some pathogens such as Mycobacterium tuberculosis have over 90 toxin-antitoxin operons. E. coli MazF cleaves free mRNA after encountering stress, and nine M. tuberculosis MazF family members cleave mRNA, tRNA, or rRNA. Moreover, M. tuberculosis MazF-mt6 cleaves 23S rRNA Helix 70 to inhibit protein synthesis. The overall tertiary folds of these MazFs are predicted to be similar, and therefore, it is unclear how they recognize structurally distinct RNAs. Here we report the 2.7-Å X-ray crystal structure of MazF-mt6. MazF-mt6 adopts a PemK-like fold but lacks an elongated β1-β2 linker, a region that typically acts as a gate to direct RNA or antitoxin binding. In the absence of an elongated β1-β2 linker, MazF-mt6 is unable to transition between open and closed states, suggesting that the regulation of RNA or antitoxin selection may be distinct from other canonical MazFs. Additionally, a shortened β1-β2 linker allows for the formation of a deep, solvent-accessible, active-site pocket, which may allow recognition of specific, structured RNAs like Helix 70. Structure-based mutagenesis and bacterial growth assays demonstrate that MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for RNase activity and likely catalyze the proton-relay mechanism for RNA cleavage. These results provide further critical insights into how MazF secondary structural elements adapt to recognize diverse RNA substrates.

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states.

During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2 to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2 reduction was found in a double conformation: Glu451a (∼7 Å from the TNC) and Glu451b (∼4.5 Å from the TNC). A positive peak of electron density above 3.5σ in an Fo - Fc map for Glu451a O(ℇ2) indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

Strained ring motif at silica surfaces: A quantum mechanical study of their reactivity towards protic molecules

The ring opening reactions of (SiO)n ring motifs (n=2, 3 and 4) at the silica surfaces by the interaction with H2O (forming two SiOH silanol groups) and HCOOH (forming a SiOH group and a SiOC(O)H surface mixed anhydride group) has been studied by means of quantum chemical methods. Results are based on a cluster approach adopting the B3LYP-D2/6-311++G(d,p) level of calculations for computing the energetics of reaction. All reactions envisage an activated complex in which the nucleophilic part of the molecules (O atom for H2O and the carbonyl O atom for HCOOH) attacks the Si atoms of the (SiO)n moiety followed by a proton transfer from the molecules towards an O atom of the ring. Opening of the most strained two-membered (SiO)2 ring by the considered molecules is highly exoergic. For the medium strained three-membered (SiO)3 ring, two different Si atoms can react, one exposed to the exterior part of the surface and the other buried in the inner region of the surface. Reaction with the outer Si atom is exoergic, whereas that with the inner Si atom is endoergic, indicating the role of possible constraints enforced on the reaction products by the silica surroundings. Opening the regular four-membered (SiO)4 ring is an endoergic process for the both probe molecules. Calculations also show that the energy barriers for reactions with HCOOH are lower than with H2O because the transition state structures involving HCOOH exhibited a 6-membered ring, far less strained than the 4-membered ring found with H2O. A further reason is the more acidic character of the HCOOH compared to H2O, favoring the proton release towards the silica surface oxygen. Furthermore, it was shown that the energy barriers of the ring opening reactions with H2O is significantly lowered by the presence of an additional H2O molecule acting through the proton relay mechanism. Reaction energies, in contrast, are less favorable with HCOOH than with H2O, thus indicating that the formation of SiOH groups is favored with respect to the SiOC(O)H moiety. Finally, population analysis indicate that the electrophilic character of the C atom in the SiOC(O)H moiety is higher than for the isolated HCOOH, thereby making it more prone to undergo nucleophilic attack bringing an easier formation of amides.

Enhanced Hydrogen Generation from Formic Acid by Half-Sandwich Iridium(III) Complexes with Metal/NH Bifunctionality: A Pronounced Switch from Transfer Hydrogenation.

By switching the catalytic function from transfer hydrogenation based on the metal/NH bifunctionality, facile dehydrogenation of formic acid was achieved by amido- and hydrido(amine)-Ir complexes derived from N-triflyl-1,2-diphenylethylenediamine (TfDPEN) at ambient temperature even in the absence of base additives. Further acceleration was observed by the addition of water, leading to a maximum turnover frequency above 6000 h(-1). A proton-relay mechanism guided by the protic amine ligand and water is postulated for effective protonation of metal hydrides.

A Metal-Free Synthesis of N-Aryl Carbamates under Ambient Conditions.

The first chemo- and site-selective process for the formation of N-aryl-carbamates from cyclic organic carbonates and aromatic amines is reported. The reactions proceed smoothly under extremely mild reaction conditions using TBD (triazabicyclodecene) as an effective and cheap organocatalyst, thus providing a sustainable and new methodology for the formation of a wide variety of useful N-aryl carbamate synthons in good to excellent yields. Computational investigations have been performed and show the underlying reason for the observed unique reactivity as related to an effective proton-relay mechanism mediated by the bicyclic guanidine base.

Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

Amino acid residues that affect the basicity of the catalytic glutamate of the hydrolytic aldehyde dehydrogenases

In the catalytic mechanism of hydrolytic aldehyde dehydrogenases (ALDHs) the role of Glu268 (mature human ALDH2 numbering) as a general base is of major relevance. Since Glu268 basicity depends on its protein environment, here we explore its interactions with other amino acid residues in the three different conformations observed in ALDH crystal-structures: “inside”, “intermediate” and “outside”. In all of them Glu268 is in a hydrophobic environment. In the “inside” conformation, the theoretical pKa estimated by PROPKA3 is the result of the effects of hydrogen bonds with the protonated thiol of the catalytic Cys302 and/or the main-chain amide nitrogen of the highly conserved Gly270, and of charge-charge interactions with neighboring side-chains—Lys178, Glu/Asp476, His465 or Glu399 depending on the enzyme. In the “intermediate” conformation Glu268-carboxyl pKa is influenced by interactions with Glu/Asp476, Arg/Lys475, Lys/Arg178, His465 or Arg459, also depending on the enzyme. In the “outside” conformation, the effects on Glu268-carboxyl pKa arise from hydrogen bonds with the side chains of the strictly conserved Thr224 and/or of Lys/Arg178, and from charge-charge interactions with Lys/Arg/Asp178, Glu476, or Arg459. The estimated pKas and interactions of Glu268-carboxyl in the “intermediate” and “outside” conformations are consistent with their previously proposed roles in activating the hydrolytic water and in a proton relay mechanism, respectively. Water channels connecting Glu268 with the bulk water were found in all hydrolytic ALDHs. In the “inside” conformation the theoretical pKas of the Glu268-carboxyl and Cys302-thiol groups suggest that the carboxyl cannot receive the proton from the thiol. We propose that a protonated Cys302 might perform the nucleophilic attack on the aldehyde, which can be facilitated by Glu268 in the “intermediate” conformation. Finally, the conservation of the residues influencing Glu268 basicity between and within ALDH families suggests that these residues, not previously studied, are important for the catalytic mechanism of many ALDH enzymes.

Mechanism of dilute-spin-exchange in solid-state NMR

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in (15)N-(15)N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in (15)N-(15)N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II

The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.