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Probing quantum mechanical tunneling (QMT) in chemical reactions is crucial to understanding and developing new transformations. Primary H/D kinetic isotopic effects (KIEs) beyond the semiclassical maximum values of 7–10 (room temperature) are commonly used to assess substantial QMT contributions in one‐step hydrogen transfer reactions, because of the much greater QMT probability of protium vs. deuterium. Nevertheless, we report here the discovery of a reaction model occurring exclusively by H‐atom QMT with residual primary H/D KIEs. 2‐Hydroxyphenylnitrene, generated in N2 matrix, was found to isomerize to an imino‐ketone via sequential (domino) QMT involving anti to syn OH‐rotamerization (rate determining step) and [1,4]‐H shift reactions. These sequential QMT transformations were also observed in the OD‐deuterated sample, and unexpected primary H/D KIEs between 3 and 4 were measured at 3 to 20 K. Analogous residual primary H/D KIEs were found in the anti to syn OH‐rotamerization QMT of 2‐cyanophenol in a N2 matrix. Evidence strongly indicates that these intriguing isotope‐insensitive QMT reactivities arise due to the solvation effects of the N2 matrix medium, putatively through coupling with the moving H/D tunneling particle. Should a similar scenario be extrapolated to conventional solution conditions, then QMT may have been overlooked in many chemical reactions. [ABSTRACT FROM AUTHOR]

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.

Olive oil phenols are recognized as molecules with numerous positive health effects, many of which rely on their antioxidative activity, i.e., the ability to transfer hydrogen to radicals. Proton-coupled electron transfer reactions and hydrogen tunneling are ubiquitous in biological systems. Reactions of olive oil phenols, hydroxytyrosol, tyrosol, oleuropein, oleacein, oleocanthal, homovanillyl alcohol, vanillin, and a few phenolic acids with a DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical in a 1,4-dioxane:water = 95:5 or 99:1 v/v solvent mixture were studied through an experimental kinetic analysis and computational chemistry calculations. The highest rate constants corresponding to the highest antioxidative activity are obtained for the ortho-diphenols hydroxytyrosol, oleuropein, and oleacein. The experimentally determined kinetic isotope effects (KIEs) for hydroxytyrosol, homovanillyl alcohol, and caffeic acid reactions are 16.0, 15.4, and 16.7, respectively. Based on these KIEs, thermodynamic activation parameters, and an intrinsic bond orbital (IBO) analysis along the IRC path calculations, we propose a proton-coupled electron transfer mechanism. The average local ionization energy and electron donor Fukui function obtained for the phenolic compounds show that the most reactive electron-donating sites are associated with π electrons above and below the aromatic ring, in support of the IBO analysis and proposed PCET reaction mechanism. Large KIEs and isotopic values of Arrhenius pre-exponential factor AH/AD determined for the hydroxytyrosol, homovanillyl alcohol, and caffeic acid reactions of 0.6, 1.3, and 0.3, respectively, reveal the involvement of hydrogen tunneling in the process. [ABSTRACT FROM AUTHOR]

Hydrogen tunneling is a type of quantum tunneling in which a hydrogen atom passes through a potential barrier without reaching the transition state governed by classical mechanics. The participation of hydrogen tunneling in a chemical reaction (stoichiometric or catalytic) can result in the formation of products that without this phenomenon would be impossible to achieve or would be formed in a very slow way. This concept paper aims to review some of the most representative examples of transition‐metal mediated chemical reactions involving hydrogen tunneling. The experimental tools to determine the possibility of the participation of quantum tunneling in a chemical reaction are presented. In addition, the theoretical methods that have been developed to calculate the effect of quantum tunneling on chemical reactions are discussed. Finally, from a personal perspective, the steps to be taken in order to predict and implement this phenomenon are proposed. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized.

Soybean lipoxygenase (SLO) is a prototype for nonadiabatic hydrogen tunneling reactions and, as such, has served as the subject of numerous theoretical studies. In this work, we report a nearly temperature-independent kinetic isotope effect (KIE) with an average KIE value of 661 ± 27 for a double mutant (DM) of SLO at six temperatures. The data are well-reproduced within a vibronically nonadiabatic proton-coupled electron transfer model in which the active site has become rigidified compared to wild-type enzyme and single-site mutants. A combined temperature-pressure perturbation further shows that temperature-dependent global motions within DM-SLO are more resistant to perturbation by elevated pressure. These findings provide strong experimental support for the model of hydrogen tunneling in SLO, where optimization of both local protein and ligand motions and distal conformational rearrangements is a prerequisite for effective proton vibrational wave function overlap between the substrate and the active-site iron cofactor.

Soybean lipoxygenase-1 (SLO-1) catalyzes the C-H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched 13C isotopologues. In this report, we present synthetic strategies for site-specific 13C labeled linoleic acid substrates. We take advantage of a Corey-Fuchs formyl to terminal 13C-labeled alkyne conversion, using 13CBr4 as the labeling source, to reduce the number of steps from a previous fatty acid 13C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme-substrate complex in lipoxygenase.

We developed a calorimeter with a vacuum container made of superconducting niobium (Nb) to study monolayers of helium adsorbed on graphite which are prototypical two-dimensional quantum matters below 1 K. Nb was chosen because of its small specific heat in the superconducting state. It is crucially important to reduce the addendum heat capacity ( C ad ) when the specific surface area of substrate is small. Here we show details of design, construction and results of C ad measurements of the Nb calorimeter down to 40 mK. The measured C ad was sufficiently small so that we can use it for heat capacity measurements on helium monolayers in a wide temperature range below 1 K. We found a relatively large excess heat capacity in C ad , which was successfully attributed to atomic tunneling of hydrogen (H) and deuterium (D) between trap centers near oxygen or nitrogen impurities in Nb. The tunnel frequencies of H and D deduced by fitting the data to the tunneling model are consistent with the previous experiments on Nb doped with H or D. [ABSTRACT FROM AUTHOR]

This Chapter describes methods for the biosynthetic substitution of the mononuclear, non-heme iron in plant and animal lipoxygenases (LOXs). Substitution of this iron center for a manganese ion results in an inactive, yet faithful structural surrogate of the LOX enzymes. This metal ion substitution permits structural and dynamical studies of enzyme-substrate complexes in solution and immobilized on lipid membrane surfaces. Representative procedures for two LOXs, soybean lipoxygenase (SLO) from plants and human epithelial 15-lipoxygenase-2 (15-LOX-2) from mammals, are described as examples., (Copyright © 2024. Published by Elsevier Inc.)

Abstract High resolution spectra, obtained for small water clusters in the Vibrational-Rotational-Tunneling spectroscopy experiments in Saykally's group, provide invaluable data that should guide us in untangling the quite elusive properties of liquid water [PNAS 98 (2001) 10533]. Recently, we suggested that the bifurcation splittings in cyclic water trimers originate from the tunneling of hydrogen bonded protons in a mean field, double well potential between two oxygen neighbors [Chem.Phys.Lett. 661 (2016) 263]. Based on the similar/corresponding pattern of splittings in cyclic water pentamers, the model developed for the trimer should be also applicable to pentamers. Assuming that pentamers are major transient components in the fluctuating hydrogen bonded network of water, the model is applied to liquid water. In this paper, we show that our 1-D model gives surprisingly good agreement with experimental 2-D spectra in the O H stretch region of liquid water, reported by Tokmakoff's group [JCP 145 (2016) 094501]. Our results strengthen the hypothesis that cyclic pentamers should be considered as transiently present in the liquid, and that their fused structures form low density liquid. We also postulate that the higher energy isomers of the pentamer contribute to high density liquid water. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Hydrogen tunneling in enzyme reactions has played an important role in linking protein thermal motions to the chemical steps of catalysis. Lipoxygenases (LOXs) have served as model systems for such reactions, showcasing deep hydrogen tunneling mechanisms associated with enzymatic C–H bond cleavage from polyunsaturated fatty acids. Here, we examined the effect of solvent viscosity on the protein thermal motions associated with LOX catalysis using trehalose and glucose as viscogens. Kinetic analysis of the reaction of the paradigm plant orthologue, soybean lipoxygenase (SLO), with linoleic acid revealed no effect on the first-order rate constants, k cat , or activation energy, E a. Further studies of SLO active site mutants displaying varying E a s, which have been used to probe catalytically relevant motions, likewise provided no evidence for viscogen-dependent motions. Kinetic analyses were extended to a representative fungal LOX from M. oryzae, Mo LOX, and a human LOX, 15-LOX-2. While Mo LOX behaved similarly to SLO, we show that viscogens inhibit 15-LOX-2 activity. The latter implicates viscogen sensitive, conformational motions in animal LOX reactions. The data provide insight into the role of water hydration layers in facilitating hydrogen (quantum) tunneling in LOX. [Display omitted] • Steady-state kinetics reveal viscosity independent k cat and E a for the SLO reaction. • Protein motions promoting hydrogen tunneling in SLO are slaved to the hydration layer of the solvent. • The human 15-LOX-2 reaction is inhibited by glucose. • Animal LOXs are associated with a viscogen sensitive conformational change. • Our results provide insights into the activation barriers of plant and animal LOX chemistry. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized.

Kinetic Isotope effects (KIEs) have long served as a probe for the mechanisms of both enzymatic and solution reactions. Here, we discuss various models for the physical sources of KIEs, how experimentalists can use those models to interpret their data, and how the focus of traditional models has grown to a model that includes motion of the enzyme and quantum mechanical nuclear tunneling. We then present two case studies of enzymes, thymidylate synthase and alcohol dehydrogenase, and discuss how KIEs have shed light on the C-H bond cleavages those enzymes catalyze. We will show how the combination of both experimental and computational studies has changed our notion of how these enzymes exert their catalytic powers.

We propose an alternative explanation of the “bifurcation” splittings observed for the water trimer in the VRT experiments of Saykally’s group [Chem. Rev. 103 (2003) 2533]. In our interpretation, the splittings originate from the quantum delocalization of hydrogen bonded protons in the mean field potential between two oxygen neighbors. The pattern and the order of our calculated splittings is in the range of experimentally observed values. Consequently, quantum delocalization of protons should be considered seriously as the origin of experimentally observed fine splittings. The presented model can be extended to a water pentamer and, hopefully, advance our understanding of liquid water. [ABSTRACT FROM AUTHOR]

Hydrogen isotope measurements of lipid biomarkers preserved in sediments are most commonly interpreted as qualitative, rather than quantitative indicators of paleoprecipitation owing to an imperfect knowledge of all factors controlling the isotopic fractionation occurring during biosynthesis. Here, we first offer a brief review of appropriate procedures for preparing enriched isotope substrates for use in tracer studies and outline the approximate δD threshold at which this transition occurs. We then present new interpretations to explain deviations from common stable isotope effects observed in our previous culture experiments and other studies. We draw particular attention to the disagreement between intercept and slope for product–substrate relationships from those predicted for isotope systems, even when R 2 values are high, and attribute it to kinetic isotope fractionation. We demonstrate that reconstructing paleoenvironmental water δD values by simply adding a Δ to measured biomarkers δD values will result in a bias toward deuterium enriched values. This applies even to implicit reconstructions in the form of qualitative interpretations of measured lipid δD values as indicators of past hydroclimate. We therefore recommend reconstructing water δD values from lipid δD values using fractionation factor ( α ). We also discuss the apparently contradictory increase in D/H fractionation observed at elevated temperature and suggest that this may be the result of the unique wave-particle duality of hydrogen isotopes, which permits isotopologues to avoid surmounting the activation energy barrier that is necessary in traditional kinetic reactions. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with Fe III OH − or Mn III OH − . Hydrogen abstraction by Fe III OH − of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13 R -LOX with catalytic manganese (13 R -MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11- 2 H 2 ]18:2 n -6 were almost temperature independent with an apparent D-KIE of ∼56 at 30 °C, which is in agreement with previous studies. In contrast, the reaction rate of 13 R -MnLOX increased 7-fold with temperature (8–50 °C), and the apparent D-KIE decreased linearly from ∼38 at 8 °C to ∼20 at 50 °C. The kinetic lag phase of 13 R -MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13 R -MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between13 R -MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor–acceptor distances, and to the metal ligands, which may not equalize the 0.7 V-gap between the redox potentials of the free metals. [ABSTRACT FROM AUTHOR]

Recent experimental and theoretical studies have proposed that enzymes involve networks of coupled residues throughout the protein that participate in motions accompanying chemical barrier crossing. Here, we have examined portions of a proposed network in dihydrofolate reductase (DHFR) using quantum mechanics/molecular mechanics simulations. The simulations use a hybrid quantum mechanics-molecular mechanics approach with a recently developed semiempirical AM1-SRP Hamiltonian that provides accurate results for this reaction. The simulations reproduce experimentally determined catalytic rates for the wild type and distant mutants of E. coli DHFR, underscoring the accuracy of the simulation protocol. Additionally, the simulations provide detailed insight into how residues remote from the active site affect the catalyzed chemistry, through changes in the thermally averaged properties along the reaction coordinate. The mutations do not greatly affect the structure of the transition state near the bond activation, but we observe differences somewhat removed from the point of CH cleavage that affect the rate. The mutations have global effects on the thermally averaged structure that propagate throughout the enzyme and the current simulations highlight several interactions that appear to be particularly important. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] Kinetic isotope effects (KIEs) are interpreted by Marcus-like models of H-tunneling. [•] KIEs’ temperature dependence reveals the nature of “tunneling ready states”. [•] Enzymes’ kinetics and structures reveal the importance of dynamics in catalysis. [ABSTRACT FROM AUTHOR]

Phosphinylidenes are an important class of organophosphorus compounds that can exhibit tautomerization between tricoordinated P(III) hydroxide (R 1 R 2 POH) and a pentacoordinated P(V) oxide (R 1 R 2 P(O)H) form. Herein we show, using the canonical variational transition state theory combined with multidimensional small-curvature tunneling approximation, the dominance of proton tunneling in the two-water-bridged tautomerizations of phosphinous acid and model phosphinylidenes comprising phosphosphinates, H-phosphonates, H-phosphinates and secondary phosphine oxides. Based on the studied system, the contribution of thermally-activated tunneling is predicted to speed up the semiclassical reaction rate by ca. threefold to as large as two orders of magnitude at 298.15 K in the gas phase. The large KIE and the concavity in the Arrhenius plots are further fingerprints of tunneling. The simulations also predicted that the rapid tunneling rate and short half-life span for the forward reaction, as opposed to the reverse reaction in fluorinated secondary phosphine oxides, would result in P(V) being elusive and only P(III) being isolable, which agrees with previous experiments where only P(III) was detected by IR and NMR spectroscopy. We also explored the role of solvent and predicted tunneling to be substantial., (© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the con-tribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of pro-tein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostat-ics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynam-ics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial. [ABSTRACT FROM AUTHOR]

Kinetic Isotope effects (KIEs) have long served as a probe for the mechanisms of both enzymatic and solution reactions. Here, we discuss various models for the physical sources of KIEs, how experimentalists can use those models to interpret their data, and how the focus of traditional models has grown to a model that includes motion of the enzyme and quantum mechanical nuclear tunneling. We then present two case studies of enzymes, thymidylate synthase and alcohol dehydrogenase, and discuss how KIEs have shed light on the C-H bond cleavages those enzymes catalyze. We will show how the combination of both experimental and computational studies has changed our notion of how these enzymes exert their catalytic powers. [ABSTRACT FROM AUTHOR]

We use quantized molecular dynamics simulations to characterize the role of enzyme vibrations in facilitating dihydrofolate reductase hydride transfer. By sampling the full ensemble of reactive trajectories, we are able to quantify and distinguish between statistical and dynamical correlations in the enzyme motion. We demonstrate the existence of nonequilibrium dynamical coupling between protein residues and the hydride tunneling reaction, and we characterize the spatial and temporal extent of these dynamical effects. Unlike statistical correlations, which give rise to nanometer-scale coupling between distal protein residues and the intrinsic reaction, dynamical correlations vanish at distances beyond 4-6 Å from the transferring hydride. This work finds a minimal role for nonlocal vibrational dynamics in enzyme catalysis, and it supports a model in which nanometer-scale protein fluctuations statistically modulate—or gate—the barrier for the intrinsic reaction. [ABSTRACT FROM AUTHOR]

The light-dependent enzyme protochlorophyllide oxidoreductase (EC:1.3.1.33) catalyzes the conversion of protochlorophyllide (PChlide) into chlorophyllide during chlorophyll synthesis. The reaction has been proposed to proceed through light-induced weakening of the C17-C18 double bond in PChlide, which then facilitates hydride transfer from a NADPH cosubstrate molecule. We have performed DFT and TDDFT computations on the reaction mechanism of this interesting enzyme. The results show that whereas in the ground state the reaction is strongly endergonic and has a very high activation free energy (38 kcal/mol), the first four excited states (corresponding to excitations within the conjugated porphyrin π-system) afford much lower activation free energies (<25 kcal/mol) and spontaneous (or only slightly endergonic) reaction paths. The sharp shape of the potential energy surface along the reaction coordinate in these excited states allows hydrogen tunneling to occur efficiently on the first few excited state surface, lowering the barrier to values closer to experiment, in agreement with recent suggestions. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2010 [ABSTRACT FROM AUTHOR]

Abstract: The investigation of C-H bond activation by enzymes over the past several decades has revealed a plethora of deviations from semi-classical kinetic models. Although the early enzymatic results were interpreted in the context of a tunneling correction, the emergent properties are now seen to be largely incompatible with this type of analysis as well. This chapter introduces some of the experimental data that form the basis for our present understanding. A vibronically nonadiabatic model, that has a number of features in common with the Marcus treatment for electron transfer, offers a robust physical picture for the hydrogen tunneling behavior seen in both native enzymes and in enzymes that have been perturbed either by site-specific mutagenesis or by perturbation of the reaction conditions. Native enzymes under optimal conditions most commonly show behavior that requires a heavy atom donor-acceptor distance that is in the range of 2.7Å. This compression beyond van der Waals distances is proposed to arise from the process of enzymatic conformational sampling. The absence of any evolutionary driving force to optimize tunneling for deuterium transfer (natural abundance < 0.02%), together with the frequent observation that the enthalpic barrier for deuterium transfer is the same or very similar to that for protium transfer, leads to the proposal that tunneling is a consequence of a generic property of enzyme function in which overall protein flexibility enables the generation of active sites that can be quite compressed. [Copyright &y& Elsevier]

The tautomerization of HNCHC(OH)NH to HNCHCONH is an important step by way of Strecker synthesis for the production of glycine in the interstellar medium (ISM) with respect to the origin of amino acids on the early Earth. Our work indicates two mechanisms for the tautomerization to occur. k (rate constant) is 45.6 s at 50 K, obtained with the small curvature tunneling (SCT) approximation and canonical variational transition state theory (CVT), to support one mechanism assisted by quantum tunneling in the ISM. Another mechanism is through the (HO)-coupled proton transfer. In the (HO)- coupled reaction, two HO molecules act as a catalyst to reduce the barrier energy by 23.7 kcal mol, and cooperatively let a proton pass a hydrogen-bonded bridge composed of HNCHC(OH)NH with (HO). k is 8.08×10 s at 100 K, calculated with TST kinetics, to demonstrate the mechanism. Therefore, the tautomerization is feasible in the ISM. HNCHC(OH)NH and HNCHCONH will be detected in the ISM and found in carbonaceous chondrites. The results present new evidence for the third theory on the origin of amino acids on the early Earth. [ABSTRACT FROM AUTHOR]

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability probed with circular dichroism, established an accessible temperature range of 10°C to 55°C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, ΔH°unf(avg). Most regions in the enzyme show ΔH°unf(avg) ⩽ 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate. [ABSTRACT FROM AUTHOR]

For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (>1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (<1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. [ABSTRACT FROM AUTHOR]

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s-1 at 25°C) and deuterium (12 to 31 s-1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085–1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of ‘barrier engineering’ by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions. [ABSTRACT FROM AUTHOR]

One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H-C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor-acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry. [ABSTRACT FROM AUTHOR]

Abstract: Tryptophylquinone cofactors are formed by posttranslational modifications that result in the incorporation of two oxygens into a tryptophan side chain, and the covalent cross-linking of that side chain to another amino acid residue. Tryptophylquinone enzymes catalyze the oxidative deamination of primary amines, and utilize other redox proteins as electron acceptors. Mechanistic and structural studies of these enzymes are providing insight into how these enzymes utilize these highly reactive protein-derived quinones in a controlled manner to facilitate biologically important catalytic and electron transfer reactions. [Copyright &y& Elsevier]

Many biological C-H activation reactions exhibit nonclassical kinetic isotope effects (KIEs). These nonclassical KIEs are too large (k H /k D > 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH /AD ) fall outside of the semiclassical range near unity. The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model. Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy-atom coordinate, from which the hydrogen transfers via nuclear tunneling. Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates the tunneling barrier, leading to a temperature-dependent KIE. These properties directly link enzyme fluctuations to the reaction coordinate for hydrogen transfer, making a quantum view of hydrogen transfer necessarily a dynamic view of catalysis. The environmentally coupled hydrogen tunneling model leads to a range of magnitudes of KIEs, which reflect the tunneling barrier, and a range of AH /AD values, which reflect the extent to which gating modulates hydrogen transfer. Gating is the primary determinant of the temperature dependence of the KIE within this model, providing insight into the importance of this motion in modulating the reaction coordinate. The potential use of variable temperature KIEs as a direct probe of coupling between environmental dynamics and the reaction coordinate is described. The evolution from application of a tunneling correction to a full tunneling model in enzymatic H transfer reactions is discussed in the context of a thermophilic alcohol dehydrogenase and soybean lipoxygenase-1. [ABSTRACT FROM AUTHOR]

Soybean lipoxygenase-1 (SLO-1) catalyzes the C-H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched 13 C isotopologues. In this report, we present synthetic strategies for site-specific 13 C labeled linoleic acid substrates. We take advantage of a Corey-Fuchs formyl to terminal 13 C-labeled alkyne conversion, using 13 CBr 4 as the labeling source, to reduce the number of steps from a previous fatty acid 13 C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme-substrate complex in lipoxygenase.

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.