Quantum Mechanical Hydrogen Tunneling Research Articles

Hydrogen Bonding Shuts Down Tunneling in Hydroxycarbenes: A Gas-Phase Study by Tandem-Mass Spectrometry, Infrared Ion Spectroscopy, and Theory.

Hydroxycarbenes can be generated and structurally characterized in the gas phase by collision-induced decarboxylation of α-keto carboxylic acids, followed by infrared ion spectroscopy. Using this approach, we have shown earlier that quantum-mechanical hydrogen tunneling (QMHT) accounts for the isomerization of a charge-tagged phenylhydroxycarbene to the corresponding aldehyde in the gas phase and above room temperature. Herein, we report the results of our current study on aliphatic trialkylammonio-tagged systems. Quite unexpectedly, the flexible 3-(trimethylammonio)propylhydroxycarbene turned out to be stable─no H-shift to either aldehyde or enol occurred. As supported by density functional theory calculations, this novel QMHT inhibition is due to intramolecular H-bonding of a mildly acidic α-ammonio C-H bonds to the hydroxyl carbene's C-atom (C:···H-C). To further support this hypothesis, (4-quinuclidinyl)hydroxycarbenes were synthesized, whose rigid structure prevents this intramolecular H-bonding. The latter hydroxycarbenes underwent "regular" QMHT to the aldehyde at rates comparable to, e.g., methylhydroxycarbene studied by Schreiner et al. While QMHT has been shown for a number of biological H-shift processes, its inhibition by H-bonding disclosed here may serve for the stabilization of highly reactive intermediates such as carbenes, even as a mechanism for biasing intrinsic selectivity patterns.

The Hydride Transfer Process in NADP-dependent Methylene-tetrahydromethanopterin Dehydrogenase

NADP-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-H4MPT to form methenyl-H4MPT+ by using NADP+ as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO2 in methylotrophic and methanotrophic bacteria. Here, we report on the crystal structures of the ternary MtdA-substrate complexes from Methylorubrum extorquens AM1 obtained in open and closed forms. Their conversion is accomplished by opening/closing the active site cleft via a 15° rotation of the NADP, relative to the pterin domain. The 1.08 Å structure of the closed and active enzyme-NADP−methylene-H4MPT complex allows a detailed geometric analysis of the bulky substrates and a precise prediction of the hydride trajectory. Upon domain closure, the bulky substrate rings become compressed resulting in a tilt of the imidazolidine group of methylene-H4MPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP+ and methenyl-H4MPT+ revealed an extremely short distance between nicotinamide-C4 and imidazoline-C14a of 2.5 Å, which demonstrates the strong pressure imposed. The pterin-imidazolidine-phenyl butterfly angle of methylene-H4MPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H2- and F420-dependent methylene-H4MPT dehydrogenases. The concept of compression-driven hydride transfer including quantum mechanical hydrogen tunneling effects, which are established for flavin- and NADP-dependent enzymes, can be expanded to hydride-transferring H4MPT-dependent enzymes.

Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer.

Previous studies showed that the L57F and F93W alcohol dehydrogenases catalyze the oxidation of benzyl alcohol with some quantum mechanical hydrogen tunneling, whereas the V203A enzyme has diminished tunneling. Here, steady-state kinetics for the L57F and F93W enzymes were studied, and microscopic rate constants for the ordered bi-bi mechanism were estimated from simulations of transient kinetics for the S48T, F93A, S48T/F93A, F93W, and L57F enzymes. Catalytic efficiencies for benzyl alcohol oxidation (V1/EtKb) vary over a range of ∼100-fold for the less active enzymes up to the L57F enzyme and are mostly associated with the binding of alcohol rather than the rate constants for hydride transfer. In contrast, catalytic efficiencies for benzaldehyde reduction (V2/EtKp) are ∼500-fold higher for the L57F enzyme than for the less active enzymes and are mostly associated with the rate constants for hydride transfer. Atomic-resolution structures (1.1 Å) for the F93W and L57F enzymes complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol are almost identical to previous structures for the wild-type, S48T, and V203A enzymes. Least-squares refinement with SHELXL shows that the nicotinamide ring is slightly strained in all complexes and that the apparent donor-acceptor distances from C4N of NAD to C7 of pentafluorobenzyl alcohol range from 3.28 to 3.49 Å (±0.02 Å) and are not correlated with the rate constants for hydride transfer or hydrogen tunneling. How the substitutions affect the dynamics of reorganization during hydrogen transfer and the extent of tunneling remain to be determined.

Hydrogen tunneling avoided: enol-formation from a charge-tagged phenyl pyruvic acid derivative evidenced by tandem-MS, IR ion spectroscopy and theory.

A charge-tagged phenyl pyruvic acid derivative was investigated by tandem-MS, infrared (IR) ion spectroscopy and theory. The tailor-made precursor ions efficiently lose CO2 in collision induced dissociation (CID) experiments, offering access to study the secondary decay reactions of the product ions. IR ion spectroscopy provides evidence for the formation of an enol acid precursor ion structure in the gas phase and indicates the presence of enol products formed after CO2 loss. Extensive DFT computations however, suggest intermediate generation of hydroxycarbene products, which in turn rearrange in a secondary process to the enol ions detected by IR ion spectroscopy. Quantum mechanical tunneling of the hydroxycarbene can be excluded since no evidence for aldehyde product ion formation could be found. This finding is in contrast to the behavior of methylhydroxycarbene, which cleanly penetrates the energy barrier to form exclusively acetaldehyde at cryogenic temperatures in an argon matrix via quantum mechanical hydrogen tunneling. The results presented here are attributed to the highly excited energy levels of the product ions formed by CID in combination with different barrier heights of the competing reaction channels, which allow exclusive access over one energy barrier leading to the formation of the enol tautomer ions observed.

Negative ion cleavages of (M-H)- anions of peptides. Part 3. Post-translational modifications.

It is now 25 years since we commenced the study of the negative-ion fragmentations of peptides and we have recently concluded this research with investigations of the negative-ion chemistry of most post-translational functional groups. Our first negative-ion peptide review (Bowie, Brinkworth, & Dua, 2002) dealt with the characteristic backbone fragmentations and side-chain cleavages from (M-H)- ions of underivatized peptides, while the second (Bilusich & Bowie, 2009) included negative-ion backbone cleavages for Ser and Cys and some initial data on some post-translational groups including disulfides. This third and final review provides a brief summary of the major backbone and side chain cleavages outlined before (Bowie, Brinkworth, & Dua, 2002) and describes the quantum mechanical hydrogen tunneling associated with some proton transfers in enolate anion/enolate systems. The review then describes, in more depth, the negative-ion cleavages of the post-translational groups Kyn, isoAsp, pyroglu, disulfides, phosphates, and sulfates. Particular emphasis is devoted to disulfides (both intra- and intermolecular) and phosphates because of the extensive and spectacular anion chemistry shown by these groups. © 2016 Wiley Periodicals, Inc. Mass Spec Rev.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

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 contribution 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 protein 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 electrostatics, 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 dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Tunneling Governs Intramolecular Proton Transfer in Thiotropolone at Room Temperature

The role of quantum mechanical tunneling has been found to be significant in a variety of reactions like oxidative additions and reductive eliminations in organometallic complexes, proton transfer and proton-coupled electron transfer (PCET) processes in molecules and enzymes, 3] 1,5-hydride shift reactions in hydrocarbons and free-radical polymerization. Schreiner et al. have recently reported hydrogen tunneling in hydroxymethylene to form formaldehyde. This tunneling reaction has a half-life of only 2 h and tunneling occurs through a large energy barrier. But its congeners, mercaptocarbene and selenocarbene, do not show quantum mechanical hydrogen tunneling. Recent experiments have confirmed computational predictions of heavy atom tunneling in the ring opening of radical clocks. However, experimental detection of tunneling in chemical reactions are confined mostly to low temperatures and hence, examples of tunneling at room temperature are rare. 11] In a recent paper, Yamabe and co-workers reported an extremely fast intramolecular proton transfer between the thione (1a) and the enethiol (1b) tautomers in thiotropolone (1), the sulfur analogue of tropolone in the solid state (see Scheme 1). The crystal structure of 1 showed no signatures of intermolecular hydrogen bonding with the molecules being almost perpendicular to each other. The S···O distance in 1 is (2.804! 0.001) ! and therefore much smaller than the sum of their van der Waals radii (3.2 !). The computed harmonic (anharmonic) IR stretching modes for C=S and C=O in 1a and 1b are 1120.4 cm1 (1102.0 cm1) and 1627.0 cm1 (1594.0 cm1), respectively, at the B3LYP/6-311G(d) level, while the measured values for C=S and C=O are 1088– 1109 cm1 and 1566–1600 cm1. For a detailed comparison of the important vibrational modes at various levels of theory see the Supporting Information. Anharmonicity is observed to be significant. Based on this observation, it was concluded that 1 exists in the solid state as a monomer with substantial intramolecular OH···S and SH···O H-bonding for the tautomers 1a and 1 b, respectively. Variable-Temperature (VT) NMR studies showed that the proton hops rapidly between the two tautomers with a measured DG< 6 kcal mol1 for 1aQ1b at 238 K. The computed free-energy barrier height at G2(MP2) level was 5.0 kcalmol1. The energy difference between the minima of 1 a and 1b is 3.4 kcalmol at MP2/TZVP (3.8 kcalmol1 at CCSD(T)/cc-pVTZ level on the MP2/TZVP geometry). The tunneling correction based on the Wigner approximation for an Eckart potential was estimated to be 0.8 kcalmol1 thereby leading to an effective computed free-energy barrier height of 4.2 kcalmol1. However, the C and O NMR spectra in 1 showed no temperature dependence at various temperatures of 143, 173, 213, and 298 K. In fact, even in the molten state at 333 K, the C NMR signals for 1 are similar to the low-temperature measurements. This we find interesting, since based on a classical over-the-barrier mechanism as predicted by the transition-state theory (TST), the reaction should have a large temperature dependence across a span of 190 K (from 143 K to 333 K). But, for a reaction occurring purely through quantum mechanical tunneling such a behavior is expected since the probability of tunneling is independent of the temperature. The significance of quantum tunneling in keto–enol tautomerization is well established in the literature. Jhonson et al. have studied the significance of tunneling in the hydrogen atom transfer reaction in 6,9-dimethylbenzosuberone. To understand and quantify the contribution of quantum mechanical tunneling for this reaction, we performed DFT calculations using the MPW1K hybrid functional. The success of the MPW1K functional has been established in many H-transfer reactions. The 6-31 + G(d,p) basis set was employed. Calculations were also done at MP2/TZVP level for comparison. CCSD(T)/ccpVTZ calculations at MP2/TZVP reference geometry have Scheme 1. Proton transfer reaction in thiotropolone and tropolone and their derivatives.

Enzymatic tunneling and kinetic isotope effects: chemistry at the crossroads

AbstractKinetic isotope effects (KIEs), originally a tool for the physical organic chemist and mechanistic enzymologist, have been instrumental in furthering our understanding of quantum‐mechanical hydrogen tunneling in enzymatic systems. This review focuses on the use of KIE studies to investigate this phenomenon in enzyme‐catalyzed reactions. A number of enzymes wherein KIEs have been used as a probe for H‐tunneling are discussed, including dihydrofolate reductase (DHFR), alcohol dehydrogenase (ADH), and formate dehydrogenase (FDH). Particular emphasis has been placed on the significance of KIE results in exposing the chemical H‐transfer step in the complex kinetic cascades typical to these systems, as well as on questions regarding the influence of protein dynamics on tunneling and, consequently, on the whole enzymatic reaction. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Phenylhydroxycarbene

Phenylhydroxycarbene (Ph-C-OH, 1), the parent of all arylhydroxycarbenes, was generated by high-vacuum flash pyrolysis of phenylglyoxylic acid at 600 degrees C and spectroscopically characterized (IR, UV-vis) via immediate matrix isolation in solid Ar at 11 K. The identity of 1 was unequivocally confirmed by the precise agreement between the observed IR bands and (unscaled) anharmonic vibrational frequencies computed from a CCSD(T)/cc-pVDZ quartic force field. The UV-vis spectrum of 1 displays a broad band with maximum absorption at 500 +/- 25 nm (2.5 +/- 0.1 eV) that extends to approximately 640 nm (1.9 eV), in full accord with combined CCSD(T)/cc-pVQZ and EOM-CCSD/cc-pVTZ computations that yield a gas-phase vertical (adiabatic) excitation energy of 2.7 (1.9) eV. Unlike singlet phenylchlorocarbene, 1 does not undergo photochemical ring expansion. Instead, 1 exhibits quantum-mechanical hydrogen tunneling to benzaldehyde underneath a formidable barrier of 28.8 kcal mol(-1), even at cryogenic temperatures. The remarkable hydrogen tunneling mechanism is supported by the temperature insensitivity of the observed half-life (2.5 h) and substantiated by a comparable theoretical half-life (3.3 h) determined from high-level barrier penetration integrals computed along the intrinsic reaction path. As expected, deuteration turns off the tunneling mechanism, so d-1 is stable under otherwise identical conditions.

Experimental Evidence for Hydrogen Tunneling when the Isotopic Arrhenius Prefactor (AH/AD) is Unity

The temperature dependence of the kinetic isotope effect (KIE) is one of the major tools used for the investigation of hydrogen tunneling in condensed phase. Hydrogen transfer reactions displaying isotopic Arrhenius prefactor ratios (A(H)/A(D)) of unity are generally ascribed to a semiclassical mechanism. Here, we have identified a double mutant of soybean lipoxygenase (SLO-1, an enzyme previously shown to follow quantum mechanical hydrogen tunneling), that displays an A(H)/A(D) of unity and highly elevated (nonclassical) KIEs. This observation highlights the shortcoming of assigning a hydrogen transfer reaction to a semiclassical model based solely on an Arrhenius prefactor ratio.

Experimental Evidence for Enzyme-Enhanced Coupled Motion/Quantum Mechanical Hydrogen Tunneling by Ketosteroid Isomerase

Although there are considerable data demonstrating that quantum mechanical hydrogen tunneling (HT) occurs in both enzymatic and nonenzymatic systems, little data exist that address the question of whether enzymes enhance the amount of HT relative to the corresponding nonenzymatic reactions. To investigate whether 3-oxo-Delta (5)-steroid isomerase (ketosteroid isomerase, KSI) enhances HT relative to the nonenzymatic (acetate-catalyzed) isomerization of Delta (5)-androstene-3,17-dione ( 1) to Delta (4)-androstene-3,17-dione ( 3), alpha-secondary deuterium kinetic isotope effects (KIE) at C-6 of the steroid were determined for both the KSI- and acetate-catalyzed isomerizations. The normal intrinsic secondary KIE for both wild type (WT) KSI (1.073 +/- 0.023) and acetate (1.031 +/- 0.010) suggest the possibility of coupled motion (CM)/HT in both the enzymatic and nonenzymatic systems. To assess the contribution of CM/HT in these reactions, the secondary KIE were also measured under conditions in which deuterium instead of hydrogen is transferred. The decrease in secondary KIE for WT (1.035 +/- 0.011) indicates the presence of CM/HT in the enzymatic reaction, whereas the acetate reaction shows no change in secondary KIE for deuterium transfer (1.030 +/- 0.009) and therefore no evidence for CM/HT. On the basis of these experiments, we propose that KSI enhances the CM/HT contribution to the rate acceleration over the solution reaction. Active site mutants of KSI (Y14F and D99A) yield secondary KIEs similar to that of WT, indicating that mutations at the hydrogen-bonding residues do not significantly decrease the contribution of CM/HT to the KSI reaction.

The chemical mechanism of nitrogenase: hydrogen tunneling and further aspects of the intramolecular mechanism for hydrogenation of η2-N2 on FeMo-co to NH3

The preceding paper (Dalton Trans., 2008, DOI: 10.1039/b806100a) describes the logical development of a chemical mechanism for the catalysis of hydrogenation of N(2) to 2NH(3) that occurs at the Fe(7)MoS(9)N(c)(homocitrate) cofactor (FeMo-co) of the enzyme nitrogenase. The mechanism uses a single replenishable path for serial supply of protons which become H atoms on FeMo-co, migrating to become S-H and Fe-H donors to N(2) and to the intermediates that follow. This chemical catalysis at FeMo-co is distinctly intramolecular: transition states and reaction profiles for the preferred 21 step pathway were presented. This paper describes a number of alternative intermediates and pathways that were considered in developing the mechanism. These results reveal further relevant principles of the reactivity of hydrogenated FeMo-co, and the reasons why these pathways are less likely to be part of the mechanism. The intramolecular character of the mechanism, and the relatively small distances over which H atoms transfer, lead to expectations of extensive quantum mechanical hydrogen tunneling as part of the catalytic rate enhancement. This possibility is supported by comparisons of reaction profiles with those for enzyme reactions for which tunneling is established.

Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction

A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate α-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the α-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.