Subangstrom crystallography reveals that short ionic hydrogen bonds, and not a His-Asp low-barrier hydrogen bond, stabilize the transition state in serine protease catalysis

To address questions regarding the mechanism of serine protease catalysis, we have solved two X-ray crystal structures of alpha-lytic protease (alphaLP) that mimic aspects of the transition states: alphaLP at pH 5 (0.82 A resolution) and alphaLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal (0.90 A resolution). Based on these structures, there is no evidence of, or requirement for, histidine-flipping during the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195 undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing the back-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionic hydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We propose that the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize the transition states. In particular, a short ionic hydrogen bond (SIHB) between His57 Nepsilon2 and the substrate's leaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimental evidence that refutes use of either a short donor-acceptor distance or a downfield 1H chemical shift as sole indicators of a LBHB.     

A critical comparison of approximation methods and models for equilibrium properties of low-barrier hydrogen bonds

Recent experimental evidence has led to the conclusion that short, strong hydrogen bonds can stabilize transition states of enzyme catalyzed biochemical reactions. Evidence for such hydrogen bonds is the low value of the isotopic fractionation factor, phi, which is defined as the equilibrium constant for the generic reaction, R-H + DOH <--> R-D + HOH, where H is the hydrogen atom participating in the low-barrier hydrogen bond in a molecule R-H. In this work we assess two approximation methods for computing the isotopic fractionation factors for single and multidimensional systems containing a low-barrier hydrogen bond. These methods are WKB and an approach that corrects the classical partition function via a quantum correction factor. We find that the latter approach is universally accurate and applicable in both single and multidimensional systems containing a low-barrier hydrogen bond. We also assess two different models for the coupling of a molecule's low-barrier hydrogen bond to other degrees of freedom, both internal and external to the molecule, and show that each leads to a lowering of the fractionation factor.     

The charge density distribution in a model compound of the catalytic triad in serine proteases

Combined low temperature (28(1) K) X-ray and neutron diffraction measurements were carried out on the co-crystallised complex of betaine, imidazole, and picric acid (1). The experimental charge density was determined and compared with ab initio theoretical calculations at the B3LYP/6-311G(d,p) level of theory. The complex serves as a model for the active site in, for example, the serine protease class of enzymes, the so-called catalytic triad. The crystal contains three short strong N-H...O hydrogen bonds (HBs) with dN...O < 2.7 A. The three HBs have energies above 13 kcalmol(-1), although the hydrogen atoms are firmly localized in the "nitrogen wells". This suggests that low-barrier hydrogen bonding in catalytic enzyme reactions may be a sufficient, but not a necessary, condition for obtaining transition-state stabilization. Structural analysis (e.g., covalent N-H bond lengthening) indicates that the hydrogen bond between H3A and 08 of imidazole and betaine respectively (HB2) is slightly stronger than the bond between H1A and O1A of imidazole and picric acid (HB1), although HB1 is shorter than HB2: (dN...O(HB1)= 2.614(1) A, dN...O(HB2) = 2.684(1) A, dH...O(HB1) = 1.630(1) A, dH...O(HB2)= 1.635(1) A, dN-H(HB1) = 1.046(1) A, dN-H(HB2) = 1.057(1) A). Furthermore, the charge density analysis reveals that HB2 has a larger covalent character than HB1, with considerable polarization of the density towards the acceptor atom. The Gatti and Bader source function (S) is introduced to the analysis of strong HBs. The source function is found to be a sensitive measure for the nature of a hydrogen bond, and comparison with low-barrier and single-well hydrogen bonding systems (e.g., benzoylacetone and nitromalonamide) shows that the low-barrier hydrogen bond (LBHB) state is characterized by an enormously increased hydrogen atom source contribution to the bond critical point in the HB. In this context, HB2 can be characterized as intermediate between localized HBs and delocalized LBHBs.     

An N⋯H⋯N low-barrier hydrogen bond preorganizes the catalytic site of aspartate aminotransferase to facilitate the second half-reaction

Pyridoxal 5′-phosphate (PLP)-dependent enzymes have been extensively studied for their ability to fine-tune PLP cofactor electronics to promote a wide array of chemistries. Neutron crystallography offers a straightforward approach to studying the electronic states of PLP and the electrostatics of enzyme active sites, responsible for the reaction specificities, by enabling direct visualization of hydrogen atom positions. Here we report a room-temperature joint X-ray/neutron structure of aspartate aminotransferase (AAT) with pyridoxamine 5′-phosphate (PMP), the cofactor product of the first half reaction catalyzed by the enzyme. Between PMP N_SB and catalytic Lys258 Nζ amino groups an equally shared deuterium is observed in an apparent low-barrier hydrogen bond (LBHB). Density functional theory calculations were performed to provide further evidence of this LBHB interaction. The structural arrangement and the juxtaposition of PMP and Lys258, facilitated by the LBHB, suggests active site preorganization for the incoming ketoacid substrate that initiates the second half-reaction.

The neutron structure of pyridoxal 5′-phosphate-dependent enzyme aspartate aminotransferase with pyridoxamine 5′-phosphate (PMP) reveals a low-barrier hydrogen bond between the amino groups of PMP and catalytic Lys258, preorganizing the active site for catalysis     

Correlated proton motion in hydrogen bonded systems: tuning proton affinities

The theorem of matching proton affinities (PA) has been widely used in the analysis of hydrogen bonds. However, most experimental and theoretical investigations have to cope with the problem that the variation of the PA of one partner in the hydrogen bond severely affects the properties of the interface between both molecules. The B3LYP/d95+(d,p) analysis of two hydrogen bonds coupled by a 5-methyl-1H-imidazole molecule showed that it is possible to change the PA of one partner of the hydrogen bond while maintaining the properties of the interface. This technique allowed us to correlate various properties of the hydrogen bond directly with the difference in the PAs between both partners: it is possible to tune the potential energy surface of the bonding hydrogen atom from that of an ordinary hydrogen bond (localized hydrogen atom) to that of a low barrier hydrogen bond (LBHB, delocalized hydrogen atom) just by varying the proton affinity of one partner. This correlation shows clearly that matching PAs are of lesser importance for the formation of a LBHB than the relative energy difference between the two tautomers of the hydrogen bond.     

Low-Barrier Hydrogen Bonds in Negative Thermal Expansion Material H3[Co(CN)6]

The covalent nature of the low-barrier N−H−N hydrogen bonds in the negative thermal expansion material H₃[Co(CN)₆] has been established by using a combination of X-ray and neutron diffraction electron density analysis and theoretical calculations. This finding explains why negative thermal expansion can occur in a material not commonly considered to be built from rigid linkers. The pertinent hydrogen atom is located symmetrically between two nitrogen atoms in a double-well potential with hydrogen above the barrier for proton transfer, thus forming a low-barrier hydrogen bond. Hydrogen is covalently bonded to the two nitrogen atoms, which is the first experimentally confirmed covalent hydrogen bond in a network structure. Source function calculations established that the present N−H−N hydrogen bond follows the trends observed for negatively charge-assisted hydrogen bonds and low-barrier hydrogen bonds previously established for O−H−O hydrogen bonds. The bonding between the cobalt and cyanide ligands was found to be a typical donor–acceptor bond involving a high-field ligand and a transition metal in a low-spin configuration.     

A simple method for the characterization of OHO-hydrogen bonds by H-solid state NMR spectroscopy

A set of OHO hydrogen bonded systems with known neutron diffraction structure has been studied by fast ¹H-MAS echo spectroscopy. It is shown that the application of a simple rotor synchronized echo sequence combined with fast MAS allows a faithful determination of the chemical shift of the proton in the hydrogen bond. Employing the empirical valence bond order model, the experimental ¹H chemical shifts of the hydrogen bonded protons are correlated to the hydrogen bond geometries. The resulting correlation between the proton chemical shift and the deviation of the proton from the center of the hydrogen bond covers a broad range of substances. Deviations from the correlation curve, which are observed in certain systems with strong hydrogen bonds, are explained in terms of proton tautomerism or delocalization in low-barrier hydrogen bonds. These deviations are a highly diagnostic tool to select potential candidates for further experimental and theoretical studies. Thus, the combination of the ¹H-MAS echo sequence with the correlation curve yields a simple and versatile tool for the structural analysis of OHO hydrogen bonds.     

The Role of Hydrogen Bond in Catalytic Triad of Serine Proteases

In order to investigate the origin of catalytic power for serine proteases, the role of the hydrogen bond in the catalytic triad was studied in the proteolysis process of the peptides chymotrypsin inhibitor 2 (CI2), MCTI-A, and a hexapeptide (SUB), respectively. We first calculated the free energy profile of the proton transfer between His and Asp residues of the catalytic triad in the enzyme-substrate state and transition state by employing QM/MM molecular dynamics simulations. The results show that a low-barrier hydrogen bond (LBHB) only forms in the transition state of the acylation of CI2, while it is a normal hydrogen bond in the acylation of MCTI-A or SUB. In addition, the change of the hydrogen bond strength is much larger in CI2 and SUB systems than in MCTI-A system, which decreases the acylation energy barrier significantly for CI2 and SUB. Clearly, a LBHB formed in the transition state region helps accelerate the acylation reaction. But to our surprise, a normal hydrogen bond can also help to decrease the energy barrier. The key to reducing the reaction barrier is the increment of hydrogen bond strength in the transition state state, whether it is a LBHB or not. Our studies cast new light on the role of the hydrogen bond in the catalytic triad, and help to understand the catalytic triad of serine proteases.     

Hydrogen-bonding partner of the proton-conducting histidine in the influenza m2 proton channel revealed from (1)h chemical shifts

The influenza M2 protein conducts protons through a critical histidine (His) residue, His37. Whether His37 only interacts with water to relay protons into the virion or whether a low-barrier hydrogen bond (LBHB) also exists between the histidines to stabilize charges before proton conduction is actively debated. To address this question, we have measured the imidazole (1)H(N) chemical shifts of His37 at different temperatures and pH using 2D (15)N-(1)H correlation solid-state NMR. At low temperature, the H(N) chemical shifts are 8-15 ppm at all pH values, indicating that the His37 side chain forms conventional hydrogen bonds (H-bonds) instead of LBHBs. At ambient temperature, the dynamically averaged H(N) chemical shifts are 4.8 ppm, indicating that the H-bonding partner of the imidazole is water instead of another histidine in the tetrameric channel. These data show that His37 forms H-bonds only to water, with regular strength, thus supporting the His-water proton exchange model and ruling out the low-barrier H-bonded dimer model.     

Strong N−H⋅⋅⋅O Hydrogen Bonding in a Model Compound of the Catalytic Triad in Serine Proteases

Low-barrier hydrogen bond (LBHB) involvement in enzyme catalysis is examined by analysis of experimental nuclear and electron densities of a model compound for the catalytic triad in serine proteases (shown schematically), which is based on a cocrystal of betaine, imidazole, and picric acid. The three short, strong N−H⋅⋅⋅O hydrogen bonds in the structure have varying degrees of covalent bonding contributions suggesting a gradual transition to the LBHB situation.     

A path integral molecular dynamics study on intermolecular hydrogen bond of acetic acid–arsenic acid anion and acetic acid–phosphoric acid anion clusters

We apply ab initio path integral molecular dynamics simulation employing ωB97XD as the quantum chemical calculation method to acetic acid–arsenic acid anion and acetic acid–phosphoric acid anion clusters to investigate the difference of the hydrogen bond structure and its fluctuation such as proton transfer. We found that the nuclear quantum effect enhanced the fluctuation of the hydrogen bond structure and proton transfer, which shows treatment of the nuclear quantum effect was essential to investigate these systems. The hydrogen bond in acetic acid–arsenic acid anion cluster showed characters related to low-barrier hydrogen bonds, while acetic acid–phosphoric acid anion cluster did not. We found non-negligible distinction between these two systems, which could not be found in conventional calculations. We suggest that the difference in amount of atomic charge of the atoms consisting the hydrogen bond is the origin of the difference between acetic acid–arsenic acid and acetic acid–phosphoric acid anion cluster. © 2018 Wiley Periodicals, Inc.     

A Mixed Quantum-Classical Approach for Computing Effects of Intramolecular Motion on the Low-Barrier Hydrogen Bond

The fractionation factor is defined as the equilibrium constant for the reaction: R – H + DOH R – D + HOH. Of interest are values of fractionation factors for reactions where reactants and/or products form intramolecular low-barrier hydrogen bonds. Experimentally measured isotopic fractionation factors are usually interpreted via a one-dimensional potential energy surface along the intrinsic proton hydrogen bond coordinate. Such a one-dimensional picture cannot be completely correct. Intramolecular motions, such as vibrations and librations, can modulate the underlying potential energy surface along the hydrogen bond coordinate and thus affect the isotopic fractionation factor. We have recently generated a picture of the motion of the proton in a low-barrier hydrogen bond as taking place in an effective single-dimensional potential, which we term the potential of mean force (PMF). In this paper, we compute the PMF for a molecule with an intramolecular hydrogen bond in order to quantify the effect of intramolecular motions on the fractionation factor. The PMF and isotopic fractionation factor are computed with a combination of high-level density functional theory and molecular dynamics simulations.     

Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid

The UV spectra of aqueous acetic acid solutions up to 2M were investigated. At these wavelengths, the carboxylic acids exhibit an absorption peak, attributed to the C=O group, which shifts when hydrogen bonds are formed.. The measured spectra were best fitted to several bands, either of Gaussian or Lorentzian shape, which can be explained as several types of structural units formed by hydrogen bonds established between acetic acid and water molecules and between acetic acid molecules themselves. Molecular dynamics simulation of these mixtures was also performed, confirming the occurrence of several types of hydrogen bonds and showing the presence of dimers at higher concentrations. The viscosity and density of these solutions were also measured at different concentrations and temperatures. These results give a more complete picture of the hydrogen bond network of the system.     

Hydrogen bonding nature between calix[6]arene and piperidine/triethylamine

We investigated the binding nature of the 1,2,3-alternate calix[6]arene with one piperidine, two piperidines, and two triethyl amines with a special emphasis on the hydrogen bonding networks by density functional theory calculations. The 1,2,3-alternate calix[6]arene strongly binds with piperidines and triethylamines at two different binding sites, exo and endo sites. In the two binding sites, the hydrogen bonding nature shows a characteristic difference. In the exo site, there formed only one hydrogen bond, while in the endo site, two hydrogen bonds except for the triethylamine. The proton transfer within the hydrogen bonding and the hydrogen bonding types, normal hydrogen bonding (NHB), short strong hydrogen bond (SSHB), and low barrier hydrogen bonding (LBHB), will be discussed in detail.     

Low-barrier hydrogen bond between phosphate and the amide group in phosphopeptide

As the conversion between the monoionic (1) and diionic (2) form of the phosphate occurs, the phosphorylated peptides or proteins can not only cause the formation of a hydrogen bond between the phosphate group and the amide group but also change the strength of the hydrogen bond to form low-barrier hydrogen bonds (LBHBs). This reversible protonation of the phosphate group, which changes both the electrostatic properties of the phosphate group and the strength of the hydrogen bond, provides a possible mechanism in regulating protein function.     

Theoretical studies of the proton transfer behaviors in molecular complexes analogous to catalytic triad of serine protease: Toward understanding the existence and significance of the low-barrier hydrogen-bond in enzymatic catalysis

A representative acetate-(5-methylimidazole)-methanol system has been employed as a model of catalytic triad in serine protease to validate the formation processes of low-barrier H-bonds (LBHB) at the B3LYP/6-311++G** level of theory, and variable H-bonding characters from conventional ones to LBHBs have been represented along with the proceedings of proton transfer. Solvent effect is an important factor in modulation of the existence of an LBHB, where an LBHB (or a conventional H-bond) in the gas phase can be changed into a non-LBHB (an LBHB) upon solvation. The origin of the additional stabilization energy arising from the LBHB may be attributed to the H-bonding energy difference before and after proton transfer because the shared proton can freely move between the proton donor and proton acceptor. Most importantly, the order of magnitude of the stabilization energy depends on the studied systems. Furthermore, the nonexistence of LBHBs in the catalytic triad of serine proteases has been verified in a more sophisticated model treated using the ONIOM method. As a result, only the single proton transfer mechanism in the catalytic triad has been confirmed and the origin of the powerful catalytic efficiency of serine proteases should be attributed to other factors rather than the LBHB. Supported by the National Natural Science Foundation of China (Grant Nos. 20633060 & 20573063), the Natural Science Foundation of Shandong Province (Grant No. Y2007B23), the Scientific Research Foundation of Qufu Normal University (Grant Nos. Bsqd2007003 and Bsqd2007008), and the State Key Laboratory of Physical Chemistry of Solid Surfaces (Xiamen University)     

Interstitial water and the formation of low barrier hydrogen bonds: A computational model study

The B3LYP/D95+(d,p) analysis of the uncharged low barrier hydrogen bond (LBHB) between 4‐methyl‐1H‐imidazole (Mim) and acetic acid (HAc) shows that uncharged LBHBs can be formed either by adding three water molecules around the cluster or by placing the Mim–HAc pair in a dielectric environment created by a polarizable continuum model with a permittivity larger than 20.7. The permittivity of environment around uncharged LBHB can be lowered significantly by including water molecules into the system. A Mim–HAc LBHB stabilized with one water molecule observed in diethyl ether (ε = 4.34), with two water molecules in toluene (ε = 2.38), and with three water molecules in vacuo (ε = 1). Solvation models with different numbers of water molecules predict average differences in the proton affinities of the hydrogen bonded bases (ΔPA) for stable uncharged LBHB systems in vacuo to be 91.5 kcal/mol being different from the ΔPA values close to zero in charge‐assisted LBHB systems. The results clearly indicate that small amounts of interstitial water molecules at the active site of enzymes do not preclude the existence of LBHBs in biological catalysis. Our results also show that interstitial water molecules provide a useful clue in the search for uncharged LBHBs in an enzymatic environment and the number of water molecules can be used as a relative measure for the polarity around the direct environment of LBHBs. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2012     

Protonation Equilibrium in the Active Site of the Photoactive Yellow Protein

The role and existence of low-barrier hydrogen bonds (LBHBs) in enzymatic and protein activity has been largely debated. An interesting case is that of the photoactive yellow protein (PYP). In this protein, two short HBs adjacent to the chromophore, p-coumaric acid (pCA), have been identified by X-ray and neutron diffraction experiments. However, there is a lack of agreement on the chemical nature of these H-bond interactions. Additionally, no consensus has been reached on the presence of LBHBs in the active site of the protein, despite various experimental and theoretical studies having been carried out to investigate this issue. In this work, we perform a computational study that combines classical and density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) simulations to shed light onto this controversy. Furthermore, we aim to deepen our understanding of the chemical nature and dynamics of the protons involved in the two short hydrogen bonds that, in the dark state of PYP, connect pCA with the two binding pocket residues (E46 and Y42). Our results support the existence of a strong LBHB between pCA and E46, with the H fully delocalized and shared between both the carboxylic oxygen of E46 and the phenolic oxygen of pCA. Additionally, our findings suggest that the pCA interaction with Y42 can be suitably described as a typical short ionic H-bond of moderate strength that is fully localized on the phenolic oxygen of Y42.     

NMR studies of coupled low- and high-barrier hydrogen bonds in pyridoxal-5'-phosphate model systems in polar solution

The 1H and 15N NMR spectra of several 15N-labeled pyridoxal-5'-phosphate model systems have been measured at low temperature in various aprotic and protic solvents of different polarity, i.e., dichloromethane-d2, acetonitrile-d3, tetrahydrofuran-d8, freon mixture CDF3/CDClF2, and methanol. In particular, the 15N-labeled 5'-triisopropyl-silyl ether of N-(pyridoxylidene)-tolylamine (1a), N-(pyridoxylidene)-methylamine (2a), and the Schiff base with 15N-2-methylaspartic acid (3a) and their complexes with proton donors such as triphenylmethanol, phenol, and carboxylic acids of increasing strength were studied. With the use of hydrogen bond correlation techniques, the 1H/15N chemical shift and scalar coupling data could be associated with the geometries of the intermolecular O1H1N1 (pyridine nitrogen) and the intramolecular O2H2N2 (Schiff base) hydrogen bonds. Whereas O1H1N1 is characterized by a series of asymmetric low-barrier hydrogen bonds, the proton in O2H2N2 faces a barrier for proton transfer of medium height. When the substituent on the Schiff base nitrogen is an aromatic ring, the shift of the proton in O1H1N1 from oxygen to nitrogen has little effect on the position of the proton in the O2H2N2 hydrogen bond. By contrast, when the substituent on the Schiff base nitrogen is a methyl group, a proton shift from O to N in O1H1N1 drives the tautomeric equilibrium in O2H2N2 from the neutral O2-H2...N2 to the zwitterionic O2-...H2-N(2+) form. This coupling is lost in aqueous solution where the intramolecular O2H2N2 hydrogen bond is broken by solute-solvent interactions. However, in methanol, which mimics hydrogen bonds to the Schiff base in the enzyme active site, the coupling is preserved. Therefore, the reactivity of Schiff base intermediates in pyridoxal-5'-phosphate enzymes can likely be tuned to the requirements of the reaction being catalyzed by differential protonation of the pyridine nitrogen.