Structural bases of hydrogen tunneling in enzymes: progress and puzzles

Accumulating experimental evidence suggests that the occurrence of hydrogen tunneling is likely to be widespread in enzyme-catalyzed reactions. The realization that hydrogen can transfer via tunneling mechanisms has far-reaching implications for our understanding of enzyme catalysis involving proton, hydride or hydrogen atom transfer reactions. The current status of the field is highlighted by three enzyme systems that have been under intensive study in recent years, including soybean lipoxygenase-1, thermophilic alcohol dehydrogenase and dihydrofolate reductase. Particular attention has been devoted to the issues of whether protein dynamics modulate hydrogen tunneling probability and whether the tunneling process contributes to the catalytic power of enzymes.     

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co-C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon-cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol(-1). We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain-Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.     

Proton tunneling and enzyme catalysis

Summary It is proposed in this paper that enzymes, by virtue of a number of correctly positioned sites of interaction with substrates, can force the compression of hydrogen bonds, increasing the probability of proton transfer by quantum mechanical tunneling. By such a catalytic mechanism a rate enhancement of many orders of magnitude may be obtained with a very low energy input requirement. The mechanism would, however, require a highly structured catalyst.Pertinent aspects of hydrogen bond theory and of tunneling theory are briefly reviewed.Work supported by NIGMS Training Grant No. GM 678-07.     

Question 9: Quantum Self-Assembly and Photoinduced Electron Tunneling in Photosynthetic Systems of Artificial Minimal Living Cells

Natural and artificial living cells and their substructures are self-assembling, due to electron correlation interactions among biological and water molecules, which lead to attractive dispersion forces and hydrogen bonds. Dispersion forces are weak intermolecular forces that arise from the attractive force between quantum multipoles. A hydrogen bond is a special type of quantum attractive interaction that exists between an electronegative atom and a hydrogen atom bonded to another electronegative atom; and this hydrogen atom exist in two quantum states. The best method to simulate these dispersion forces and hydrogen bonds is to perform quantum mechanical non-local density functional potential calculations of artificial minimal living cells consisting of around 1,000 atoms. The cell systems studied are based on peptide nucleic acid and are 3.0–4.2 nm in diameter. The electron tunneling and associated light absorption of the most intense transitions, as calculated by the time dependent density functional theory method, differs from spectroscopic experiments by only 0.2–0.3 nm, which is within the value of experiment errors. This agreement implies that the quantum mechanically self-assembled structures of artificial minimal living cells very closely approximate realistic ones.     

Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

Effect of energy status of hydrogen bond protons on the rate of electron transfer in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination which takes place in Rhodobacter sphaeroides reaction centers between a primary quinone (Q(A)) and a bacteriochlorophyll dimer. Taking the energy of interaction between hydrogen bond protons and an excessive electron into account, we described qualitative by this nonmonotonous dependence. We considered a molecular model of the primary quinone from Rb. sphaeroides reaction centers. In addition to the primary quinone, the model includes two reaction center fragments that form hydrogen bonds with Q(A). One of these fragments is His(M219), and the other is the peptide [Asn(M259) - Ala(M260)]. We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/absorbed during the electron tunneling is determined by a relative shift of the donor and the acceptor energy levels, the detuning of levels. The value of level detuning was shown to be temperature dependent in a nonmonotonous manner in the case of hydrogen bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depends on temperature parametrically. The dependence is nonmonotonous and is in qualitative agreement with the experimental one.     

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ～1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen–phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.     

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.     

Radical mechanisms in adenosylcobalamin-dependent enzymes

Adenolsylcobalamin-dependent enzymes catalyze free radical mediated reactions of their substrates. Stereochemical methods have been used to establish the nature of the primary radical initiation step in ribonucleoside triphosphate reductase. Kinetic isotope effects have been used to establish a kinetic coupling between cobalt-carbon bond cleavage and hydrogen atom abstraction from the substrate. Isotope effects have also been used to identify rate-limiting steps with wild type and mutant forms of the enzymes and in model reactions to assess tunneling contributions to hydrogen atom transfer steps. Computational methods have been employed to explore the pathways for functional group migration in the radical pathways. Analogs of substrates and of adenosylcobalamin have been used to explore the fidelity of the enzyme active sites and the radical pathways.     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

Hydrogen-mediated Stone-Wales isomerization of dicyclopenta[de,mn]anthracene

The mechanism of transformation of two radicals (R1p and R1i) obtained by addition of a hydrogen atom to an external and internal carbon atom of dicyclopenta[de,mn]anthracene (P1) was investigated. Two pathways were revealed. The first mechanism is a one-step process, whereas the second mechanism includes two transition states and a cyclobutyl intermediate. The formation of R1p and R1i and the homolytic cleavage of the radicals obtained during the isomerization processes were also examined. In both pathways the addition of a hydrogen atom to the internal carbon significantly lowers the activation energy for hydrogen-mediated isomerization of P1 to acefluoranthene. This finding could be explained by the specific electronic structures of the transition states and intermediates participating in the isomerization processes.     

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 alpha-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 alpha-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.     

Reduction of 1,4-quinone and ubiquinones by hydrogen atom transfer under UVA irradiation

1,4-Benzoquinone, coenzyme Q 0 and Q 10 were reacted with a series of hydrogen donors in the ESR cavity in the presence or absence of UVA irradiation. The signals of the radicals generated from the hydrogen donors or of those of the semiquinones were detected. The reaction mechanism was interpreted by a hydrogen atom transfer instead of the usual electron transfer mechanism on the basis of the redox potentials of the reactants and the Marcus theory. The hydrogen atom transfer is explained by the excited triplet state of quinones, which, on the basis of quantum mechanic calculations, may be reached even under visible light. In some cases, hydrogen atom transfer was also observed without irradiation, although to a lesser extent.     

Reduction of 1,4-quinone and Ubiquinones by Hydrogen Atom Transfer under UVA Irradiation

1,4-Benzoquinone, coenzyme Q 0 and Q 10 were reacted with a series of hydrogen donors in the ESR cavity in the presence or absence of UVA irradiation. The signals of the radicals generated from the hydrogen donors or of those of the semiquinones were detected. The reaction mechanism was interpreted by a hydrogen atom transfer instead of the usual electron transfer mechanism on the basis of the redox potentials of the reactants and the Marcus theory. The hydrogen atom transfer is explained by the excited triplet state of quinones, which, on the basis of quantum mechanic calculations, may be reached even under visible light. In some cases, hydrogen atom transfer was also observed without irradiation, although to a lesser extent.     

Quantum chemical studies for oxidation of morpholine by Cytochrome P450

Since morpholine oxidation has recently been shown to involve Cytochrome P450, the study on its mechanism at molecular level using quantum chemical calculations for the model of cytochrome active site is reported here. The reaction pathway is investigated for two electronic states, the doublet and the quartet, by means of density functional theory. The results show that morpholine hydroxylation occurs through hydrogen atom abstraction and rebound mechanism. However, in the low spin state, the reaction is concerted and hydrogen atom abstraction yields directly ferric-hydroxy morpholine complex without a distinct rebound step while in quartet state the reaction is stepwise. The presence of nitrogen in a morpholine heterocycle is postulated to greatly facilitate hydrogen abstraction. The hydroxylated product undergoes intramolecular hydrogen atom transfer from hydroxy group to nitrogen, leading to the cleavage of the C-N bond and the formation of 2-(2-aminoethoxy) acetaldehyde. The cleavage of the C-N bond is indicated as the rate-determining step for the studied reaction. The assistance of explicit water molecule is shown to lower the energy barrier for the C-N bond cleavage in enzymatic environment whereas solvent effects mimicked by COSMO solvent model have minor influence on relative energies along the pathway.     

Yeast nucleosomes allow thermal untwisting of DNA.

Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.     

Vibrationally enhanced tunneling as a mechanism for enzymatic hydrogen transfer.

We present a theory of enzymatic hydrogen transfer in which hydrogen tunneling is mediated by thermal fluctuations of the enzyme's active site. These fluctuations greatly increase the tunneling rate by shortening the distance the hydrogen must tunnel. The average tunneling distance is shown to decrease when heavier isotopes are substituted for the hydrogen or when the temperature is increased, leading to kinetic isotope effects (KIEs)--defined as the factor by which the reaction slows down when isotopically substituted substrates are used--that need be no larger than KIEs for nontunneling mechanisms. Within this theory we derive a simple KIE expression for vibrationally enhanced ground state tunneling that is able to fit the data for the bovine serum amine oxidase (BSAO) system, correctly predicting the large temperature dependence of the KIEs. Because the KIEs in this theory can resemble those for nontunneling dynamics, distinguishing the two possibilities requires careful measurements over a range of temperatures, as has been done for BSAO.     

Effect of trypsinization and histone H5 addition on DNA twist and topology in reconstituted minichromosomes.

Free DNA in solution exhibits an untwisting of the double helix with increasing temperature. We have shown previously that when DNA is reconstituted with histones to form nucleosome core particles, both the core DNA and the adjacent linker DNA are constrained from thermal untwisting. The origin of this constraint is unknown. Here we examine the effect of two modifications of nucleosome structure on the constraint against thermal untwisting, and also on DNA topology. In one experiment, we removed the highly positively charged histone amino and carboxy termini by trypsinization. Alternatively, we added histone H5, a histone H1 variant from chick erythrocytes. Neither of these modifications had any major effect on DNA topology or twist in the nucleosome.