Linus O. Johannissen

Hydrogen tunnelling in enzyme-catalysed H-transfer reactions : flavoprotein and quinoprotein systems

It is now widely accepted that enzyme-catalysed C–H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data—especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs—are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.

The kinetic effect of internal hydrogen bonds on proton-coupled electron transfer from phenols: a theoretical analysis with modeling of experimental data.

Proton-coupled electron transfer (PCET) was studied in two biomimetic covalently linked Ru(bpy)(3)-tyrosine complexes with the phenolic proton hydrogen-bonded to an internal carboxylate group. The phenolic group is either a salicylic acid (o-hydroxybenzoic acid, SA) or an o-hydroxyphenyl-acetic acid (PA), where the former gives a resonance-assisted hydrogen bond. Transient absorption data allowed direct determination of the rate constant for these intramolecular, bidirectional, and concerted PCET (CEP) reactions, as a function of temperature and H/D isotope. We found, unexpectedly, that the hydrogen bond in SA is in fact weaker than the hydrogen bond in the complex with PA, which forced us to reassess an earlier hypothesis that the proton coupling term for CEP with SA is increased by a stronger hydrogen bond. Consequently, the kinetic data was modeled numerically using a quantum mechanical rate expression. Sufficient experimentally determined observables were available to give robust and well-determined parameter values. This analysis, coupled with DFT/B3LYP and MP2 calculations and MD simulations, gave a detailed insight into the parameters that control the CEP reactions, and the effect of internal hydrogen bonds. We observed that a model with a static proton-tunneling distance is unable to describe the reaction correctly, requiring unrealistic values for the equilibrium proton-tunneling distances. Instead, when promoting vibrations that modulate the proton donor-acceptor distance were included, satisfactory fits to the experimental data were obtained, with parameter values that agree with DFT calculations and MD simulations. According to these results, it is in fact the weaker hydrogen bond of SA which increases the proton coupling. The inner reorganization energy of the phenolic groups is a significant factor contributing to the CEP barriers, but this is reduced by the hydrogen bonds to 0.35 and 0.50 eV for the two complexes. The promoting vibrations increase the rate of CEP by over 2 orders of magnitude, and dramatically reduce the kinetic isotope effect from ca. 40 for the static case to a modest value of 2-3.

Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes.

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression-an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression-through fast promoting vibrations, or other means-could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.

How does pressure affect barrier compression and isotope effects in an enzymatic hydrogen tunneling reaction

The shape of the reaction barrier is crucial for enzymatic hydrogen tunneling reactions. Thus, the concept of barrier compression is of fundamental importance. Such compression is believed to arise from “promoting vibrations”—rapid, subpicosecond donor–acceptor (DA) vibrational modes that transiently compress the reaction barrier, thus enhancing the probability of tunneling and of over-the-barrier transfers and hence the rate of H-transfer. For reactions that are dominated by H-tunneling, this has experimentally observable impact on the kinetic isotope effect (KIE) and its temperature-dependence, DDH : by shortening the tunneling distance, DA compression decreases the KIE, while the temperature-dependence of the vibration gives rise to an elevated DDH . However, the role of promoting vibrations in H-tunneling reactions is highly contentious. 7–11] We present a computational study on the effect of pressure on DA compression in an enzymatic H-tunneling reaction, demonstrating that experimental observations are directly coupled to the promoting vibration. The hydride transfer in the reductive half-reaction of morphinone reductase (MR) has been well characterized, with both experimental and computational studies suggesting that a promoting vibration is required to achieve tunneling. 17] Two recent experimental studies on MR have focused on the effect of mutation and hydrostatic pressure on the equilibrium donor–acceptor distance (DAD). These revealed a complex, even counter-intuitive, effect on the KIE which can be explained by concomitant changes to the promoting vibration. However, such changes cannot be measured directly. Hay et al. observed an increase in primary KIE with pressure, no change in DDH , and a modest increase in rate constant, and accounted for these effects by invoking an ad-hoc increase in the force constant for a “soft” (low-frequency) promoting vibration accompanied by a decreased average DAD. The kinetics could thus be modeled using a nonadiabatic, vibronic tunneling formalism, but this required a very large decrease in DAD of 0.7 from 1 bar to 2 kbar, which is inconsistent with molecular dynamics (MD) simulations. A subsequent phenomenological model allowed for a more realistic decrease in DAD of 0.07 , although this model overestimates the increase in rate constant by several orders of magnitude. Also, the hypothesized increase in force constant with pressure has recently been challenged by Warshel and co-workers. Here, we computationally identify the promoting vibration for this H-transfer and analyze how it changes with pressure, and use this information to reproduce the experimentally observed trends in kinetics. We ran constant-pressure MD simulations of MR with bound coenzyme nicotinamide adenine dinucleotide (NADH) and cofactor flavin mononucleotide (FMN) at 1 bar, 1 kbar, and 2 kbar. The decrease in DAD with increasing pressure is similar to that observed previously (Supporting Information, Figure S1). To analyze DA fluctuations at each pressure, spectral densities for DA compression over a 3 ns window were calculated (Figure 1). These

The photochemical mechanism of a B12-dependent photoreceptor protein.

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

Pressure Effects on Enzyme-Catalyzed Quantum Tunneling Events Arise from Protein-Specific Structural and Dynamic Changes

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.

The enzyme aromatic amine dehydrogenase induces a substrate conformation crucial for promoting vibration that significantly reduces the effective potential energy barrier to proton transfer

The role of promoting vibrations in enzymic reactions involving hydrogen tunnelling is contentious. While models incorporating such promoting vibrations have successfully reproduced and explained experimental observations, it has also been argued that such vibrations are not part of the catalytic effect. In this study, we have employed combined quantum mechanical/molecular mechanical methods with molecular dynamics and potential energy surface calculations to investigate how enzyme and substrate motion affects the energy barrier to proton transfer for the rate-limiting H-transfer step in aromatic amine dehydrogenase (AADH) with tryptamine as substrate. In particular, the conformation of the iminoquinone adduct induced by AADH was found to be essential for a promoting vibration identified previously—this lowers significantly the ‘effective’ potential energy barrier, that is the barrier which remains to be surmounted following collective, thermally equilibrated motion attaining a quantum degenerate state of reactants and products. When the substrate adopts a conformation similar to that in the free iminoquinone, this barrier was found to increase markedly. This is consistent with AADH facilitating the H-transfer event by holding the substrate in a conformation that induces a promoting vibration.

Atomistic insight into the origin of the temperature-dependence of kinetic isotope effects and H-tunnelling in enzyme systems is revealed through combined experimental studies and biomolecular simulation

The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts. Knowledge of enzyme catalysis is predominantly descriptive, gained from traditional protein crystallography and solution studies. Our goal is to understand catalysis by developing a complete and quantitative picture of catalytic processes, incorporating dynamic aspects and the role of quantum tunnelling. Embracing ideas that we have spearheaded from our work on quantum mechanical tunnelling effects linked to protein dynamics for H-transfer reactions, we review our recent progress in mapping macroscopic kinetic descriptors to an atomistic understanding of dynamics linked to biological H-tunnelling reactions.

Vertebrate Cryptochromes are Vestigial Flavoproteins

All cryptochromes are currently classified as flavoproteins. In animals their best-described role is as components of the circadian clock. This circadian function is variable, and can be either light-dependent or -independent; the molecular origin of this difference is unknown. Type I animal cryptochromes are photoreceptors that entrain an organism’s clock to its environment, whereas Type II (including mammals) regulate circadian timing in a light-independent manner. Here, we reveal that, in contrast to Type I, Type II animal cryptochromes lack the structural features to securely bind the photoactive flavin cofactor. We provide a molecular basis for the distinct circadian roles of different animal cryptochromes, which also has significant implications for the putative role of Type II cryptochromes in animal photomagnetoreception.