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The development of non-natural photoenzymatic systems has reinvigorated the study of photoinduced electron transfer (ET) within protein active sites, providing new and unique platforms for understanding how biological environments affect photochemical processes. In this work, we use ultrafast spectroscopy to compare the photoinduced electron transfer in known photoenzymes. 12-Oxophytodienoate reductase 1 (OPR1) is compared to Old Yellow Enzyme 1 (OYE1) and morphinone reductase (MR). The latter enzymes are structurally homologous to OPR1. We find that slight differences in the amino acid composition of the active sites of these proteins determine their distinct electron-transfer dynamics. Our work suggests that the inside of a protein active site is a complex/heterogeneous dielectric network where genetically programmed heterogeneity near the site of biological ET can significantly affect the presence and lifetime of various intermediate states. Our work motivates additional tunability of Old Yellow Enzyme active-site reorganization energy and electron-transfer energetics that could be leveraged for photoenzymatic redox approaches.

Morphinone reductase (MR) is an important model system for studying the contribution of protein motions to H-transfer reactions. In this research, we used quantum mechanical/molecular mechanics (QM/MM) simulation together with transition path sampling (TPS) simulation to study two important topics of current research on MR: the existence of multiple catalytic reaction pathways and the involvement of fast protein motions in the catalytic process. We have discovered two reaction pathways for the wild type and three reaction pathways for the N189A mutant. With the committor distribution analysis method, we found reaction coordinates for all five reaction pathways. Only one wild-type reaction pathway has a rate-promoting vibration from His186, while all of the other four pathways do not involve any protein motions in their catalytic process through the transition state. The rate-promoting vibration in the wild-type MR, which comes from a direction perpendicular to the donor-acceptor axis, functions to decrease the donor-acceptor distance by causing a subtle "out-of-plane" motion of a donor atom. By comparing reaction pathways between the two enzymes, we concluded that the major effect of the N189A point mutation is to increase the active site volume by altering the active site backbone and eliminating the Asn189 side chain. This effect causes a different NADH geometry at the reactant state, which very well explains the different reaction mechanisms between the two enzymes, as well as the disappearance of the His186 rate-promoting vibrations in the N189A mutant. The unfavorable geometry of the NADH pyridine ring induced by the N189A point mutation is the potential cause of multiple reaction pathways in N189A mutants.

The enzymatic reduction of C=C bonds in allylic alcohols with Old Yellow Enzymes represents a challenging task, due to insufficient activation through the hydroxy group. In our work, we coupled an alcohol dehydrogenase with three wild-type ene reductases-namely nicotinamide-dependent cyclohex-2-en-1-one reductase (NCR) from Zymomonas mobilis, OYE1 from Saccharomyces pastorianus and morphinone reductase (MR) from Pseudomonas putida M10-and four rationally designed β/α loop variants of NCR in the bienzymatic cascade hydrogenation of allylic alcohols. Remarkably, the wild type of NCR was not able to catalyse the cascade reaction whereas MR and OYE1 demonstrated high to excellent activities. Through the rational loop grafting of two intrinsic β/α surface loop regions near the entrance of the active site of NCR with the corresponding loops from OYE1 or MR we successfully transferred the cascade reduction activity from one family member to another. Further we observed that loop grafting revealed certain influences on the interaction with the nicotinamide cofactor.

The role of dynamical effects in enzyme catalysis is both complex and widely debated. Understanding how dynamics can influence the barrier to an enzyme catalyzed reaction requires the development of new methodologies and tools. In particular compressive dynamics-the focus of this study-may decrease both the height and width of a reaction barrier. By making targeted mutations in the active site of morphinone reductase we are able to alter the equilibrium of conformational states for the reactive complex in turn altering the donor-acceptor (D-A) distance for H-transfer. The sub-A changes which we induce are monitored using novel spectroscopic and kinetic "rulers". This new way of detecting variation in D-A distance allows us to analyze trends between D-A distance and the force constant of a compressive dynamical mode. We find that as the D-A distance decreases, the force constant for a compressive mode increases. Further, we demonstrate that-contrary to current dogma-compression may not always cause the magnitude of the primary kinetic isotope effect to decrease.

This paper is a response to an invitation to share my viewpoint by writing an opinion piece (not a review) on proton tunneling, especially from the point of view of whether it has a greater importance in enzymatic reactions than in other reactions. The paper begins with a discussion of the emergence of a conceptual framework for including tunneling in reaction rate calculations; the framework is general enough to include not only transfer of protons but also transfer of hydrogen atoms and hydride ions and their isotopes, and not only enzymatically catalyzed reactions but also nonenzymatic reactions. Then the paper discusses the special issues that arise when the reaction rate under consideration is for an enzyme-catalyzed reaction. The emphasis is on physical considerations in reaction rate calculations, not on system-specific comparison of results for different modes of reaction. It is argued that enzymatic and nonenzymatic reactions may be treated within the same basic framework except that ensemble averaging, which is not usually required for gas-phase reactions, is essential for treating enzyme reactions. Enzymes explicitly discussed include methylamine dehydrogenase, aromatic amine dehydrogenase, E. coli dihydrofolate reductase, hyperthermophilic dihydrofolate reductase, liver alcohol dehydrogenase, methylmalonyl-CoA mutase, soybean lipoxygenase, copper amine oxidase, pentaerythritol tetranitrate reductase, morphinone reductase, enolase, xylose isomerase, and 4-oxalocrotonate tautomerase. Copyright © 2010 John Wiley & Sons, Ltd.

Four flavoproteins from the old yellow enzyme (OYE) family, pentaerythritol tetranitrate (PETNR) reductase, N-ethylmaleimide reductase (NEMR), morphinone reductase (MorR) and estrogen-binding protein (EBP1), exhibited a broad substrate tolerance by accepting conjugated enals, enones, imides, dicarboxylic acids and esters, as well as a nitroalkene and therefore can be employed for the asymmetric bioreduction of carbon-carbon double (C=C) bonds. In particular, morphinone reductase and estrogen-binding protein often showed a complementary stereochemical preference in comparison to that of previously investigated OYEs.

We report the first study of the effects of hydrostatic pressure on α-2° KIEs for an enzyme-catalysed H-transfer reaction that occurs by ‘deep’ tunnelling. High pressure causes a significant decrease in the observed α-2° KIE on the pre-steady-state hydride transfer from NADH to FMN in the flavoprotein morphinone reductase. We have recently shown that high pressure causes a reduction in macroscopic reaction barrier width for this reaction. Using DFT vibrational analysis of a simple active site model, we posit that the decrease in α-2° KIE with pressure may arise due to a decrease in the vibrational coupling between the NADH primary (transferred) and secondary hydrogens in the ‘tunnelling ready configuration’, which more closely resembles the reactant state than the transition state. Copyright © 2010 John Wiley & Sons, Ltd.

Using alpha-secondary kinetic isotope effects (2 degrees KIEs) in conjunction with primary (1 degrees ) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2 degrees KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2 degrees KIEs, unlike 1 degrees KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2 degrees KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1 degrees KIE. The work emphasizes the benefit of combining studies of 1 degrees and 2 degrees KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.

The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased approximately 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.

Morphinone reductase (MR) catalyzes the NADH-dependent reduction of alpha/beta unsaturated carbonyl compounds in a reaction similar to that catalyzed by Old Yellow Enzyme (OYE1). The two enzymes are related at the sequence and structural levels, but key differences in active site architecture exist which have major implications for the reaction mechanism. We report detailed kinetic and solution NMR data for wild-type MR and two mutant forms in which residues His-186 and Asn-189 have been exchanged for alanine residues. We show that both residues are involved in the binding of the reducing nicotinamide coenzyme NADH and also the binding of the oxidizing substrates 2-cyclohexen-1-one and 1-nitrocyclohexene. Reduction of 2-cyclohexen-1-one by FMNH(2) is concerted with proton transfer from an unknown proton donor in the active site. NMR spectroscopy and flavin reoxidation studies with 2-cyclohexen-1-one are consistent with His-186 being unprotonated in oxidized, reduced, and ligand-bound MR, suggesting that His-186 is not the key proton donor required for the reduction of 2-cyclohexen-1-one. Hydride transfer is decoupled from proton transfer with 1-nitrocyclohexene as oxidizing substrate, and unlike with OYE1 the intermediate nitronate species produced after hydride transfer from FMNH(2) is not converted to 1-nitrocyclohexane. The work highlights key mechanistic differences in the reactions catalyzed by MR and OYE1 and emphasizes the need for caution in inferring mechanistic similarities in structurally related proteins.

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP + -dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.

Putting the squeeze on: Hydrostatic pressure causes a shortening of the charge-transfer bond in the binary complex of morphinone reductase and NADH(4) (see diagram). Molecular dynamics simulations suggest that pressure reduces the average reaction barrier width by restricting the conformational space available to the flavin mononucleotide and NADH within the active site. The apparent rate of catalysis increases with pressure.

Background: Old yellow enzyme (OYE) was the first flavoenzyme purified, but its function is still unknown. Nevertheless, the NADPH oxidase activity, the flavin mononucleotide environment and the ligand–binding properties of OYE have been extensively studied by biochemical and spectroscopic approaches. Full interpretation of these data requires structural information. Results The crystal structures of oxidized and reduced OYE at 2 A resolution reveal an α / β –barrel topology clearly related to trimethylamine dehydrogenase. Complexes of OYE with p –hydroxybenzaldehyde, β –estradiol, and an NADPH analog show all three binding at a common site, stacked on the flavin. The putative NADPH binding mode is novel as it involves primary recognition of the nicotinamide mononucleotide portion. Conclusion This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate. It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution.

The morphine alkaloid hydromorphone (dihydromorphinone) was identified as an intermediary metabolite in the degradation of morphine by Pseudomonas putida M10. A constitutive NADH-dependent morphinone reductase capable of catalyzing the reduction of the 7,8-unsaturated bond of morphinone and codeinone, yielding hydromorphone and hydrocodone, respectively, was shown to be present in cell extracts. The structures have been identified by 1H nuclear magnetic resonance and mass spectrometry. Morphinone reductase has been partially purified by anion-exchange and gel filtration chromatography. This enzyme has potential applications as a biocatalyst for the synthesis of the highly potent analgesic hydromorphone and the antitussive hydrocodone.

The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound beta-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK(a) of the reduced FMN N1 is 7.4+/-0.7, based on the pH-dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H-transfer as both the rate and temperature-dependence of the reaction are insensitive to changes in solution pH above and below this pK(a). Further, the solvent kinetic isotope effect is approximately 1.0 and both the 1 degrees and 2 degrees KIEs are insensitive to solution pH. The effect of the solvent's dielectric constant is investigated and the rate of H-transfer is found to be unaffected by changes in the dielectric constant between approximately 60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H-tunnelling reaction is insensitive to such changes.

The secondary kinetic isotope effect for hydride transfer from NADPH to dihydrofolate catalyzed by dihydrofolate reductase (see traces) is neither temperature dependent nor exalted. In environmentally coupled models of H-tunneling, the secondary isotope effects do not report on promoting motions, but reflect the active site geometry attained immediately prior to H transfer (i.e. the ‘tunnelling ready configuration′).

Using the gene mapping membrane technique, we identified a gene (nemA) that encodes N-ethylmaleimide reductace in Escherichia coli. The open reading frame encodes a polypeptide of 365 amino acids with a molecular mass of 39, 514 Da. The deduced amino acid sequence showed a high degree of homology (87% identical) with the pentaerythritol tetranitrate reductase of Enterobacter cloacae and the morphinone reductase of Pseudomonas putida (52% identical).

Morphinone reductase from Pseudomonas putida M10, a flavoprotein involved in the degradation of morphine alkaloids, was purified from an overexpressing strain of Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.5 Angstrom. The I-centred orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular replacement calculations have been performed using Old Yellow Enzyme as the search model.

Semisynthetic derivatives of morphine and related alkaloids are in widespread clinical use. Due to the complexity of these molecules, however, chemical transformations are difficult to achieve in high yields. We recently identified the powerful analgesic hydromorphone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Here we describe the construction of recombinant strains of Escherichia coli that express morphine dehydrogenase and morphinone reductase. These strains are capable of efficiently transforming the naturally occurring alkaloids morphine and codeine to hydromorphone and the antitussive hydrocodone, respectively. Our results demonstrate the potential for recombinant DNA technology to provide biological routes for the synthesis of known and novel semisynthetic opiate drugs.

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 degrees C; this is analogous to previous findings with mesophilic ADH at 25 degrees C. Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli , and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.

The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy. Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps. In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH. The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4. 8 x 10(5) M-1 s-1 at pH 7.0 and 5 degrees C. In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form. The rate of flavin reduction (23.4 s-1 at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm. The oxidative half-reaction proceeds in three kinetically resolvable steps. The first is due to the formation of a reduced enzyme-codeinone charge-transfer complex and is observed at long wavelength (about 650 nm). The rate of charge-transfer complex formation is dependent on codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s-1 at pH 7.0 and 5 degrees C. The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s-1) which is independent of codeinone concentration. The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s-1. This step most likely represents hydrocodone release from the oxidized enzyme. Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR. The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme. Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes.

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.

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.

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.