MINIREVIEW Structural and mechanistic aspects of flavoproteins: probes of hydrogen tunnelling Sam Hay, Christopher R Pudney and Nigel S Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, UK Keywords high pressure; H-tunneling; kinetic isotope effect; kinetic isotope fractionation; multiple reactive conformations; Old Yellow Enzyme; promoting vibration; protein dynamics; quantum mechanics; stopped-flow kinetics Correspondence N S Scrutton, Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK Fax: +44 161 306 8918 Tel: +44 161 306 5152 E-mail: nigel.scrutton@manchester.ac.uk At least half of all enzyme-catalysed reactions are thought to involve a hydrogen transfer In the last 10 years, it has become apparent that many of these reactions will occur, in part, or in full, by quantum mechanical tunnelling We are particularly interested in the role of promoting vibrations on H transfer, and the Old Yellow Enzyme family of flavoproteins has proven to be an excellent model system with which to examine such reactions In this minireview, we describe new and established experimental methods used to study H-tunnelling in these enzymes and we consider some practical issues important to such studies The application of these methods has provided strong evidence linking protein dynamics and H-tunnelling in biological systems (Received 23 December 2008, revised 28 April 2009, accepted May 2009) doi:10.1111/j.1742-4658.2009.07121.x Introduction There is now fairly widespread recognition that enzyme-catalysed C–H bond cleavage reactions can occur by quantum mechanical tunnelling [1–5] The role of protein dynamics in these reactions is still hotly debated and it has been proposed that promoting vibrations, nonequilibrated fast (sub-ps) dynamics, could modify the reaction barrier and profoundly influence the reaction rate [4,6–12] In recent years, we have investigated H-transfer reactions in a number of enzymes, primarily quinoprotein [4,13,14] and flavoprotein [8,15–20] systems Using a combination of experimental and computational approaches, we have shown that H-transfer reactions can occur by ‘deep’ tunnelling and the reaction can be enhanced by local- ized dynamics in the enzyme active site – putative promoting vibrations Although it is fairly well established that enzymatic H transfers often involve tunnelling, the role of promoting vibrations remains contentious [21] In this minireview, we describe experimental methods we have recently employed and developed to probe the role of environmental coupling ⁄ promoting vibrations in H-transfer reactions in the Old Yellow Enzyme (OYE) family of flavoproteins Hydrogen tunnelling Because of wave ⁄ particle duality, electrons and light atoms have appreciable (de Broglie) wavelengths The Abbreviations DHFR, dihydrofolate reductase; EIE, equilibrium isotope effect; ET, electron transfer; GO, glucose oxidase; KIE, kinetic isotope effect; MR, morphinone reductase; OYE, Old Yellow Enzyme; PETNR, pentaerythritol tetranitrate reductase; RHR, reductive half-reaction 3930 FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS S Hay et al Hydrogen tunnelling in biological systems wavelength of H (used here to denote H+, H• and H)) ˚ is $1 A and thus similar to a typical bond length, whereas thepffiffiffi wavelength of deuterium is shorter by a factor of $ As a consequence, the position of H (and to a lesser extent, D) is somewhat diffuse, and H transfer may involve an appreciable degree of quantum mechanical tunneling, in which H or D transfer occurs by ‘tunnelling’ through part of the reaction barrier rather than by passing over the barrier as is the case in a classical transition-state reaction [22] It is accepted that long-range electron transfer (ET) reactions occur by tunnelling [23,24] and we now have nearly 20 years of both experimental and computational evidence demonstrating that H-tunnelling reactions can also occur during enzyme-catalysed reactions [1–5] It is possible to computationally estimate the extent to which tunnelling plays a role during an H-transfer reaction In dihydrofolate reductase, hydride transfer occurs by tunnelling $50% of the time [25,26], with the remainder of the H transfer occurring by an over-the-barrier mechanism Conversely, in soybean lipoxygenase-1 [27], aromatic amine dehydrogenase [4] and the flavoprotein morphinone reductase (MR) [28], calculations have shown that at least 99% of H transfer occurs by tunnelling These reactions can be thought of as ‘deep’ tunnelling reactions because the H tunnels a relatively long way below the top of the reaction barrier The rate of a nonadiabatic (deep tunnelling) H transfer can be described using modified Marcus theory [23]: ! rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2p DGz ð1Þ ktun ¼ jV j ðF:C:Þ exp À  4pkkB T h kB T where V is the electronic coupling, F.C is the Frank– Condon nuclear wave function overlap (related to the de Broglie wavelength of the H or D) and DGà is the Marcus activation energy The activation energy is described by the driving force, DG0, and reorganization energy in the standard way: DGz ẳ DG0 ỵ k =4k ð2Þ The driving force dependence of H transfer in the flavoprotein glucose oxidase (GO) was investigated by Brinkley & Roth [29] The endogenous FAD was substituted with other chemically modified flavins with differing redox potentials, and these authors showed that the apparent rate of H transfer obeys Eqns (1) and (2) and the reorganization energy of this reaction appears to be large ($280 kJỈmol)1) We have since shown a driving force dependence during the reduction of the quinoprotein aromatic amine dehydrogenase with p-substituted phenylethylamine substrates [30] and estimated the reorganization energy for the reaction of this enzyme with tryptamine to be 250–300 kJỈ mol)1 [9] From the little experimental evidence currently available, it appears that it is appropriate to describe H-tunnelling reactions using Marcus theory, and that a general feature of these reactions may be a large reorganization energy The Frank–Condon term in Eqn (1) has been described by Kuznetsov & Ulstrup: Z r0  À Áà exp Àli xi Dr2 =2 expEX =kB T ịdX h F:C:0;0 ẳ ð3Þ where l and x are the mass and frequency of the transferred H or D and Ex is the environmental energy or promoting vibration, which reduces the H-transfer distance from an equilibrium distance, r0, by Dr = (r0 ) rX) [9,31,32] The kinetic isotope effect (KIE = kH ⁄ kD) arises because of differences in the mass, frequency and consequently the transfer distance of H and D Experimentally, the identification of promoting vibrations is extremely challenging and, as yet, there is no method to directly visualize such vibrations because they occur in the relatively inaccessible THz region The first experimental evidence for a role of environmental coupling during H-tunnelling reactions in enzymes [5,13] was inferred from observations of KIEs with aberrant temperature dependencies that not conform to the predictions of semiclassical transition state theory or Bell-type quantum correction models [33] However, we have recently shown that the KIE temperature dependence (DDHà, see below) is not a reliable diagnostic of environmental coupling [9] and other experiments are required to corroborate predictions based solely on DDHà values Measurement of H-transfer reactions in OYEs The OYE family of flavoproteins comprise a large group of FMN-containing NAD(P)H-dependent oxidases We have concentrated our studies on two homologous OYEs: MR isolated from Pseudomonas putida M10 and pentaerythritol tetranitrate reductase (PETNR) from Enterobacter cloacae For reference, in Table 1, we summarize the flavoproteins for which a specific H-tunnelling study has been performed Generally, the KIEs of H transfer in flavoproteins are fairly modest (< 10) but the temperature dependencies of these KIEs are quite varied (Table 1) FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS 3931 Hydrogen tunnelling in biological systems S Hay et al Table Kinetic isotope effects observed in selected flavoproteins CO, choline oxidase; DD, human class dihydroorotate dehydrogenase; FDTS, flavin dependent thymidylate synthase; GO, glucose oxidase; MAO A ⁄ B, monoamine oxidase A ⁄ B; MR, morphinone reductase; PAO, L-phenylalanine oxidase; PETNR, pentaerythritol tetranitrate reductase; TMADH, trimethylamine dehydrogenase; TSOX, heterotetrameric sarcosine oxidase; nd, not determined or reported The KIEs are for pre-steady-state flavin reduction by the denoted substrate unless otherwise stated Isotope effects are H ⁄ D unless otherwise stated Enzyme PETNR MR MR GO CO MAO A MAO B FDTS DD TMADH PAO TSOX Substrate DDHà (kJỈmol)1) 1° KIE b-NADPH b-NADH cylohexen-1-one 2-deoxyglucose choline benzylamine p-methoxy-benzylamine b-NADPH dihydroorotate trimethylamine L-phenylalanine sarcosine a a 7.0 ± 0.04 6.8 ± 0.1a 3.5 ± 0.2 $10b 10.6 ± 0.6b 8.0e 8.9 ± 1.6c 25 ± 6d 3.77 ± 0.08 4.6 ± 0.4 5.4 ± 0.3 7.3e 6.5 ± 2.76 7.4 ± 1.5a )0.5 ± 1.8 $0b 1.0 ± 0.3b n.d $9.0c,e n.d n.d 0.5 ± 5.2 f 0.2 ± 0.03 0.6 ± 2.1 AàH : AàD Ref 0.51 ± 0.04 0.12 ± 0.09 4±2 $10g 14 ± n.d $0.2g n.d n.d 7.8 ± 1.2 5.2 ± 0.2 5.4 ± 0.4 [15,18] [15,18] [15] [73] [74] [75] [76] [77] [78] [16] [72] [17] a Revised from previously reported, manuscript in preparation b Data from kcat ⁄ Km measurements c Data not corrected for the calculated commitment to catalysis d Data from H ⁄ T isotope effect e No error given f Data for the H172Q mutant g Calculated from the KIE and DDHà values The reductive half-reaction (RHR) of MR and PETNR occurs in three steps: A binding Eox ỵ NADPịH ẵEox NADPịHCT ! reduction= Htransfer ! product release 4ị Ered NADPịỵ Ered ỵ NADPịỵ ! MR reacts only with NADH, whereas PETNR has a preference for NADPH It is sometimes possible to trap the binary CT complex in OYEs by substituting NAD(P)H with the nonreactive analogue 1,4,5,6-tetrahydroNAD(P)H (NAD(P)H4) [19] MR binds NADH4 with Kd = 0.35 mm [34] and we have recently solved the X-ray crystallographic structure of MR in complex ˚ with NADH4 to a resolution of 1.3 A [19] The structure of the active site in MR is shown in Fig 1, as is the proposed mechanism of hydride transfer ⁄ FMN reduction In MR and PETNR, the H transfer is stereospecific because the NAD(P)H nicotinamide moiety can only bind in one orientation within the active site, which places the pro-R (transferred) primary hydrogen (Hp) directly over the FMN N5 acceptor atom (labelled in Fig 1) The reduced OYEs can be reoxidized by molecular oxygen (k $ s)1) [35] or with various oxidative substrates – one generic substrate being cylcohexen-1-one With many of the oxidative substrates tested, the oxidative half-reactions of MR and PETNR are fully rate limiting during steady-state turnover Consequently, the steady-state KIE on the RHR hydride transfer is 3932 B Fig (A) A model of the active site of NADH-bound morphinone reductase based on pdb 2R14 [19] Residues which form hydrogen bonds (dotted lines) to the bound NADH are shown, as are the NADH nicotinamide pro-R (Hp, the transferred H) and pro-S (Hs) hydrogens and the FMN N5 (acceptor) (B) The proposed reaction mechanism of hydride transfer ⁄ FMN reduction during the reductive half reaction in old yellow enzymes unity and steady-state analysis is clearly not appropriate to study these reactions However, in MR, we have measured a KIE of 3.5 ± 0.2 on kcat for the hydride transfer from the reduced FMN to cylcohexen-1-one FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS S Hay et al Hydrogen tunnelling in biological systems ð5Þ and in the case of the oxidative half-reaction of MR, the observed double KIE should be: 3.5 · 2.3 = 8.0, which is in agreement with the observed value of 8.2 ± 1.4 [15] Although it has been argued that violation of the rule of geometric mean may be used as evidence for H-tunnelling [36–38], the oxidative KIEs in MR are not measurably temperature dependent – a diagnostic of ground state H-tunnelling [15] Using a stopped-flow spectrometer, it is possible to determine most of the rate constants for the steps in the OYE RHR reaction (Eqn 4) above However, care must be made to keep the samples anaerobic by either using an anaerobic glove box or by adding glucose ⁄ GO The binary complex has a characteristic p-p charge transfer (CT) absorbance and NAD(P)H binding and dissociation can be measured by following the formation of this CT absorbance at, for example, 555 nm while performing a concentration dependence [35,39]: kobs ẳ koff ỵ kon ½NADðPÞHŠ ð6Þ Similarly, the rate of hydride transfer can be determined because H transfer is concomitant with FMN reduction By following the bleaching of FMN absorbance at $465 nm while performing a concentration dependence [15,18,35,39], it is possible to characterize the RHR: kobs ẳ kox ỵ kred ẵNADPịH koff =kon ị ỵ ẵNADPịH 7ị In both MR and PETNR, at room temperature, kon, the apparent rate of coenzyme binding is $106 m)1Ỉs)1 The rate of NAD(P)H dissociation, koff, is $102 s)1 and the apparent rate of H transfer, kred, is 56 and 33 s)1 in MR and PETNR, respectively [18,35,39] Product (NAD(P)+) inhibition of MR and PETNR is very weak suggesting that NAD(P)+ rapidly dissociates from the active site once FMN reduction occurs We have been unable to measure the reverse rate of hydride transfer, kox, in either enzyme and it appears to be close to zero [18,40] We have also determined the driving force for hydride transfer during the RHR of MR with NADH to be $60 kJỈmol)1 [40], which is also consistent with an effectively irreversible H transfer 0.20 0.20 Absorbance KIEa;b ¼ KIEa  KIEb We have mutated various amino acid residues within the active site of MR and PETNR [19,41,42] In the wild-type enzymes, FMN reduction occurs as a monoexponential process (Fig 2) – greatly simplifying the stopped-flow analysis In the H186A and N189A active-site mutants in MR (Fig 1A), FMN reduction kinetics become more complex – i.e multi-exponential [41] As an extreme example, we have measured at least four kinetically resolved components of FMN reduction in the N189A mutant of MR, each with a significant KIE (Fig 2) [19], and each able to interconvert [42a] We have attributed this complexity to the formation of multiple reactive configurations in the mutant enzyme because of improper binding of the NADH nicotinamide moiety within the active site Of concern is that, had we performed a steady-state analysis of this mutant, we would not have observed this heterogeneity and the mutant enzyme would have appeared to be quite similar to the wild-type enzyme Caution is therefore needed when using steady-state approaches to analyse tunnelling, especially with mutant enzymes During the RHR of MR and PETNR, when pro-R deuterated NAD(P)H (denoted (R)-[4-2H]-NAD(P)H) is used in place of the protiated coenzyme, a significant KIE is observed on hydride transfer (kred) but not on koff ⁄ kon (Table 1) [15,18] Because the H transfer is effectively irreversible and kinetically resolved from coenzyme binding (no KIE on koff ⁄ kon), the observed KIE is essentially the intrinsic KIE Using stoppedflow methods, we have measured the temperature dependence of the rate of H transfer in both MR and PETNR For convenience, we tend to measure kobs (in Absorbance [15] There is also a solvent KIE of 2.3 ± 0.3 and a double isotope effect (measured with deuterated FMN in D2O) of 8.2 ± 1.4 The rule of geometric mean [36– 38] states that multiple KIEs should be described by: 0.15 0.15 0.10 0.05 0.00 0.10 0.0 0.1 t·s–1 20 40 0.05 N189A wt 0.00 0.01 0.1 t·s–1 10 100 Fig Multiple reactive conformations during an H-transfer reaction Stopped-flow traces of the observed FMN reduction during the reaction of the wild-type (wt) and N189A mutant of MR with NADH The wt trace is fit to a single exponential and the N189A trace to a 4-exponential function – see the main text for more details (Inset) The same data on a split-axis linear time scale Adapted from Pudney et al [19] FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS 3933 Hydrogen tunnelling in biological systems S Hay et al the presence of saturating [NAD(P)H]) rather than kred (Eqn 7), although they give equivalent results [8,15,18,19,35] We define saturating coenzyme concentrations as [NAD(P)H] > 10 · KS, where KS = koff ⁄ kon Care must be taken because KS can be quite temperature dependent [15,19,35] We typically analyse these data in terms of Eyring (transition state) theory:     kobs kB DSz DH z À ¼ ln þ ln T RT T h ð8Þ lar observation has been observed in the Escherichia coli DHFR [45] Consequently, it would appear that inflated a-2° KIEs may be indicative of a tunnelling contribution to the H transfer, but normal KIEs not rule out tunnelling [43] A similar argument can be made for 1° KIEs – although KIEs £ can be explained using transition state theory, the KIE arising from a full tunnelling reaction (described by Eqns 1–3) can be any value [10,46] Preparation of coenzymes with KIEobs ¼ on the KIE: H D kobs =kobs and the temperature dependence DDH z ¼ DH zD À DH zH ¼ DEa ð9Þ In wild-type MR and PETNR it is generally possible to determine observed rate constants with an accuracy of $1% (measured with different samples on different days) It is then possible to determine KIEs to an accuracy of 2-5% and DDHà with an error of 1-2 kJỈ mol)1 [18] The use of KIE analyses relies heavily on the ability to obtain isotopically pure substrates or coenzymes One of the reasons OYEs are particularly attractive to study is the ability to create stereospecifically labelled isotopologues of NAD(P)H (described in the next section) This allows the measurement of 1° KIEs, 2° KIEs and double KIEs – where both Hp and Hs are deuterated [18] We have been able to use stopped-flow methods to measure quite accurately both the magnitude and temperature dependence of a-2° KIEs during the RHR in MR and PETNR [18,40], and for hydride transfer in the thermophilic dihydrofolate reductase (DHFR) from Thermotoga maritima [43] The equilibrium isotope effect (EIE) on NAD(P)H oxidation was measured by Cook & Cleland to be 1.13 [44] The observation of a-2° KIEs values larger than the EIE was rationalized by Huskey & Schowen [36] because of coupling of the motion between the 2° hydrogen (labelled in Fig 1A) and the 1° (transferred) hydrogen during an H-tunnelling reaction We have measured identical a-2° KIE values of $1.2 in MR and PETNR, which are significantly larger than the EIE [18,40] We have also measured the double KIE in MR [18] and shown that in this reaction, the rule of geometric mean (Eqn 5) is most likely violated [39] We have shown computationally that the H transfer in MR occurs by deep tunnelling [28] so Huskey & Schowen’s [36] interpretation of inflated 2° KIEs would seem plausible However, we have measured a normal (KIE £ EIE) and temperature-independent a-2° KIE in TmDHFR, yet this reaction proceeds by 50-80% tunneling, depending on the temperature [25,43] A simi3934 The methods of coenzyme deuteration are well described [47–50], but are typically for microscale syntheses, $1 mg This can be an issue for stopped-flow experiments, substrate-binding titrations and crystallographic studies when a very large amount of the substrate may be required; typical NAD(P)H saturation constants for OYEs are 0.1-1 mm and as an example, a typical measurement, by stopped-flow, of the temperature dependence of the 1° KIE of an OYE may require $100 mg of isotopically pure substrate For reference, we briefly describe our preferred methods of synthesis for large-scale ($1 g) preparations of all three deuterated NADH and NADPH isotopologues: (R)-[4-2H]-NAD(P)H, (S)-[4-2H]-NAD(P)H and (R,S)[4,4-2H2]-NAD(P)H These syntheses typically yield > 95% isotopologue purity (based on 1H NMR spectra, see Pudney et al [18] for examples), with the corresponding impurity being the protiated coenzyme Kohen [49] recently developed syntheses for extremely high-purity NADPH isotopologues and the method of McCracken et al [49] has been reported to yield > 99% isotopologue purity – a purity necessary when performing competitive isotope experiments We also describe our synthesis of 1,4,5,6-tetrahydroNAD(P)H We typically prepare (R)-[4-2H]-NADH by the stereospecific reduction of NAD+ (500 mg) with 1-[2H6]ethanol (1 g) using yeast alcohol dehydrogenase (200 U) and aldehyde dehydrogenase (100 U) in 20 mm Taps pH 8.5 (20 mL) at room temperature This method is a slight modification of the procedure reported in Viola et al [48] (R)-[4-2H]-NADPH is prepared through a stereospecific reduction of NADP+ (300 mg) with 1-[2H6]-isopropanol (1 g) using NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brokii (100 U) in 20 mm Taps pH 8.5 (50 mL) at 42 °C These reactions are deemed complete when A340 stopped increasing and A260:A340 < 3, typically after h (S)-[4-2H]-NADH and (S)-[4-2H]NADPH are prepared through stereospecific reduction of NAD+ (500 mg) and NADP+ (500 mg), respectively, with 1-[2H]-glucose (150 mg) using glucose FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS S Hay et al deuterated product This fractionation also leads to an overestimation of kD and consequently an underestimation of the KIE It appears that it is possible to correct for incomplete deuteration using a simple linear relationship in an analogous fashion to that used to correct steady-state data: D kobs ¼ kH fD ị ỵ kD fD 10ị where fD is the fraction of substrate deuteration, which can usually be determined quite accurately by H NMR [18,48,52] or possibly MS [18,53] In Fig 3, we use Eqn (10) to model the effect of partial deuteration on the observed rate and KIE of an H transfer reaction If kD is underestimated then so too will be DDHà and the effect of fD on the apparent temperature dependence of the KIE is also shown in Fig We determined Eqn (10) empirically and this relationship is quite approximate Nevertheless, we have been able to correct the RHRs of MR and PETNR and also the RHR of aromatic amine dehydrogenase with benzylamine using Eqn (10) [39] However, further studies are required to confirm the general validity of this correction method That fractionation can occur emphasizes the need for: (a) care in preparing highpurity coenzymes, and (b) correction for small isotope impurities in the analysis of tunnelling kinetics using stopped-flow single turnover measurements Hydrostatic pressure Hydrostatic pressure offers an alternative or complementary method to temperature with which to study Kobs/s–1 A 4 2 10 ΔΔH ‡ / kJ·mol–1 B KIEobs dehydrogenase (150 U) in Taps pH 8.5 (10 mL) at room temperature This method is a slight modification of the procedures reported in Ottolina et al [50] and the reactions typically take h (R,S)-[4,4-2H2]NADH is prepared by stereospecific oxidation of (S)-[4-2H]-NADH (300 mg) with 100 mm cyclohexen1-one catalysed by 10 lm MR in Taps pH 8.5 (10 mL) The deuterated NAD+ is purified in the same manner as for (R)-[4-2H]-NADH (R,S)-[4,4-2H2]NADH is then prepared through a further stereospecific reduction of [4-2H]-NAD+ with 1-[2H]-glucose (100 mg) and glucose dehydrogenase (100 U) in Taps pH 8.5 (10 mL) (R,S)-[4,4-2H2]-NADPH can be prepared in the same manner as (R,S)-[4,4-2H2]-NADH However, an NADPH-specific enzyme such as PETNR must be used in place of MR NADH4 and NADPH4 are prepared by maintaining a slight pressure ($1.2 bar) of hydrogen (> 99%) over a solution of NAD(P)H (500 mg) and palladium-activated charcoal (30 mg) in Tris ⁄ Cl pH 9.0 (5 mL) stirred on ice [19] The reaction is stopped when no absorbance at 340 nm is observed and typical A266 ⁄ A288 ratios of $1.06 are obtained We purify the coenzymes using anion-exchange (QSepharose) chromatography, eluting NADH and NADPH isotopologues (including the tetrahydro forms) in $200 and $500 mm ammonium bicarbonate, respectively [18] All of the enzymes used in these syntheses (excluding MR) are available from Sigma-Aldrich (St Louis, MO, USA) and the coenzymes are available from Melford Laboratories (Chelsworth, UK) We use extinction coefficients of 6.22 mm)1Ỉcm)1 at 340 nm for NAD(P)H isotopologues and 16.8 mm)1Ỉcm)1 at 289 nm for NAD(P)H4 [34] Usually the enzymatic synthesis of (R)-[4-2H]-NAD(P)H does not proceed to completion We have observed that freezing or freeze-drying the reaction volume before purification usually leads to the formation of a significant impurity of undeuterated NAD(P)H Consequently, on this scale, it is important to purify the reaction volume as quickly as possible after synthesis has ceased Also, one must take care to maintain the pH at $8.5 over the course of the enzymatic synthesis, because acid catalysed decomposition of NAD(P)H may be a significant contributor to substrate (in)activity [51] As an aside, we have recently investigated the effect of incomplete coenzyme ⁄ substrate deuteration on the observed KIE measured using stopped-flow methods [39] We found that, if there is a reversible chemical step preceding H transfer and the reverse rate of this step (koff in Eqn 4) is comparable with the rate of H transfer, then kinetic isotope fractionation can occur, leading to the formation of more protiated than Hydrogen tunnelling in biological systems 0.0 0.2 0.4 0.6 0.8 Fraction deuteration 1.0 Fig The effect of substrate isotopic purity on the observed rate of deuterium transfer (filled squares) and the corresponding KIE (open circles) (A), and on the temperature dependence (B) of a modelled H-transfer reaction The data are modelled using Eqn (10) with kH = s)1, a KIE of and various values of DDHà FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS 3935 Hydrogen tunnelling in biological systems S Hay et al where r0 is the average H-transfer distance, Dr is the change in this distance with pressure, j0 is the force constant describing the promoting vibration and Dj is the change in this force constant with pressure Equation (11) can be used as a fitting function with four adjustable parameters and the KIE can either increase or decrease with increasing pressure when Dr and ⁄ or Dj become significant (Fig 4) Although this model is oversimplistic, it is possible to use Eqn (11) to describe a reaction in which both the apparent rate and KIE increase (or decrease) with pressure [61] The model 3936 A 10 KIE 0.0 0.02 0.5 0.01 1.0 bar 0.00 1.5 ure/k dr/ –0.01 Åk s ba – –0.02 2.0 Pres r B 10 KIE 1.0 0.0 0.5 0.5 dκ 0.0 1.0 ar kb 1.5 ure/ /J·m –0.5 s –2 –1.0 2.0 es ·kb Pr ar –1 C 10 KIEobs enzymatic reactions Semiclassical transition-state theory states that pressure effects on isotope effects arise because of differences in vibrational frequencies [54–56] and stretching vibrations are insensitive to pressures of a few kbar [54] Consequently, the KIEs of purely transition state reactions are expected to be insensitive to pressure Conversely, several chemical systems with inflated KIEs indicative of a tunnelling contribution to the H transfer have been shown to exhibit a significant pressure-dependence of both rate and KIE [57] Thus, in principle, the pressure-dependence of an isotope effect provides an excellent method for distinguishing between transition state and tunnelling reactions The use of pressure to study enzymatic H-tunnelling reactions was pioneered by Northrop, who, 10 years ago, developed a model [58] for the pressure-dependence of H-transfer reactions based on the Bell correction [33,58] This model was then used quite successfully to model the pressure dependence of steady-state H-transfer reactions in alcohol dehydrogenase and aldehyde dehydrogenase [57–59] We recently performed a high-pressure stopped-flow study of the hydride transfer during the RHR of MR with NADH The apparent rate of hydride transfer increased by approximately twofold per kbar increase in pressure and the 1° KIE also showed a small but significant increase in magnitude with pressure (Fig 4C) Together, these observations could not be explained using Northrop’s model [58], nor with a simple nonadiabatic H-tunnelling model (e.g Eqns 1–3) when pressure simply causes a compression of the reaction barrier [60] However, we found that we could qualitatively model the data by invoking a promoting vibration that changes frequency with pressure [8] We have since refined this analysis and recently described a simple nonadiabatic H-tunnelling model which explicitly includes pressure as a variable [61]: n o À Á2 h kH =kD $ exp ½lD xD À lH xH Š r0 ỵ Dr:p =2 11ị h expfẵlD xD lH xH kB T= j0 ỵ Dj:pịg 3.6 3.5 3.4 3.3 10 3.2 /T· K –1 2.0 1.5 r 1.0 ba 0.5 e/k ur 0.0 s es Pr Fig A variable pressure H-tunnelling model (Eqn 3) [61] The KIE pressure dependence is modeled when (A) the frequency of the promoting motion or (B) the H-transfer distance changes with pressure Positive values of Dj and Dr reflect increases in frequency and distance with pressure, respectively The data are modeled ˚ with j = JỈm)2, r0 = 0.52 A (KIE0 = 5) and only one parameter in each plot is varied It is possible for both Dj and Dr to vary with pressure (as we have modelled in MR) [61], causing curvature in the KIE versus pressure plots We have also plotted (C) the combined pressure and temperature dependence of the observed KIE on hydride transfer during the reductive half reaction of morphinone reductase The data are taken from Hay et al [8] We have not plotted error bars for clarity but the average error in the KIE for this data set is ±5% and the minimum and maximum error is 1% and 18%, respectively FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS S Hay et al should only be used qualitatively, but is useful to estimate whether: (a) the tunnelling distance changes with pressure, and (b) to confirm that there is environmental coupling and to determine whether the frequency of this vibration is likely to change with pressure Although it seems intuitive that hydrostatic pressure will ‘squeeze’ the enzyme and thus compress the H-transfer distance (achieved by increasing the population of enzyme–substrate conformers with shorter H-transfer distances in an equilibrium distribution of conformational states), until recently, this assumption remained untested Ewald [62] has shown that increasing pressure causes a progressive shortening of the CT bond in synthetic p-p complexes with an accompanying shift to red wavelengths and increase in absorbance We have recently shown that increasing pressure also causes a shortening of the CT bond (increase in CT absorbance) in NADH4-bound MR, which we have interpreted as pressure-induced barrier compression [34] Using variable pressure molecular dynamics simulations of NADH-bound MR, we were able to corroborate this finding [34] We found that the heavy atom transfer distance has an approximately Gaussian distribution that both narrows and shifts to shorter distances at elevated pressures It appears, at least in MR, that pressure does not physically squeeze the microscopic reaction barrier, but rather reduces the average barrier width, the macroscopic barrier, by restricting the conformational space available to the NADH and FMN moieties within the active site Further studies are required to determine whether this is a general phenomenon Other experimental probes In addition to temperature and hydrostatic pressure, it is possible to experimentally probe enzymatic H-tunnelling reactions using additional experimental parameters and we briefly discuss the use of varying the solvent composition to probe the effect of solvent dielectric and viscosity on H transfer chemistry It is predicted from von Smoluchowski’s theory [63] that the rate of a diffusion-controlled (bimolecular) reaction will be inversely proportional to the bulk solution viscosity The effect of viscosity on a unimolecular reaction is more complicated but can be described in combination with the Eyring equation according to Ansari et al [64]: ! !   kB T ỵ r DSz DH z exp exp 12ị kobs ẳ h gỵr R RT where r, in units of viscosity, is the contribution of the protein friction to the total friction of the system Hydrogen tunnelling in biological systems The activation entropy and enthalpy can be determined independently from the temperature dependence of the reaction [65] Solution viscosity has been used to probe the role of dynamics in interprotein [65–68] and intraprotein [69] ET reactions and protein rearrangement after carbon monoxide dissociation from myoglobin [64] In general, the rates of ET reactions that are conformationally gated decrease upon an increase in solvent viscosity The viscosity dependence of several enzymatic H-transfer reactions has now been investigated Protein dynamics can be affected by surface glycosylation and this approach has been used by Klinman and coworkers to study the viscosity dependence of the rate of hydride transfer in GO [70,71] These authors studied various glycoforms of the enzyme (varying in the extent of glycosylation) [70] and also replaced the native polysaccharide with different polymeric forms of polyethylene glycol [71] A decrease in the ‘fitness’ of GO was observed when the apparent surface viscosity increased or decreased relative to the wild-type enzyme Fitness was defined as a reduction (away from unity) in the Arrhenius pre-exponential ratio (AD : AT) [70,71] In a more conventional study, we found that the magnitude and temperature dependence of the presteady-state rate and KIE for proton tunnelling during the RHR of the quinoprotein methylamine dehydrogenase are unchanged following the addition of 30% glycerol – an increase in solvent viscosity of approximately two- to threefold [13] Conversely, a decrease in KIE and increase in apparent enthalpy for the RHR of l-phenylalanine oxidase upon the addition of 30% glycerol has been reported [72] In a more systematic study, we recently showed that the rate of coenzyme capture decreases, whereas the rate and KIE of hydride transfer during the RHR in MR are invariant over a 10-fold increase in solution viscosity [20] We found it was possible to use a conventional stoppedflow to make these measurements by varying the viscosity between $0.9 and cP at 25 °C with the addition of 0–60% w ⁄ w glycerol Addition of > 60% glycerol leads to mixing artefacts that precluded further measurements The addition of glycerol to the solvent will also reduce the solvent dielectric We independently probed the role of solvent dielectric on the RHR of MR by measuring the temperature dependence in this reaction in the presence of ethanol Neither the rate nor enthalpy significantly changed upon the addition of 20% v ⁄ v ethanol – a change in dielectric from $80 to $65, but NADH binding was significantly compromised with an increase in KS from 0.2 to 2.7 mm observed Unfortunately, no clear trends have emerged as to the effect of viscosity (or dielectric) FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS 3937 Hydrogen tunnelling in biological systems S Hay et al on enzymatic H-transfer reactions and more systematic studies are probably required to determine whether this is a useful probe of H transfer dynamics Future perspectives It is now fairly well established that many enzyme H-transfer reactions involve a degree of quantum mechanical H-tunnelling The role of promoting vibrations, which couple protein dynamics to the H transfer reaction coordinate, remains contentious Although there is now a growing body of compelling experimental and computational evidence for such 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temperature dependence of the presteady-state rate and KIE for proton tunnelling during the RHR of the quinoprotein methylamine dehydrogenase are unchanged following the addition of 30%... AF (1989) Enzymatic-synthesis of 4R-H-2NAD(P)H and 4S-H-2NAD(P)H and determination of the stereospecificity of 7-alpha-hydroxysteroid and 12-alpha-hydroxysteroid dehydrogenase Biochim Biophys Acta... arises because of differences in the mass, frequency and consequently the transfer distance of H and D Experimentally, the identification of promoting vibrations is extremely challenging and, as yet,