Solvent-Slaved Motions in the Hydride Tunneling Reaction Catalyzed

Research Article pubs.acs.org/acscatalysis

Solvent-Slaved Motions in the Hydride Tunneling Reaction Catalyzed by Human Glycolate Oxidase Elvira Romero,§,† Safieh Tork Ladani,§,∥ Donald Hamelberg,§,∥ and Giovanni Gadda*,§,¶,∥,# §

Department of Chemistry, ¶Department of Biology, ∥Center for Biotechnology and Drug Design, and #Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302-3965, United States S Supporting Information *

ABSTRACT: Enzyme motions facilitate many hydride-transfer reactions involving quantum mechanical (QM) tunneling. The evidence mainly comes from the determination of kinetic isotope effects (KIEs) and their temperature dependence that have been used to reveal interesting characteristics of human glycolate oxidase (HsGOX). Previous studies have shown that HsGOX oxidizes glycolate to glyoxylate via a hydridetransfer mechanism to an enzyme-associated FMN. Here, we investigate the temperature effect on the anaerobic rate of flavin reduction (kred) for HsGOX with glycolate and [2R-2H]glycolate. While the kred values for HsGOX are temperature-dependent, their KIEs on the kred values (Dkred) do not change as the temperature is varied. This is consistent with the involvement of QM hydride tunneling in the highly optimized active site of HsGOX. We show that the enzyme motions are slaved by the fluctuations in the bulk solvent after determining the kred and Dkred for HsGOX at various solvent viscosities and constant temperature. These results are interpreted in the context of an extension of the Transition State Theory (TST) previously described for adiabatic processes. These experiments demonstrate for the first time that the solvent viscosity modulates the rate of hydride transfer in an enzyme-catalyzed reaction. Furthermore, molecular dynamics simulations show that an increase in the collision frequency of only the bulk solvent from 0.8 ps−1 to 3.8 ps−1 slows down the dynamics of the HsGOX-glycolate complex in the active site, suggesting a direct coupling between solvent motions and the active site dynamics. KEYWORDS: enzyme motion, glycolate oxidase, hydride tunneling, internal friction, kinetic isotope effect, molecular dynamics simulation, solvent-slaved motion, solvent viscosity

E

Some enzymes present a poorly reorganized TRS with a wide range of DADs at thermal equilibrium.3 This yields temperature-dependent KIEs because the lighter isotope can tunnel over longer distances than the heavier one.2 At elevated temperatures, thermally activated DAD fluctuations populate shorter DADs leading to lower KIEs. Besides temperature, other properties of the bulk solvent may also affect enzyme motions and catalysis. Kramers’ Theory (KT),4 in contrast to TST, includes a diffusion coefficient that modulates the rate constant of chemical reactions. The diffusion coefficient is determined by the friction coefficient of the reactants moving in a solvent, which is proportional to the solvent viscosity based on the Stokes’ law.5 Frauenfelder et al.6 described two types of protein motions, which they named slaved and nonslaved motions. The rate of slaved protein motions is proportional to the rate of fluctuation of the solvent, which is mainly determined by the viscosity of the solvent.6 In addition to the solvent viscosity, intramolecular interactions in the protein may contribute to the friction coefficient.7−9

xperiments and simulations suggest that enzymes are able to optimize their conformation for each step along the reaction coordinate taking advantage of their adequate structural flexibility.1 This involves fast changes, either local or covering a large portion of the protein, in the hydrogen bond network, electrostatics, and the distribution of water molecules in the protein.1 Protein motions facilitate many hydride-transfer reactions involving quantum mechanical (QM) tunneling.2 The tunneling probability is higher for light than heavy isotopes.2 Enzyme reactions are often investigated using an Arrhenius plot displaying the effect of temperature on the kinetic isotope effect (KIE) associated with the reaction rate constants.2 These experimental data, in the case of adiabatic processes, can be interpreted using the extension of the Transition State Theory (TST) recently described in ref 3. We have used this model to understand an enzyme-catalyzed hydride-transfer reaction. In terms of this model, many enzyme reactions involve conformational sampling to bring the donor and acceptor closer to each other, resulting in a very narrow distribution of donor−acceptor distances (DADs) at the tunneling-ready-state (TRS) that is not influenced by changes in temperature.3 This leads to temperature-independent KIEs, despite the individual rate constants maintaining a dependence on the temperature.2,3 © 2016 American Chemical Society

Received: December 17, 2015 Revised: January 30, 2016 Published: February 11, 2016 2113

DOI: 10.1021/acscatal.5b02889 ACS Catal. 2016, 6, 2113−2120

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Figure 1. Crystal structure and UV−visible absorption spectra of HsGOX. Panel A shows the cartoon representation of the crystal structure (PDB ID: 2RDU). Panel B shows the stick representation of the active site residues conserved in the LHAD enzymes, glyoxylate, and FMN (purple, cyan, and yellow carbons, respectively). Panel C shows the absorption spectrum of HsGOX before and after the anaerobic reaction of the enzyme (11 μM) with glycolate (4 mM) in the presence of 12% glycerol (m/m), at pH 7.0 and 30 °C (GOXox and GOXred, respectively).

Scheme 1. Reaction Catalyzed by HsGOXa

Among the most important causes of this additional source of friction, known as internal friction, are likely the formation and breaking of amino acid contacts and dihedral angle rotations of the side chains in the protein (see ref 10 and refs therein). We show here the effect of temperature and solvent viscosity on the rate constant for anaerobic flavin reduction in human glycolate oxidase (HsGOX; EC 1.1.3.15; isozyme A; HAOX1 gene product), which belongs to the L-α-hydroxyacid dehydrogenase (LHAD) family. All members of this family present a β8α8-barrel fold, also known as TIM-barrel, a highly conserved buried active site (Figure 1A,B) that contains noncovalently bound FMN, and use L-α-hydroxyacids as organic substrates.11 In liver peroxisomes, HsGOX catalyzes the oxidation of glycolate to glyoxylate, which is released to the solvent from the reduced enzyme (Scheme 1).12 The subsequent reaction of the reduced enzyme with dioxygen generates oxidized flavin and hydrogen peroxide, thereby completing the catalytic cycle. HsGOX is a potential drug target in diseases characterized by an excessive renal excretion of oxalate leading to renal failure, such as in primary hyperoxaluria, since glyoxylate is an immediate precursor of oxalate.13 A mechanism involving hydride transfer from the substrate C2 atom to the FMN N5 atom (Scheme 1) has been previously established for various LHAD enzymes including GOX, mainly based on KIE and computational studies.11,14,15 The present work shows for the first time the participation of QM hydride tunneling in an LHAD enzyme. In addition, our experiments and molecular dynamics (MD) simulations show also for the first time that solvent viscosity directly modulates

a

For FMN, R is ribitol phosphate.

the rate constant for hydride transfer in an enzyme-catalyzed reaction.

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MATERIALS AND METHODS Materials. Recombinant pure HsGOX was obtained as published.16 [2R-2H]Glycolate and glycolate were synthesized in our laboratory as described in the Supporting Information (SI). All other chemicals were of the highest purity commercially available. Rapid Kinetics. The anaerobic reduction of HsGOX was monitored with an Hi-Tech KinetAsyst SF-61DX2 stoppedflow spectrophotometer (TgK Scientific, Bath, U.K.) using a xenon lamp and a photomultiplier tube. The enzyme, substrate, and instrument were made anaerobic as described in SI. The experiments were performed in triplicate by mixing equal 2114

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the Ornstein−Uhlenbeck (OU) process27 which leads to the autocorrelation function (eq 8). Data Analysis. The equations below were used as specified in the corresponding section of the manuscript. The parameters and variables in these equations have the standard meaning, as described in SI. The software Kinetic Studio (TgK Scientific, Bath, U.K.) was used for eq 1, and the software KaleidaGraph (Synergy Software, Reading, PA) was used for eq 2, 3, 4, and 6.

volumes of HsGOX and either glycolate or [2R-2H]glycolate, and monitoring the decrease in absorbance at 450 nm due to the reduction of the flavin bound to HsGOX (Figure 1C). The final substrate concentration (0.08−4.0 mM) was at least five times greater than that of enzyme (15−20 μM) to ensure pseudo-first-order conditions. The effect of isotopically labeled substrate on the enzyme reduction was determined by alternating substrate isotopologues. Sodium phosphate buffer (100 mM) was used in the pH range of 6.0−8.0. When a pH value of 9.0 or 10.0 was required, the buffer contained both 100 mM sodium phosphate and 10 mM sodium pyrophosphate. Data were collected at various temperatures (10−40 °C) in the presence of an appropriate glycerol percentage (0−24%, m/m) to keep the viscosity constant (η = 1.3 cP) as the temperature was changed. In other experiments, the temperature was kept constant (30 °C) and the solvent viscosity was varied (η = 0.8− 3.8 cP) by adding different percentages of glycerol (17−49%, m/m; η = 1.3−3.8 cP) or sucrose (14−36%, m/m; η = 1.2−3.4 cP), or 10% Ficoll 400 (η = 3.8 cP). Before mixing in the stopped-flow reaction chamber, the enzyme and substrate solutions contained the same viscosigen concentration to prevent uneven mixing or turbidity. The absolute (or dynamic) viscosity (η, cP) and dielectric constant (ε) values, at different temperatures, of glycerol or sucrose solutions were taken or interpolated from refs 17−22. The kinematic viscosity (v, cSt) and the density (ρ, kg/m3) of various Ficoll 400 solutions (6− 13%, m/m) at 30 °C were measured in triplicate using a Cannon-Ubbelohde Semi-Micro viscometer (size 75) and a pycnometer (2.104 mL), respectively. The average values of these determinations (3 replicates) were used to calculate the absolute viscosity (η = νρ). Using these data, the percentage of Ficoll 400 required to obtain a solvent viscosity of 3.8 cP was calculated by interpolation. Computational Simulations. The starting crystal structure for running MD simulations was that of HsGOX including glyoxylate (PDB ID: 2RDU; 1.65 Å resolution). The glyoxylate in this complex was replaced with glycolate. Parametrization of the glycolate and FMN were carried out using Antechamber program in the AMBER 10 suite of programs (University of California, San Francisco, CA). The xleap module in AMBER 10 was used to add the missing hydrogen atoms and solvate the complex with explicit TIP3P water model in a periodic octahedron box.23 A total of 2050 TIP3P water molecules were added to obtain a box dimension of approximately 81 Å × 81 Å × 81 Å. Two sodium ions were added to obtain electrostatic neutrality. All simulations were run using the pmemd program in AMBER 10 with the modified version of the parm99 (ff99SB) all-atom force field (ref 24 and refs therein). The entire system was minimized and equilibrated at 300 K, by applying a Langevin thermostat with a collision frequency of 1 ps−1, and 1 bar. A cutoff of 10.0 Å was applied for nonbonded short-range interactions, and long-range electrostatic interactions were treated using the particle mesh Ewald method.25 A time step of 2 fs was used to numerically integrate the equation of motion. The simulations were carried out with two values of the solvent collision frequency, 0.8 and 3.8 ps−1. No collisions were applied to the protein−substrate complex during data collection. A previously developed model26 was used here to describe the changes in the distance between the glycolate C2 atom and the FMN N5 atom as a Brownian motion on an effective one-dimensional energy profile. These fluctuations can be modeled as a special case of

A450 = B1e−kobs1t + B2 e−kobs2t + C

(1)

kobs = k redS /(Kdapp + S)

(2)

ln k red = ln A − Ea /RT

(3)

ln D k red = ln AH /AD − [Ea(D) − Ea(H)]/RT

(4)

k red = [A /(σ + η)]e−ΔEa / RT

(5)

1/k red = η /Ae−ΔEa / RT + σ /Ae−ΔEa / RT

(6)

‡

k = C(T )e−ΔG /(RT )

∫0

∞

P(m ,DAD)e−(E(DAD)/(kBT ))dDAD

(7)

C(t ) = / = e−tDK / kBT

■

< d 2 > = kBT /K

(8)

RESULTS Temperature Dependence. To assess the role of QM tunneling in the hydride-transfer reaction catalyzed by HsGOX, the anaerobic reduction of the enzyme-bound flavin with either glycolate or [2R-2H]glycolate was investigated using a stoppedflow spectrophotometer between 10 and 40 °C. These studies were performed in the presence of various glycerol amounts up to 24% (m/m) to maintain constant the viscosity of the solvent (η = 1.3 cP) as the temperature was changed. The oxidized enzyme was mixed with varying substrate concentrations at pH 7.0 and the decrease in absorbance at 450 nm due to the twoelectron reduction of the flavin bound to HsGOX was monitored (Figure 1C). In all cases, the stopped-flow traces at 450 nm were best fit with a double exponential function, with a fast phase accounting for >95% of the absorption change (eq 1; Figure S1A). The rate constant for the slow phase was independent of substrate concentration, did not show a substrate deuterium KIE and was considerably smaller than the rate constant for the overall turnover (kcat) of the enzyme under the same conditions (e.g., kobs ∼ 2.0 s−1 vs kcat = 16 s−1 at pH 7.0 and 25 °C).16 Thus, the slow phase was deemed not to be on the catalytic pathway of the enzyme and was not investigated further. The fast phase of flavin reduction observed in the stoppedflow spectrophotometer exhibited a hyperbolic dependence of the observed rate constant (kobs) on substrate concentration under all assayed conditions (Figure S1B), allowing for accurate determination of the limiting rate constant for anaerobic flavin reduction (kred) at saturating substrate. Upon changing the temperature from 10 to 40 °C, the kred value increased 7.5 times, irrespective of whether glycolate or [2R-2H]glycolate was used as the substrate (Figure 2A and Table 1 and S1). The resulting Dkred value was temperature-independent and significantly different from unity (Figure 2B, Table 1 and S1). Temperature-independent Dkred values and temperature-de2115

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(m/m) increases the viscosity by 5 times, there was no change in the kred value (Figure S2A). A linear dependence was found between the reciprocal of the kred values, i.e., relaxation times, and the viscosity of the solvent exerted by microviscosigens, as illustrated in Figure 3A,B. Fitting of these data to eq 6 yielded internal friction values (σ) of σglycerol‑glycolate = −0.2 ± 0.3 cP; σglycerol‑[2R‑2H]‑glycolate = −0.1 ± 0.2 cP; σsucrose‑glycolate = 0.8 ± 0.2 cP; and σsucrose‑[2R‑2H]‑glycolate = 0.9 ± 0.5 cP. The resulting Dkred values were instead independent of the microviscosigen concentration (Figure 3C-D). The Kapp d value was not influenced by the presence of sucrose in the HsGOX reactions (Table S2). In contrast, glycerol decreased the Kdapp of HsGOX for either glycolate or [2R-2H]glycolate (e.g., (Kapp d )H is increased 3-fold by including 49% (m/m) glycerol in the reaction; Table S2). These results suggested that glycerol might act as a competitive inhibitor in the HsGOX reaction. This hypothesis was ruled out on the basis of the fact that the HsGOX Km value with glycolate (0.05 mM at pH 7.0, 30 °C, and atmospheric oxygen) does not change in the presence of 6, 14, 21, and 28% (m/m) glycerol (unpublished data obtained for ref 16). Alternatively, the interactions in the HsGOX-glycolate complex may be weaker due to the presence of glycerol since this compound can affect various solvent and enzyme properties besides viscosity.28 The effect of glycerol on Kapp d was not studied in further detail, since glycerol and sucrose present a similar significant effect on kred. A DKapp d value of 1.3−1.6 was determined in the presence of either glycerol or sucrose (Table S2). It is likely that the DKapp d value arises from the hydride-transfer step, since the HsGOX Kapp d includes both substrate binding and the hydride-transfer step.16 Alternatively, binding kinetic isotope effects that have been previously reported in various enzymes could be the 29 origin of the observed DKapp d . We will not discuss them further in this manuscript since they are not directly related to the catalytic step chemistry.29 pH Dependence. Due to its multistep nature enzyme catalysis can present kinetic complexity, which occurs when kinetic steps other than chemical steps contribute to the overall rate of enzyme turnover. Despite being typically considered in steady-state kinetics,30 kinetic complexity may also affect rapid kinetics, for example, if a slow isomerization of the enzyme− substrate complex is required for fast flavin reduction.31 To establish whether kinetic complexity was responsible for the inverse dependence of the kred value on the viscosity of the solvent, the Dkred values for HsGOX were determined as a function of pH between 6.0 and 10.0. Since the Dkred values of HsGOX were insensitive to changes in temperature and viscosity of the solvent (Figure 2B and 3C−D), the effect of pH on the Dkred values was studied under an arbitrary condition of 12% (m/m) glycerol and 30 °C. Both the (kred)H and (kred)D values were pH-independent between pH 6.0 and 10.0 (Figure S4 and Table S3), in agreement with previous results with glycolate showing lack of ionizable groups relevant for the flavin reduction reaction in HsGOX.16 A pH-independent value for D kred of 1.9 ± 0.1 was determined here, which was similar to the temperature-independent Dkred value of 1.8 determined between 10 and 40 °C (see above) and the intrinsic Dkred of 2.4 calculated for plant GOX.15 Alterations in conditions, such as pH and temperature, affect the kinetic complexity of enzymecatalyzed reactions yielding pH- and temperature-dependent KIEs.32 Thus, the lack of pH and temperature effects on the D kred value is consistent with the reductive half-reaction catalyzed by HsGOX not having a rate-limiting viscosity-

Figure 2. Temperature dependence of the kred (A) and Dkred (B) values for HsGOX. HsGOX was anaerobically reacted with increasing concentrations of either glycolate (blue) or [2R-2H]glycolate (red) at solvent viscosity of 1.3 cP, pH 7.0, and 10−40 °C. Each reaction was assayed in triplicate. The error bars represent the standard error. When no error bar is visible, it is smaller than the symbol.

Table 1. Kinetic and Thermodynamic Parameters for the HsGOX Reductiona parameter

value

(kred)H, s−1 (kred)D, s−1 D kredb Ea(H), kcal/mol Ea(D), kcal/mol Ea(D)-Ea(H), kcal/mol AH, s−1 AD, s−1 AH/AD

8−66 5−36 1.8 ± 0.2 13 ± 1 12 ± 1 −1.2 ± 1.0 (70 ± 4) × 109 (60 ± 3) × 108 13 ± 1

a

Conditions: anaerobic reactions at solvent viscosity of 1.3 cP, pH 7.0, and 10−40 °C. Glycolate and [2R-2H]glycolate were the substrates. b The average of the Dkred values measured at different temperatures is reported.

pendent kred values are consistent with QM tunneling of the hydride in the reaction catalyzed by HsGOX. The kred and Dkred values determined at various temperatures were fitted to the Arrhenius eq (eq 3 and 4, respectively) to obtain the thermodynamic parameters listed in Table 1. Viscosity Dependence. To study the effect of solvent viscosity on the kred value for HsGOX, various amounts of the microviscosigens glycerol or sucrose were added to the reaction mixtures for anaerobic flavin reduction at pH 7.0 and 30 °C. It was found that increased viscosities of the solvent yielded decreased kred values (Figure S2 and Table S2). For example, a 5-fold increase in viscosity achieved by including 49% (m/m) glycerol in the reaction mixture resulted in a 5-fold decrease in the kred value (Figure S3A). Similarly, the presence of 36% (m/ m) sucrose, which increased the viscosity by 4 times, resulted in a 3 times decrease in the kred value (Figure S3B). In contrast, in the presence of a macroviscosigen like Ficoll 400, which at 10% 2116

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Figure 3. Solvent viscosity dependence of the reciprocal of the kred values (A,B) and the Dkred values (C,D) for HsGOX. HsGOX was anaerobically reacted with increasing concentrations of either glycolate (blue) or [2R-2H]glycolate (red) at pH 7.0 and 30 °C. Glycerol (17−49%, m/m; A,C) or sucrose (14−36%, m/m; B,D) were used as viscosigens. Each reaction was assayed in triplicate. The error bars represent the standard error. When no error bar is visible, it is smaller than the symbol.

that in the more viscous solution [(2.2 ± 0.3 and 0.9 ± 0.2) × 10−4 Å2 ps−1, respectively], resulting in faster dynamics in the active site at lower viscosity of the solvent.

dependent step occurring before the chemical step, thereby establishing that the solvent viscosity directly modulates the hydride-transfer rate. Computational Simulations. MD simulations were carried out to directly probe the effect of solvent viscosity on the active site dynamics of HsGOX in complex with glycolate. The trajectories of the simulations with two different solvent collision frequencies of the solvent, and thus two different solvent viscosities, were analyzed to calculate the autocorrelation of the distance between the glycolate C2 atom and FMN N5 atom. Figure 4 shows the autocorrelation functions, C(t), of

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DISCUSSION QM Hydride Tunneling. The determination of the temperature dependence of the KIE values is one of the major tools used for the detection of QM tunneling in enzymecatalyzed reactions.2 Because changes in temperature cause changes in solvent viscosity (η = 1.3 cP at 10 °C and 0.7 cP at 40 °C), a few studies have investigated the effect of temperature on the KIE values in the presence of the proper percentage of an additive to keep the solvent viscosity constant as the temperature is changed.33 This approach has been used here to study the anaerobic reduction of HsGOX. The results are interpreted within the formalism of an extension of the TST recently described in.3 The kred values for HsGOX increased with increasing temperature using either glycolate or [2R-2H]glycolate. By fitting these data to the Arrhenius equation, similar activation energies were determined for the transfer of protium and deuterium from the substrate C2 atom to the flavin N5 atom in HsGOX (Table 1). This resulted in a temperature-independent D kred value, whereas the deuterium isotope effect on the Arrhenius prefactor was significantly larger than one (Table 1). In contrast to the semiclassical TST, the Bell tunneling correction models, and Marcus-like models for nonadiabatic reactions,34,35 the model described in ref 3 (eq 7), can account for temperature-dependent rates with temperature-independent KIEs in adiabatic reactions like the hydride transfer catalyzed by HsGOX. According to this model, the observation of temperature-dependent kred values for HsGOX is due to the participation of thermally activated heavy atom motions, occurring in the μs−ms time scale and bringing the system to the TRS. These heavy atom motions, which are not sensitive to the mass of the transferred particle, are influenced by

Figure 4. Effect of solvent viscosity on the autocorrelation function of distance between the glycolate C2 atom and the FMN N5 atom in HsGOX. MD simulations were run in water with collision frequency of 0.8 (blue) and 3.8 (red) ps−1. The averages of three replicates are shown. The inset shows the first 5 ps.

the distance between these atoms. The diffusion coefficient of the dynamics along the distance was estimated by fitting the tail of the autocorrelation functions in Figure 4 to a single exponential (eq 8). This analysis showed that the autocorrelation of the simulation with a solvent collision frequency of 0.8 ps−1 decayed faster than that of 3.8 ps−1. The diffusion coefficient value in the former solution was 2.5-fold higher than 2117

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work of Ansari et al. by studying, at various solvent viscosities, an isomerization that occurs in myoglobin after photodissociation of carbon monoxide from the heme iron.7 Subsequently, various studies have supported the importance of internal friction in protein isomerizations8 and folding.9,42,44 The reported internal friction values of different enzyme reactions vary from −0.6 to 4300 cP.8 In the case of HsGOX, a linear dependence between the reciprocal of the kred values and the solvent viscosity was observed, resulting in close to zero internal friction values (Figure 3A,B). These results suggest that the kred value for HsGOX is mainly influenced by friction caused by collisions with solvent molecules, rather than by friction due to intramolecular interactions in the protein.7 This, in turn, is consistent with the lack of kinetic complexity of the enzyme in the reductive half-reaction. Therefore, the internal friction effects do not exist in HsGOX or, more likely, the effects of solvent viscosity on the protein motions obscure the effects of the internal friction. Therefore, while these experiments clearly show that the solvent viscosity modulates the rate of hydride transfer for HsGOX, they do not rule out the contribution of the friction arising from the protein itself. Effect of Solvent Properties on Electron-, Proton-, and Hydride-Transfer Enzyme Reactions. In some interprotein and intraprotein electron-transfer reactions, a rate-limiting viscosity-dependent isomerization preceding the electron transfer has been reported.33,45−47 In the case of HsGOX, the D kred value does not decrease when the solvent viscosity is increased (Figure 3C,D and Table S2) as it would be expected if there were an increment in the degree of rate-limitation of a viscosity-dependent step occurring before or after the chemical step.14 Therefore, this result rules out the presence of a viscosity-dependent isomerization of the enzyme−substrate complex preceding the hydride transfer in HsGOX. Besides electron-transfer reactions, proton-transfer reactions have been also investigated by varying the solvent viscosity. It was found that the rate constant for proton transfer via QM tunneling in NADPH:protochlorophyllide oxidoreductase, from two cyanobacterial species, decreases as the solvent viscosity increases.48 These results suggested that the proton transfer in these enzymes is coupled to viscosity-dependent motions involving a large region of the protein.48 To confirm this hypothesis, the absorbance changes due to proton transfer and the fluorescence changes associated with fluctuations in the bulk solvent were simultaneously measured.49 In contrast, the kred value for the QM proton tunneling reaction catalyzed by methylamine dehydrogenase was not sensitive to the presence of 30% glycerol.50 In the case of the hydride transfer catalyzed by HsGOX, the kred values decreased in the presence of glycerol or sucrose. The solvent viscosity was changed 1.5−4.8 times by including these viscosigens in the reactions (η = 0.8−3.8 cP), while the solvent dielectric constant only decreased 1.1−1.2 times due to the presence of glycerol or sucrose (ε = 77−63). These data suggest that the observed decrease in kred for HsGOX is the result of the difference in solvent viscosity, rather than in solvent dielectric, between reactions that contain the additives and those that do not. Besides the study presented here, there are only a limited number of studies showing the effect of the solvent properties on hydride-transfer enzyme reactions. The reaction catalyzed by NADPH:protochlorophyllide oxidoreductase, besides QM proton tunneling, involves a viscosity independent QM hydride tunneling step.48 Similarly, the kred values for hydride transfer in morphinone reductase are

changes in solvent viscosity. The temperature-independent KIE observed for HsGOX suggest that this enzyme presents, at all assayed temperatures, fast femtosecond to picosecond fluctuations that populate short DADs at the TRS. The distribution of DADs at the TRS is not influenced by the solvent viscosity, since the D k red values are constant at all viscosigen concentrations assayed. Since a particle’s ability to tunnel through a barrier decreases with its increasing mass,2 larger KIEs than those expected from semiclassical theory are indicative of tunneling. However, a small Dkred value (2.0) was determined here for HsGOX. Previously, a small intrinsic D kred of 2.4 was calculated for plant GOX15 using a different approach, and small KIEs (