Influence of the Environment on the Oxidative Deamination of p

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Influence of the Environment on the Oxidative Deamination of p-Substituted Benzylamines in Monoamine Oxidase Roland K Zenn, Enrique Abad, and Johannes Kästner J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp512470a • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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The Journal of Physical Chemistry

Influence of the Environment on the Oxidative Deamination of p-Substituted Benzylamines in Monoamine Oxidase Roland K. Zenn,† Enrique Abad,∗,†,‡ and Johannes Kästner† Institute of Theoretical Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany E-mail: [email protected]

Phone: +49 2461 61 9560. Fax: +49 2461 61 4823

∗

To whom correspondence should be addressed Institute of Theoretical Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany ‡ Present Address: Computational Biophysics Group, German Research School for Simulation Sciences, Wilhelm-Johnen-Strasse, 52425 Jülich, Germany †

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Abstract The flavin-containing enzyme monoamine oxidase (MAO) is essential for the enzymatic decomposition of amine neurotransmitters. The exact mechanism of the oxidative deamination of amines to aldehydes by the enzyme has not yet been fully understood despite extensive research on the area. The rate limiting step is the reductive half reaction where the Hα together with two electrons of the amine substrate is transferred to the flavin cofactor. However, it is still not known whether the hydrogen is transferred as a proton or a hydride. Experimental results cannot be fully explained by either of those mechanisms. In our previous work, theoretical results based on QM/MM calculations of the full enzyme show an intermediate situation between these two cases. In this paper we report on an in-depth computational analysis concerning the role of the enzymatic environment for the reaction mechanism of human MAO-B with different psubstituted benzylamines as substrates. Our results show that steric and electrostatic effects from the active site environment turn the mechanism closer to an asynchronous polar nucleophilic mechanism. We found indications that the protein environment of MAO-A enhances the polar nucleophilic character of the mechanism compared to that of MAO-B. Keywords: Theoretical chemistry, QM/MM, MAO, reaction mechanism

INTRODUCTION The flavoenzyme monoamine oxidase (MAO, EC 1.4.3.4) is found at the outer membrane of mitochondria in various eukaryotic cells. Two different isozymes exist in mammals: 1 MAOA and MAO-B, with 70 % homology 2 but slightly different substrate specificities. 1,3,4 The isozymes show significant similarities in the spatial geometry and the primary structure of the active site. 5 MAOs contribute to the regulation of endo- and exogenous concentrations of amines in the brain and peripheral tissues. They catalyze the oxidative deamination of neurotransmit-

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ters such as adrenaline, noradrenaline, dopamine and serotonin. 3 For this reason they have been a pharmacological target in drug treatment of neurological diseases such as depression, Alzheimer’s and Parkinson’s disease for 60 years. 6–10 The pharmacological significance led to a lively interest by the scientific community, demonstrated by the large number of about 40,000 scientific papers about MAO that have been published since 1950. 11 The amine substrates are first oxidized to the corresponding imines. MAO uses a flavin adenine dinucleotide (FAD) cofactor, which is thereby reduced to FADH2 . Subsequently, the imine is hydrolyzed non-enzymatically to the corresponding aldehyde. 12,13 The re-oxidation of the cofactor, and thus the regeneration of the enzyme to its active form is accomplished by the reduction of molecular oxygen (O2 ) to hydrogen peroxide (H2 O2 ). The overall reaction is summarized in scheme 1. α H2 C R

NH2

α

FAD -FADH2

RHC

Amine

NH

H2O -NH3

RHC

Imine

O

Aldehyde

Scheme 1: Reaction scheme of the oxidative deamination catalyzed by MAO. Crystal structures of MAO-A and MAO-B have been solved by means of X-ray diffraction at refinements of 3.0 5,14 to 1.7 15 Å. The active site is known as the “aromatic cage”, a hydrophobic binding pocket in which the FAD cofactor is located. FAD is covalently bound to a cysteine residue of the protein via an 8α-thioether linkage (Scheme 2a). Catalytic activity of the two tyrosine residues Tyr398 and Tyr435 (notation of human MAO-B) has been suggested as they polarize the amine N lone pair of the substrate. 16 Enzyme

S 8a

7a

8

9 9a

R N

1 10a

10

5a

7

N 5

6

α-pro-R

N

2

O

4

NH

H

H α

4a

α

NH2

3

O

a)

b)

Scheme 2: Chemical structure of the flavin ring (a), and the substrate (benzylamine) (b). Because of the simple synthesis of analogues or isotopically labeled forms an often used 3



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model substrate in mechanistic studies was benzylamine (compound (b) in scheme 2). The catalytic mechanism is still not fully understood. It is generally accepted, however, that the rate-determining step of the oxidative deamination is the transfer of the pro-R Hα of the amine substrate to the N5 atom of the FAD cofactor. 17–19 Several different mechanisms have been proposed for the hydrogen transfer (see Scheme 3): a) A polar nucleophilic mechanism, where the benzylamine nitrogen Nam attacks the C4a atom of the flavin as a nucleophile to form a Nam -C4a-flavin-substrate adduct. The adduct then decomposes and the bonding electrons remain on flavin. The hydrogen is transferred as a proton concerted with either the formation or decomposition of the adduct; 19–21 b) A direct hydride transfer; 22–24 c) A two-step hydride transfer. 25 The first step consists of a hydride transfer concerted with the formation of a N5-Cα -flavin-substrate adduct. The second step involves the transfer of a proton from the iminium cation to N1 via two water molecules and the decomposition of the adduct. 22,25 d) Based on QM/MM calculations our group proposed a concerted asynchronous polar nucleophilic mechanism. 26 In the reactant state we found a bonding interaction between the nucleophilic Nam and the electrophilic C4a which can be represented through the two resonance structures shown in (d) of Scheme 3. This interaction facilitates the reductive half reaction in three respects: 1. It causes a small distance between C4a and Nam which enables the transfer of negative charge from the amine lone pair to flavin in the reactant state (during the reductive half-reaction in total two electrons need to be transferred), 2. the interaction increases the basicity of N5 (see resonance structure on the right hand side) which in return can abstract the pro-R Hα from the benzylamine more easily, 3. it leads to a correct positioning of the substrate close to the enzyme. Starting from this reactant state the hydrogen is then transferred concerted with the remaining negative 4


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charge from benzylamine to flavin. Due to the quantum nature of electrons it can not be solved in principle whether these electrons originate from the Nam lone pair (polar nucleophilic mechanism), or from the Cα -H bond (hydride mechanism). a)

R N

Enzyme

Enzyme N

R N

O NH

N

Enzyme

H

Ph Enzyme

R N

N

NH

N

N

b)

O NH

O

O

NH2

NH2

H Ph

H

H

Enzyme

R N

Enzyme

N

NH2 Ph C H

O NH

N

O H Ph

N H

H

R N

O

R N

O NH2

NH2

H Ph

O NH

N H

O

Enzyme

N

N

O NH

N

R N N H

O

N

O NH

NH2 + Ph C H

O

H H Ph

c) R N

Enzyme

N

NH2

Enzyme

O NH

N

H O

O H H

NH2

Ph

O H

R N

H

R N

NH

N

H Ph

O

O NH2 H

O H

Ph

Enzyme N

O NH2

H

R N

H O

H H

H

R N

O NH

O NH2

H Ph

H

O NH

N δ+

H N

O NH

H O H

O

N C H Ph

O H H

Enzyme N

δ−

O NH2

R N N H

Enzyme N

N

H Ph

Enzyme

O NH

N H

H

d) Enzyme

N

H

R N N H

N

O NH

O

NH2 Ph C H

Scheme 3: Proposed reaction mechanisms for MAO: a) polar nucleophilic mechanism, 21 b) direct hydride transfer, 22,24,27 c) two step hydride transfer, 22,25 d) the mechanism proposed by our group, an intermediate situation between a polar nucleophilic mechanism and a hydride transfer. 26 Previously, a radical mechanism, in which the hydrogen atom is transferred as a proton 5



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and the oxidation of the amine substrate is accomplished by two consecutive single electron transfers, 28 was proposed. However, experimental evidence (lack of electron paramagnetic resonance signal, 19,29 reduction potential 30 ), as well as theoretical results 26,31 ruled out this option. Higher resolution crystal structures also ruled out a previously proposed polar mechanism where a protein residue acts as a base for the proton abstraction. 32,33 Kinetic studies found large kinetic isotope effects (KIE): kH /kD = 11.5 ± 0.6 (steady state), 19 kH /kD = 9.3 ± 1.2 (stopped flow kinetics for the rate limiting step) in MAO-A at 10.9◦ C 19 and kH /kD = 4.7 ± 1.0 (steady state) at 25◦ C in MAO-B. 34 It was suggested that quantum tunneling of hydrogen atoms may play an important role in the enzyme activity. 35 Recent theoretical results by our group confirm that tunneling is indeed necessary to explain the large values of KIEs. 26

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N KIE measurements suggest that the amine-N rehybridization

and the H transfer are not concerted. 21 MAO has been studied by theoretical methods at different levels of accuracy. Energy barriers of p-substituted benzylamines obtained by means of gas-phase semiempirical PM3 calculations hint at a polar nucleophilic mechanism. 20 Other gas-phase calculations, where the active site was modeled at different levels of theory, claim against the biradical mechanism. 31 The effect of Tyr398 and Tyr435 was also studied at the HF/MM level. 36 The two-step hydride transfer has been proposed after gas-phase calculations at the DFT-M06 level of theory. 25 This study has been extended recently 37 to take into account the steric effects of the environment by means of the empirical valence bond method. However, their study focused on the energetics of the reaction, and not on the reaction mechanism. QM/QM calculations, with the active site treated by DFT-M06 and the surrounding residues by the semiempirical PM6 method are in agreement with the direct hydride mechanism. 22 Recently, our group performed QM/MM calculations where configurational sampling of the whole protein was taken into account. 26,38 Our results suggest a mechanism with a mixture of polar nucleophilic and hydride character. 26 Here, we will show how the balance between these two limiting cases depends on the protein environment of the active site.

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The ChemShell suite was used throughout our calculations. 39,40 It interfaces several QM and MM codes, controlling the information flux between the QM and the MM parts, and it can be used for energy minimization and transition state search thanks to the DL-FIND geometry optimizer. 41 The QM part was considered at the density functional theory (DFT) level. For geometry optimization, we used the BP86 exchange-correlation functional 42,43 with a def2-TZVP basis set 44,45 and the resolution of the identity. Those calculations were made with the turbomole code version 6.3. 46 It has been reported by some of us 47 that geometries obtained with the BP86 functional are in good agreement with the ones found with the B3LYP functional in enzymes. The difference of distances d(Cα −Hα )−d(Hα −N5) was used as the reaction coordinate in potential energy surface (PES) scans. Energies were obtained after constrained optimization. In order to find the transition state, the implementation of the dimer method 48 in DLFIND 49 was used. The two starting geometries for the transition state search were the two ones with the highest energies in the PES scan. Single point energy calculations at stationary points with the M06 functional 50 and the same basis set def2-TZVP were performed with the molpro code. 51 This protocol (geometries optimized with BP86 functional and single point energies calculated with M06 functional) has already been successfully applied in our group. 26,52 In order to analyze the chemistry of the reaction, we calculated NBO charges, 53 as implemented in turbomole, and IBO charges 54 as implemented in molpro. Both charge projection techniques give very similar results. The protein environment was studied at different levels of theory to investigate its influence on the reaction. In a first approximation we modeled the protein as a continuum with a dielectric constant of εr = 4 and employed the COnductor-like Screening MOdel (COSMO), as implemented in turbomole 6.3. To include atomic detail we used the CHARMM27 force field 55–61 in DL_POLY 62 for the protein and water, as in our previous study. 26 With respect to the interaction between the QM and the MM part, we considered both mechanic and electrostatic embedding. The difference between these schemes is that only in electro-

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static embedding the protein and water environment can polarize the QM part. Regarding the connection between the QM and the MM part, all the frontier bonds were C–C single bonds, where H link atoms were used to saturate the dangling bonds of the QM carbons. The charge shift scheme was used for the charges close to the frontier. A water molecule close to the flavin ring was included in the QM part. The molecules and protein residues closer than 8 Å to the active site were optimized, as in Ref. 26. In order to estimate reaction rate constants of the p-substituted benzylamines, we calculated a two point difference Hessian, considering all QM atoms, but not the water molecule. The latter is important for the electronic structure level, but not for the vibrations. The water can move almost freely. The low-frequency modes can not be treated as harmonic oscillators in the rate expression with satisfactory accuracy. We found only real frequencies in reactant geometries and a single imaginary frequency in transition states, which validates our calculations (see Supporting information). Reaction rates were calculated within the transition state theory and we considered the vibrational degrees of freedom as harmonic oscillators. The vibrational analysis was done at a BP86 level of theory, but the barrier at the M06-level was used. In our previous work 26 we showed the quantitative importance of quantum tunneling in determining the reaction rate of MAO-B, therefore we consider this effect in an approximate manner in our calculations. Our reaction path was fitted to a symmetric Eckart barrier, 63 as in our previous work, 26 since the quantum transmission for this barrier can be calculated analytically. A molecular dynamics (MD) run of benzylamine attached to MAO-A was carried out in order to understand the effect of the environment in the active site dynamics and compare it to MAO-B. The initial molecular geometry was taken from the crystal structure of human MAO-A with a reversible inhibitor (harmine hydrochloride) determined at a resolution of 2.2 Å (PDB entry 2Z5X). 64 The harmine molecule was replaced by a benzylamine molecule. The protocol for equilibration and MD using NAMD 65 and the CHARMM27 force field was equivalent to our previous work. 26

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RESULTS AND DISCUSSION As pointed out in the introduction, there is still some controversy about the reaction mechanism of MAO, both from the theoretical 20,22,25,26 and experimental 16,24,66 point of view. Theoretical and experimental findings don’t point conclusively towards a polar nucleophilic mechanism 19–21 or hydride mechanism (in one or two steps). 22,25,66 To shed light on this issue, we studied the effects of the environment on the geometries, charge distribution and energy barriers. QM/MM calculations of p-substituted benzylamines were carried out in order to rationalize published experimental data.

Effects of the environment We carried out calculations where the environment has been taken into account with different degrees of accuracy. In particular, we used: (1) the active site embedded in a continuum environment modeled via COSMO (see calculation details), (2) QM/MM calculations with mechanic embedding (i. e. where the electrostatic influence of the environment on the electronic structure of the active site is neglected) and (3) QM/MM with electrostatic embedding (as in our previous work 26 ). The results of the different methods will provide understanding of the effects caused by the environment. Table 1: Equilibrium distances and NBO charges in the reactant state for different approximations of the environment. Distances are in Å, charges in atomic units. COSMO, flat flavin COSMO, bent flavin QM/MM (mech. embed.) QM/MM (electros. embed.)

1

d(Nam –C4a) (Å) 2.93 2.72 2.60 2.46

charge N5 charge C4a charge amino1 −0.32 +0.09 −0.06 −0.34 +0.11 −0.03 −0.37 +0.13 −0.03 −0.44 +0.11 +0.15

Sum of charges of Nam and its two bonded H

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The flavin geometry is planar in a continuum environment, as found in other proteins or in solution. 67,68 However, QM/MM calculations on MAO show a bent flavin, as found in the crystal structure. 14,15 We checked the effect of the flavin bending by considering both the flat and bent flavin in the COSMO calculations. In order to keep the flavin bent, the flavin atoms remained fixed during the geometry optimization. Bent flavin geometry was taken from QM/MM calculations with electrostatic embedding. 100 COSMO COSMO (bent flavin) QM/MM (Mech. embed.) QM/MM (Electros. embed.)

80 -1

Energy (kJ mol )

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60 40 20 0 1.5

2.0

2.5

3

3.5

d(Nam-C4a) (Å)

Figure 2: PES along the Nam –C4a distance for the reactant with the environment modeled at different levels of theory. Arrows indicate the positions of the respective minima. The distance of the nucleophilic Nam (benzylamine) and the electrophilic C4a (flavin) in the reactant state indicates the character of the reaction mechanism. The polar nucleophilic mechanism relies on a short Nam –C4a distance, in order to transfer the amine lone pair to the flavin ring. The Nam –C4a distance of the reactant state varies for different models of the environment. Results can be seen in Figure 2 and in Table 1. The weakest interaction between Nam and C4a is found in the COSMO calculations, where the flavin is flat. We get a Nam –C4a distance of 2.93 Å, in good agreement with previous calculations. 22 Forcing the flavin to bend in a COSMO description of the environment reduces the Nam –C4a distance to 2.72 Å. This can be rationalized the following way: when flavin is bent, the configuration of the N5 atom is closer to sp3 than to sp2 . As a consequence, there is somewhat more electron density at N5 and less at C4a, which results in a stronger interaction between the nucleophilic Nam and the electrophilic C4a (see Table 1). Explicit inclusion of the environment via the 11



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QM/MM approach increases the electron density at N5 even more and reduces it at C4a (the effect is larger with electrostatic embedding than with mechanic embedding). Thus, the interaction between the nucleophilic amine lone pair and the electrophilic C4a is enhanced when the protein environment is taken explicitly into account (Nam –C4a distance is 0.33 Å smaller for mechanic embedding and 0.47 Å smaller for electrostatic embedding, than in flat flavin with COSMO). The protein favors the right hand side resonance structure shown in Scheme 3d. The negative charge on N5 is significantly higher when all environmental effects are taken into account (see Table 1). A higher electron density at N5 should facilitate the abstraction of the Hα . On the other hand, the positive charge of the amino group of benzylamine increases with more environmental effects taken into account. The amino group of an isolated benzylamine molecule has a negative charge (−0.10 e). By contrast, it is positively charged (+0.15 e) in the enzyme (QM/MM, electrostatic embedding). These results confirm the existence of an Nam –C4a interaction which can be represented by the resonance structure on the right side in Scheme 3d, in which the amino group carries a positive charge and the N5 a negative charge. In order to characterize the Nam –C4a interaction, we calculated intrinsic bond orbitals following the scheme proposed by Knizia. 54 We found a bonding orbital connecting the Nam and the C4a atoms (see Figure 3). This confirms that there is a Lewis acid-base interaction between these atoms, and that this is a possible path for the amine lone pair to be transferred to C4a. In all environments, reducing the Nam –C4a distance leads to a monotonic increase in energy. This rules out a pure polar nucleophilic mechanism in which an Nam -C4a-flavinsubstrate adduct needs to be formed. In order to check how the Nam –C4a distance influences the transfer of charge from benzylamine to flavin we calculated the charge of benzylamine for different Nam –C4a distances. The results are shown in Figure 4 for the different degrees of approximation of the environment. Our results clearly show that charge transfer between benzylamine and flavin increases when the Nam –C4a distance decreases. The charge/distance

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been suggested previously, with the main contribution expected from the dipole interaction with Tyr398 and Tyr435. 16 However, when we artificially remove the electrostatic interaction with the side chains of these residues, the charge in benzylamine and flavin hardly changes. Although the electrostatic interaction with the environment does contribute to the transfer of the amine lone pair, the contribution of the tyrosines from the aromatic cage is negligible. 1.4 Charge of benzylamine (e)

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1.2

COSMO QM/MM (Mech. embed.) QM/MM (Electros. embed.) Pure hydride transfer

1.0

TS

TS

0.8

TS

TS

0.6 0.4 0.2 0 -1.5

RS RSRS -1

-0.5 0 0.5 1.0 d(Cα-Hα)-d(Hα-N5) (Å)

1.5

2.0

Figure 5: NBO charges of benzylamine along the reaction coordinate d(Cα –Hα ) − d(Hα – N5) for different approximations of the environment. Orange: pure hydride mechanism, as obtained from the snapshot 3 of reference 26. Curves start in the reactant (for hydride mechanism, d(Cα –Hα ) − d(Hα –N5) = −4.49 Å). Arrows indicate the transition state. The charge distribution during Hα abstraction may help to differentiate between the hydride and the polar nucleophilic mechanism. In a pure hydride transfer, where the two electrons are transferred together with Hα , the charge transfer between benzylamine and flavin is intimately related with the H transfer. The charge of benzylamine should be zero on the reactants and increase to one during the H transfer. In a polar nucleophilic mechanism, since the electrons and the proton follow different paths, the electron and proton transfer may be asynchronous. Benzylamine NBO charges (Hα has been considered as part of benzylamine throughout all the PES) for the different approximations of the environment are shown in Figure 5. Since a frozen flavin cannot adapt its geometry during the H transfer, this case was not considered here. It can be seen that some charge has already been transferred in the reactant state (before Hα is abstracted); as much as 0.31 electrons in the case of QM/MM calculations with electrostatic embedding. In order to compare this with a pure hydride 14


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transfer, we also plot the results obtained in our previous work 26 for snapshot 3. In snapshot 3 the geometric arrangement with a great Nam –C4a distance inhibited a charge transfer via the Nam –C4a interaction. It is clear that in the most accurate description (QM/MM with electrostatic embedding) a significant amount of charge is transferred already prior to the proton transfer, demonstrating a partially nucleophilic character of the mechanism. Regarding the energetics, the energy barrier for the full QM/MM electrostatic embedding is the smallest one (see Table 2). The barrier reduction by the environment cannot be properly recovered by a dielectric continuum. This is, again, coherent with the proposal of Li et al. 16 that the protein environment polarizes the active site, thus decreasing the energy barrier. Table 2: Energy barriers for Hα abstraction. ∆E ‡ (kJ mol−1 ) COSMO, flat flavin 98.4 QM/MM (mechanic embedding) 151.8 QM/MM (electrostatic embedding) 75.5

These results show that the reaction mechanism has, at least partly, a polar nucleophilic character. The hydride character becomes more important as the Nam –C4a distance increases. Previous calculations found a strong hydride character of the reaction, 22,25 but this could be related to the fact that the protein environment was treated with implicit solvent there, causing larger Nam –C4a distances.

Effects of the para-substituents Usually, physical organic chemists change the para-substitution of aromatic substrate analogues in order to distinguish between hydride and proton transfer mechanisms, by determining the effect of electron withdrawing or donation on the reaction rates. However, in enzyme reactions the picture is not so simple, since steric and hydrophobic effects can override the 15



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electronic effect caused by a substituent. There have been several experimental studies with p-substituted benzylamines on MAO. 16,19,66 In order to rationalize these results, we have modeled the effect of p-substituents on the reaction, by using QM/MM with electrostatic embedding. In particular, p-OH (electron donating group) and p-NO2 (electron withdrawing group) were used. Our geometries for p-NO2 benzylamine are compatible with the solved X-ray structure at 2.0 Å resolution —PDB entry 2C70— (see next section). In Table 3 we show the energy barriers and the calculated reaction rates. Table 3: Experimental vs. calculated reaction rate constants of the reductive half reaction. Substituent p-H p-OH p-NO2

kred (min−1 ) ∆E ‡ d(Nam –C4a) 69 −1 (Exp.) (Theory) (kJ mol ) (Å) 760 ± 2 35 75.5 2.46 117 ± 3 2 82.0 2.48 3.8 ± 0.2 0.01 94.3 2.49

Our results qualitatively agree with the experimental results of bovine MAO-B in terms of the relative changes. 69 Surprisingly, an electron donating group such as p-OH and an electron withdrawing group such as p-NO2 both greatly reduce the reaction rate. In the case of a pure polar nucleophilic mechanism, one would expect a higher reaction rate for pNO2 -benzylamine and a smaller reaction rate for p-OH-benzylamine, since the p-NO2 group would stabilize and the p-OH group would destabilize the negative charge at Cα . For the hydride transfer, the influence of the substituents on the reaction rates should be reversed. The fact that both substituents reduce the reaction rate indicates that the mechanism in MAO is neither a text-book hydride transfer nor a text-book polar nucleophilic mechanism. Our results suggest that the reaction rate is mainly influenced by the Nam –C4a distance and not by electronic effects of the substituents. As we found earlier, the Nam –C4a distance determines the Nam –C4a interaction and the amount of charge already transferred from benzylamine to flavin in the reactant state. Since both substituents, p-NO2 and p-OH, 16


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cause a greater Nam –C4a distance (see Table 3) compared to p-H it is not surprising that they both reduce the reaction rate. Thus, the experimental results can be explained with the concerted asynchronous polar nucleophilic mechanism. The increased Nam –C4a distance for p-NO2 -benzylamine and p-OH-benzylamine could be due to steric and/or hydrophobic effects.

Different mechanisms in MAO-A and MAO-B? The results shown so far point out that the reaction mechanism of MAO has some characteristics from a polar nucleophilic mechanism and some characteristics from a hydride mechanism. Recently, it was proposed that MAO-A and MAO-B could have different mechanisms: 66 MAO-A would have a polar nucleophilic mechanism and MAO-B a hydride mechanism. As we saw, the environment somehow modulates the degree of polar nucleophilic or hydride character of the reaction. So the environment of the active site could differentiate between the polar nucleophilic or hydride character, respectively.

2000 0.14 Å

MAO-A MAO-B

1500 Counts

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1000

500

2.5

3

3.5 4 d(C4a-Nam) (Å)

4.5

5

Figure 6: Distribution of the Nam –C4a distance from MD simulations of MAO-A and MAOB. The arrows indicate the mean distance.

17



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To investigate this claim, we performed classical MD simulations for both systems with the same force field setup. The distribution of d(Nam –C4a) obtained by the MD is shown in Figure 6. On average, the nucleophile Nam and electrophile C4a are closer by 0.14 Å in MAO-A than in MAO-B. Figure 6 also show that small values of d(Nam –C4a) are more likely also on MAO-A than on MAO-B. Since these geometries are more representative in reaction calculations, 38 our results point towards a MAO-A environment decreasing the Nam –C4a distance, and thus enhancing the polar nucleophilic character.

CONCLUSIONS In this paper we have demonstrated the influence of the enzymatic environment on the reaction mechanism of MAO-B. In our previous work, we concluded that the reaction mechanism is an intermediate situation between a polar nucleophilic mechanism and a hydride mechanism. In this work we have shown how the environment polarizes the Nam –C4a interaction, and decreases the distance between the two atoms, thus enhancing the degree of polar nucleophilic character of the reaction. MD calculations on MAO-A suggest that this effect can be even more important in MAO-A. This is in line with recent experimental results. 66 We have also analyzed the apparently contradictory experimental studies of p-substituted benzylamines in MAO-B. Our calculations confirm that both electron donating and withdrawing substitutions reduce the reaction rate. This indicates that the mechanism does not follow the textbook examples of hydride or polar nucleophilic mechanisms. In our calculations both substitutions caused an increased Nam –C4a distance, which lead to a decreased Nam –C4a interaction and a smaller charge transfer from benzylamine to flavin in the reactant state. This suggests that the reaction rate is significantly influenced by the Nam –C4a interaction and that the electronic effects of the substituents play only a minor role. This confirms our recently proposed mechanism. Comparison of the dynamics of MAO-B and MAO-A indicates that the mechanism in MAO-A is closer to an asynchronous polar nucleophilic

18


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mechanism than the one in MAO-B.

Supporting information Optimized reactant state geometries (in pdb format), frequency of vibrational modes, vectors of the imaginary-frequency mode and partial charges at important atoms in the PES scans for p-H benzylamine at several degrees of approximation of the environment (cosmo, QM/MM mechanic embedding, QM/MM electrostatic embedding) and for p-H, p-OH and p-NO2 benzylamine at the QM/MM electrostatic embedding level, charge density difference on the reactant of benzylamine between QM/MM calculations with mechanic and electrostatic embedding. This material is available free of charge via the Internet at http://pubs.acs.org/

Acknowledgments This work was financially supported by the Alexander von Humboldt foundation as well as by the Baden-Württemberg Stiftung.

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α 29 of 38 FAD H2O Page The Journal ofαPhysical Chemistry H2C NH2 RHCPlus Environment NH ACS Paragon -FADH2 -NH3 R 1Amine

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100

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80

-1

Energy (kJ mol )

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60 40 20 0 1.5

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0.8

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Charge of benzylamine (e)

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COSMO COSMO (bent flavin) QM/MM (Mech. embed.) QM/MM (Electros. embed.)

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Charge of benzylamine (e)

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Influence of the Environment on the Oxidative Deamination of p

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