Enolization as an Alternative Proton Delivery Pathway in Human Aromatase (P450 19A1)

Enolization as an Alternative Proton Delivery Pathway in HumanAromatase (P450 19A1)Balazs Kramos and Julianna Olah*

Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gellert ter 4, H-1111Budapest, Hungary

*S Supporting Information

ABSTRACT: Human aromatase catalyzes the last step ofestrogen biosynthesis, the aromatization of ring A ofandrostenedione (ASD) and testosterone leading to estroneand estradiol. The enolization of the substrate molecule has beensuggested to play an essential role in this process. In this workusing quantum mechanical and hybrid QM/MM calculations, thereaction mechanism of enolization was investigated. It is shownthat the energetically unfavorable enolization of andostenedioneoccurs in a coupled process with the energetically favorableprotonation of the ferrous superoxo complex (traditionally called ferric peroxo complex) via a low barrier of about 5 kcal/mol.This mechanism implies an alternative way for protonation of the ferrous superoxo complex to form compound 0, which occursvia the Asp309−water−ASD proton delivery pathway instead of the Asp−water−Thr pathway suggested for other P450 enzymes.It is also shown that Thr310, which is known experimentally to be important for catalysis, plays a key role in the conversion ofcompound 0 to compound I.

1. INTRODUCTION

Human aromatase (P450 19A1) is a hemoprotein belonging tothe superfamily of cytochrome P450 enzymes (P450s). Theactive site of P450 enzymes contains an iron ion which istethered to the protein via a thiolate ligand derived from acysteine residue. Human aromatase plays a key role in theregulation of sex steroids in the human body1 as it isresponsible for the last and rate-limiting step of estrogenbiosynthesis. It converts androstenedione (ASD) and testoster-one to estrone and estradiol, respectively, by aromatizing ringA2,3 (Scheme 1). As estrogens are known to play a major role inthe development of hormone-dependent cancers, e.g., breastcancer, aromatase has become a particularly attractive target fortheir treatment4−6 and several aromatase inhibitors (anastro-zole, exemestane, letrozole) are already on the market.Understanding the catalytic mechanism of aromatase couldcontribute to the development of newer and more efficaciousdrugs to prevent estrogen production in patients suffering frombreast cancer.The conversion catalyzed by aromatase consists of three

catalytic subcycles. In the first and second oxidation stepshydroxylation of the 19-methyl group occurs most likely by theclassical P450-mediated hydrogen atom abstraction−hydroxylradical rebound mechanism originally proposed by Groves andco-workers.7 In the second subcycle a 19-gem-diol is generated,which may dehydrate to the 19-aldehyde.8,9 In the thirdsubcycle the oxidative cleavage of the C10−C19 bond occurs andthe aromatized steroid and one molecule of formic acid aregenerated. Several mechanisms including 2β-hydroxyla-tion,10−12 4,5-epoxidation,13,14 Baeyer−Villiger oxidation of

C19, and 10β-hydroxyestr-4-ene-3,17-dione formation,15 havebeen put forward to explain this mysterious step; however, allof them were proven experimentally to be unlikely.3 Althoughseveral of these intermediates are known to be convertedspontaneously10 or by aromatase16 into estrone, none of themis currently accepted as lying upon the major pathway foraromatization primarily, because they contradict the available18O labeling studies which indicate that the third oxygen atomis incorporated into formate.3,15 According to the mostaccepted mechanism a peroxohemiacetal is formed after thenucleophilic attack of the ferrous superoxo complex on the C19

carbon,15 but several unanswered questions remain inconnection with this reaction. It is experimentally proven thatthe 1β- and 2β-hydrogen atoms are eliminated selectively.17,18

Furthermore, based on model reaction studies the formation ofthe 2,3-enol is a prerequisite for aromatization,19,20 as theenergy barrier for 2β-hydrogen abstraction is significantly lowerin the 2,3-enol form because of the increased stability of theresulting intermediate.21 The stereoselectivity of the 2β-hydrogen atom removal indicates that the enzyme mediatesthe enolization process.22 Before the crystallization of humanaromatase, homology modeling methods were used to studythe enolization reaction and the roles of Asp309 as protonacceptor and Lys473 as proton donor were emphasized.23

However, based on the crystal structure of aromatase (PDB ID,3EQM),24 the position of these side chains does not allow them

Received: July 24, 2013Revised: December 19, 2013Published: December 26, 2013

Article

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© 2013 American Chemical Society 390 dx.doi.org/10.1021/jp407365x | J. Phys. Chem. B 2014, 118, 390−405

pubs.acs.org/JPCB

to facilitate the conversion. Therefore, Ghosh and co-workerssuggested that Asp309 may be the proton donor in theenolization reaction and a network, consisting of the backboneoxygen of Ala306, the side chain of the highly conserved Thr310,and a water molecule, may serve as proton acceptor.24 In arecent quantum mechanics/molecular mechanics (QM/MM)

study, the mysterious third catalytic subcycle was thoroughlyinvestigated.25 According to this multistep mechanism first aperoxo hemiacetal is formed by the nucleophilic attack of theferrous superoxo complex on the formyl group of the substrate.The enolization occurs in the final step after the fission of theC10−C19 bond and the elimination of the 1β-hydrogen. The

Scheme 1. Conversion of Androstenedione to Estrone Catalyzed by Aromatase

Figure 1. Consensus catalytic cycle of P450s. The two protonation steps are highlighted and the electron configurations of the states involved areshown.

The Journal of Physical Chemistry B Article

dx.doi.org/10.1021/jp407365x | J. Phys. Chem. B 2014, 118, 390−405391

enolization is catalyzed by the ferric hydroxy complex as protonacceptor and the Asp309 as donor. According to their results theperoxo hemiacetal is formed in a barrierless process, whichcould suggest the elusive nature of the ferrous superoxocomplex. In an experimental study the reduction of the oxyferrous complex was carried out with γ-irradiation at 77 K, andthe intermediates were studied with EPR spectroscopy, whichsuggested the formation of ferrous superoxo complex bycryoreduction.26 In contrast, in the case of P450cam theprotonation of ferrous superoxo complex occurred even at 77K.27 These experiments suggest indirectly the increased stabilityof ferrous superoxo complex in aromatase. The seemingcontradiction between two different conclusions mightoriginate from the fact that the first catalytic subcycle wasinvestigated experimentally and the third one was investigatedtheoretically. However, these EPR spectral measurementsindicate that the protonation of the ferrous superoxo complexis more hindered in aromatase than in most other P450s. Webelieve that it is worth considering other possible pathways forthe third subcycle besides peroxo hemiacetal formation, and toinvestigate the role of Thr310 especially in this subcycle usingsite-directed mutagenesis, since it is not essential in themechanism suggested by Sen et al.25 but it has a crucial role incompound I formation mentioned below.A closer look at the consensus catalytic cycle of P450s drew

our attention to the two protonation steps observed in thecycle: first the ferrous superoxo complex (traditionally calledferric peroxo complex) is protonated to form the ferrichydroperoxo complex (compound 0, Cpd 0), which in asecond protonation step could be converted to compound I(Cpd I, see Figure 1). As these protonation steps have tohappen during the catalytic cycle, the idea occurred to uswhether any of these two species (the ferrous superoxocomplex or Cpd 0) may serve as the proton acceptor in theenolization process, while an acidic amino acid side chain wouldact as proton donor.28

The electronic structure of the ferrous superoxo complex hasbeen studied both theoretically and experimentally, and thedoublet state was identified as its electronic ground state29,30

with an unpaired electron on the π*O−O orbital shown in Figure1. We note that based on the electronic structure the name of“ferrous superoxo complex” is correct but the name of “ferricperoxo complex” is commonly used in the literature for thisstate.29,30 In the consensus catalytic cycle the ferrous superoxocomplex becomes protonated on the distal oxygen and isconverted to Cpd 0. After a second proton shift and eliminationof a water molecule the ultimate active species Cpd I can beobtained, which has low-lying doublet and a quartet states closein energy (see Figure 1).29,30 The protonation of the ferroussuperoxo complex in P450eryF enzyme was studied with densityfunctional theory (DFT), MM, and DFT(ROB3LYP)/MMtechniques. Based on the calculated proton affinity of thiscomplex, its very strong basic character was concluded. In theproposed model Ser246 was able to protonate the ferroussuperoxo complex via two water molecules in an exothermicprocess (−10.7 kcal/mol).31−33 Others found that in P450camthe acidic Asp251 and Glu366 may be more suitable protonsources,29,34 and Guallar’s and Friesner’s DFT(ROB3LYP)/MM calculations confirmed the presence of a proton deliverypathway from the Glu366 to the ferrous superoxo complexthrough a low energy barrier of about 4.0 kcal/mol.35

However, later experimental works showed that the mutationof Glu366 did not have a significant effect on the activity of the

P450cam enzyme, but that Asp251 could be essential.36 Thehighly increased kinetic solvent isotope effect (KSIE) value inthe D251N mutant enzyme (wild type, 1.7; D251N, 10)37

suggested a different proton delivery pathway in the mutant,which should be longer and mediated by more water molecules.It was also hypothesized that Asp251 might work as a gate forthe protons between bulk water and the active site. The newestresults suggest that Asp251, which is hydrogen bonding toArg186, can move out from this position through a low energybarrier (<4 kcal/mol)38,39 and after rotation around its Cα−Cβ

bond by about 40° can protonate the ferrous superoxo complexvia a water molecule and the highly conserved Thr252 residue.The calculated barriers were about 2.5 kcal/mol, and thereaction was highly exothermic (−33.6 kcal/mol).40 In humanaromatase site-directed mutagenesis studies suggest a similarlyhigh importance of Asp309, which is analogous to Asp251 inP450cam.

41

Cpd 0 is less basic than the ferrous superoxo complex.31 Thequestion of the protonation of the ferrous superoxo complexseems to be settled by now, but there are still numerouscontroversial theories regarding the protonation of Cpd 0 in theliterature. The existence of protonated Cpd 0 and the extent ofthe exothermicity of the formation of Cpd I have been muchdebated.30 The homolysis of the O−O bond beforeprotonation leading to the formation of Cpd II and a hydroxylradical was also considered.42 Cpd II is the one-electron-reduced form of Cpd I with a filled a2u orbital and a tripletground state. On the basis of the most recent results derivedfrom theoretical investigation of P450cam, protonated Cpd 0does not exist, and Cpd I is generated in the course of theproton-coupled electron transfer (PCET) process. In the PCETprocess the homolysis of the O−O bond is followed by anelectron transfer from the porphyrin a2u orbital to the departinghydroxyl radical, and a proton shift occurs from Asp251 via asingle water molecule and Thr252 in a simultaneous process.39

Experimental studies showed that the mutation of the Thr310,which is analogous with the Thr252 in the P450cam, causessimilar changes to the catalytic activity.43 Davydov and co-workers investigated experimentally the role of the highlyconserved Thr252 in P450cam with EPR/ENDOR measure-ments. In the T252A mutant instead of camphor hydroxylationH2O2 formation was observed via Cpd 0, implying that thisresidue is essential especially in the second protonation step,but other pathways may exist for the first one.27

In our group we have been studying various aspects of P450chemistry, e.g., the properties of Cpd I,44 the ultimate oxidizingagent of P450s, and the factors affecting the regioselectivity ofP450s.45 In this work we studied the enolization reaction ofandrostenedione in the active site of human aromatase usingquantum chemical and QM/MM calculations. We investigatedthe possibility whether actors of the consensus catalytic cycle ofP450s, more specifically, the ferrous superoxo complex andCpd 0, could serve as a base to catalyze the enolization process,and we sought to identify the most likely proton source servingas acid in the reaction. In order to get a thoroughunderstanding of the process, first we studied small gas-phasemodels to get insight into the factors that influence theenolization process. In the second part of the study, weperformed QM/MM calculations on the X-ray structure ofaromatase using various models to determine the reactionmechanism and assess the viability of our hypothesis thatenolization of andostenedione can occur in a coupled processwith the protonation of the ferrous superoxo complex. In our



model the 2β-hydrogen of the substrate is shifted to the ferroussuperoxo complex as a proton and the oxo group is protonatedby the conserved Asp309 residue directly or via a watermolecule. We also compared two mechanisms for theprotonation of the ferrous superoxo complex leading to Cpd0: (1) the enolization reaction and (2) the mechanism (protontransfer chain from an aspartic acid via a water molecule and aThr residue) proposed in the consensus catalytic cycle ofP450cam. Finally, we tested the reliability of our results bystudying the effect of the applied DFT functional and comparedthe g tensor values calculated for the complexes obtained in thisstudied to the experimentally determined values.

2. METHODS2.1. QM Calculations. In order to get insight into the

electronic structure of the studied systems and to identify thefactors influencing the enolization reactions, first small modelsystems were studied. An unsubstituted porphine ring was usedinstead of a porphyrin IX ring, as it was earlier shown to be agood model of it.46 The axial Cys437 ligand was represented byan SH− group, which performs better in vacuum calculationsthan the bigger SMe− group.29,30 Only ring A of the substratemolecule was modeled with the C19 carbon attached to it,which belonged to either a methyl (ASDQM), a hydroxymethyl(ALCQM), or a formyl group (ALDQM). The calculations wereperformed with the Gaussian 09 program package47 with theB3LYP hybrid functional48 using an unrestricted formalism,which has become the general method for the modeling of theactive site of P450s, and is known to give correct spin stateenergetics for the studied species.29,30 Altun et al. investigatedthe Cpd 0, the Cpd I, and the hydroxo intermediate, and theyshowed that DFT(B3LYP)/MM calculations reproduce therelative energies from correlated ab initio QM/MM treatmentsquite well, except for the splitting of the lowest A1u−A2u radicalstates in Cpd I.49 The ferrous superoxo complex and Cpd 0were studied in the experimentally established doublet groundstate.29,30 For geometry optimizations the SDD basis set andSDD effective core potential were used for Fe in conjunctionwith the 6-31G* basis set for other atoms similarly to otherstudies.50 This basis set composition will be called “B1”. Theidentities of the located stationary points were checked usingsecond derivative calculations, minima were characterized byonly positive frequencies, and transition states were charac-terized by a single imaginary frequency. Zero-point-energycorrection was also calculated at this level. The stability of allwave functions was checked with the stable=opt option ofGaussian 09. In order to obtain more reliable energies, single-point calculations were carried out on the obtained geometriesat the B3LYP/6-311+G* and B3LYP/cc-pVTZ levels and theempirical dispersion correction was calculated with the DFT-D3 program51 developed by Grimme and co-workers. Naturalcharges and spin densities were calculated with the NBO 5.9program.52

2.2. System Setup: Molecular Mechanics (MM)calculations and Molecular Dynamics (MD) Simulations.The crystal structure of human aromatase in complexandrostenedione (PDB ID, 3EQM)24 was used as a startingstructure. The PROPKA53−56 and H++57−59 programs, whichuse empirical parameters and functions to evaluate the pKavalues of ionizable amino acid side chains, were employed todetermine the protonation state of the enzyme. PROPKAestimated the pKa value of Asp309 to be 7.69, indicating its weakacidic character in the active site; for this reason, it was

originally protonated. His480 was doubly protonated, and allother histidines were protonated in the ε position. Hydrogenatoms were added according to the standard CHARMMprotocol and their positions optimized. Residues farther than25 Å from the iron were removed. Charged side chains wereneutralized using “patch” residues in the region of 21−25 Åaway from the iron center in order to avoid the unrealisticeffects of truncation. The structure was solvated within a 60 Åbox with 8000 pre-equilibrated water molecules, represented bythe TIP3P model,60 centered on the heme iron. The watermolecules containing oxygen atom within the 2.8 Å area ofother non-hydrogen atoms or being more than 25 Å from theheme iron were removed. The total electric charge of oursystem was 4 electrons in the molecular dynamics (MD)simulations, and no counterions were used to neutralize thesystem. The presence of nonzero charge on the system in MDsimulation with stochastic boundary conditions is much lessproblematic than in the case of simulations with periodicboundary conditions. At the same time the way of position ofcounterions can have a large artificial effect on QM/MMbarriers, so in some cases the counterions are not used.44,40

Furthermore, an extended study on the relationship betweenneutralizing the charge of the system and reaction barriers inthe case of the condensation reaction in β-ketoacyl-ACPsynthase has showed that no neutralization leads to a loweractivation energy and for QM/MM calculations it may notalways be preferred to neutralize the charge of the system.61

The added water was then equilibrated by stochastic boundaryMD at 310 K over 20 ps with respect to the substrate boundenzyme structure and minimized. In the buffer region (between21 and 25 Å from the center), increasing restraints were appliedto restrain the movement of heavy atoms around theircrystallographic positions. All atoms were minimized, followedby stochastic boundary MD simulation of the whole systemwhich was heated to 310 K over 60 ps. Subsequent MDequilibrations at 310 K were carried out over 2000 ps.Stochastic boundary MD simulations were run on a systemwith rotated Asp309 for the C1

QM/MM models (see section 3.2)in the same way. Atoms within 20 Å of the heme iron weretreated with full Newtonian dynamics, and the remainder weretreated with Langevin dynamics. The water atoms were keptwithin 25 Å of the heme iron by the application of a deformableboundary potential (wat25.pot file in CHARMM). Frictioncoefficients used in the Langevin dynamics were 62 ps−1 forwater oxygen atoms and 250 ps−1 for protein heavy atoms. Thetopology file of ASD and nonstandard parameters are given inthe Supporting Information. We did not find any good forcefield parameters for the ferrous superoxo complex, and theparameters used for the MD simulations of the ferroussuperoxo complex led to unrealistic distortion of the Fe−O−O moiety. This is why we decided to use the Cpd I state forMD simulations, for which reliable MM parameters exist,62 andcomplete the selected structures to ferrous superoxo complexbefore QM/MM optimization. As the ligand and the porphyrinring are rigid structures, most likely this approach did not leadto false results. However, a critical question remained inconnection with the MD simulations: the hydrogen bondinteraction of Thr310 residue with the superoxo complex couldnot be studied using the Cpd I state. For this reason weinvestigated the role of the Thr310 residue separately in theC3

QM/MM model system. For all simulations the CHARMM27force field63 and the CHARMM software package64 were used.We did not find any unrealistic distortion of the active site of



the enzyme in the course of the MD simulation. (SupportingInformation, Figure S1).2.3. QM/MM Calculations. Starting structures for QM/

MM calculations were selected from the MD trajectories basedon geometric criteria described under Results and Discussion.In some cases the structures were modified before the QM/MM optimization. The water molecule belonging to the QMregion was manually positioned between the Asp309 and the oxooxygen of the substrate in the C2

QM/MM, C3QM/MM, and FQM/MM

models and between Asp309 and the Thr310 in the GQM/MMmodel. In order to test various hypotheses regarding themechanism of enolization, various models of the QM regionwere designed, which will be described in detail in section 3.2.The QM region in all cases contained the unsubstitutedporphine ring, the whole substrate and various amino acid sidechains depending on the model (indicated in the heads ofTable S6 and Table S7 in the Supporting Information), and/ora water molecule. The Asp309, Thr310, and Cys437 residues wererepresented by their side chain broken between their Cα and Cβ

atoms. Hydrogen-type link atoms were used to cap the openvalences of QM atoms. The charges of MM atoms (and thegroups they belong to) bonded to QM atoms were set to zeroto avoid unphysical effects due to the strong polarization of theQM wave function due to the proximity of large point charges.The atoms further than 20 Å from the iron center were fixed.The geometry of the QM region was optimized at theUB3LYP/B1 level, and single-point calculations were carriedout in minima and transition states at the UB3LYP/6-311+G*level. The MM region was described with a CHARMM27 forcefield.63 For these simulations the QoMMMa 8.02 program65

was used, which uses the Gaussian 09 program65 for describingthe QM region and the TINKER program66,67 for the MM

region. As dispersion effects are important in enzymaticreactions,68−71 empirical dispersion correction of energy andforces were applied in each optimization steps using DFT-D2parameters as implemented in the QoMMMa 8.02 program.72

Unless otherwise stated, three parallel energy profiles weregenerated by adiabatic mapping using the difference betweenthe breaking and forming bond lengths as reaction coordinateusing a step size of 0.1 Å. Minima were optimized withoutconstraints, and the maxima of the energy profiles wereconsidered as transition states. NBO charges and spin densitieswere calculated with the NBO5.9 program.52 The MOLDEN73

and VMD74 programs were used for visualization.2.4. Additional Calculations. We carried out single-point

calculations on the CQM, C2QM/MM (profile III), and GQM/MM

(profile II) model systems in order to check the dependence ofthe calculated relative energies on the applied DFT functional.In these calculation the M06,75 the TPSSh,76 the OLYP,77 andthe long-range and dispersion corrected ωB97X-D78 functionalswere used, in conjunction with the cc-pVTZ basis set in thecase of CQM and the 6-311+G* basis set in the other cases. Firstwe calculated EPR parameters (g tensor), as implemented inGaussian,79,80 for two simple gas-phase models (ferroussuperoxo complex and Cpd 0) at the B3LYP/cc-pVTZ level,and then for the reactant and product states of all profiles ofour best model (C2

QM/MM) at the B3LYP/6-311+G* level inthe presence of point charges. In these calculations the “NMR”,“prop=EPR”, and “integral=(grid=ultrafine)” options were usedin Gaussian 09.47 The calculated relative g shifts were convertedfrom parts per million to absolute g values, for instance, for thezz component with eq 1, where ge is the g value of the freeelectron (2.002 319 3):

Figure 2. Transition states of model systems for QM-only calculations (carbon, brown; oxygen, red; iron, yellow; nitrogen, blue; hydrogen, white;sulfur, lighter brown.



= +g gg

g10zz zz(ppm)

e6 e (1)

The protein environment was taken into account as pointcharges with the “charge” option in these single-pointcalculations. In the case of C2

QM/MM (profile III), we carriedout further calculations with an extended QM region, where theside chains of the heme residue were included in the QMregion in order to check the effect of porphyrin side chains forcalculated EPR parameters.

3. RESULTS AND DISCUSSION3.1. QM calculations. In order to assess the feasibility of

our hypothesis that the enolization of androstenedione couldbe catalyzed by the ferrous superoxo species of aromatase, firstsmall model systems (shown in Figure 2) were designed to getsome insight into the factors that can influence the reaction.Our calculations show that in the gas phase the enol form of themodel of ASD lies about 17 kcal/mol higher in energy than theoxo form (see also Table S1 in the Supporting Information),indicating that enolization of ASD alone is thermodynamically astrongly unfavorable process. However, when it is coupled toanother energetically favored process, such as the protonationof the ferrous superoxo complex, which can and has to beprotonated according to the consensus catalytic cycle,29,30 itmay still take place.In our first model (AQM) the reaction between ASD and the

ferrous superoxo complex was considered. Using this base ascatalyst to remove the 2β-hydrogen atom, the barrier of theenolization was reduced to around 12.4 kcal/mol (see Figure3), thus lower than the energy difference between the oxo andenol forms of ASD. Of course, this reaction cannot beeffectively productive, because for the enolization process to becompleted a proton should be donated to the oxo group ofASD. However, this reaction does indicate that the ferroussuperoxo complex may catalyze the reaction.Therefore, in model BQM a proton transfer chain was

designed, similar to what might be present in the enzyme, inwhich the ferrous superoxo complex would act as a base toaccept the 2β-hydrogen as a proton and a water molecule actingas acid could donate its proton to the oxo group. Unfortunately,the geometry optimization of the product state of this and thefollowing reactions was not possible because the system had atotal charge of −2 electrons (e) that was originally located on

the ferrous superoxo complex, but after the proton transfertook place the electrostatic repulsion between the two singlynegatively charged residues hindered the geometry optimiza-tion of the product in a vacuum. In the BQM model the barrierof enolization was further decreased to 7.9 kcal/mol, but it isobvious from the geometrical parameters of the transition state(Supporting Information, Table S1) that water may be a too-weak acid to donate its proton to ASD. The characteristicdistances involving the proton (Hd) donated by water hardlydiffer between the reactant and transition states, but the 2β-hydrogen remains closer to ASD than in model AQM.For this reason in model CQM a stronger acid (acetic acid,

HOAc) was chosen as proton donor, and the barrier of thereaction was reduced to 5.6 kcal/mol, which is already very low,indicating that the reaction may proceed at a reasonable rate. Inthis model system the donated hydrogen ion (Hd) is alreadycloser to ASD than in the previous systems, and the 2β-hydrogen of ASD is found halfway between the donor andacceptor molecules. In this case the geometry of the transitionstate is more similar to the reactant state than in the case of theAQM or BQM model, because the H2β−O2 distance issignificantly longer. Although we were not able to optimizethe product state as mentioned above, based on theHammond−Leffler postulate and on the geometry of thetransition states our results suggest that the stronger protondonor can decrease not only the barriers, but the reactionenergy as well.In the DQM and EQM models we have replaced the methyl

group involving C19 by hydroxymethyl and formyl groups,respectively, and obtained similar transition state structures andactivation energies as in the CQM model. The slightly higherenergy barrier observed in the DQM models is due to the factthat the hydroxyl group of the substrate makes a hydrogenbond with the O2 atom of ferrous superoxo complex,decreasing its proton affinity. The fact that the C19 substituenthardly influences the feasibility of the enolization reaction isvery important for our study. As mentioned in the Introduction,the catalytic cycle of aromatase consists of three oxidativesubcycles; therefore, the results obtained with the CQM, DQM,and EQM models indicate that enolization might take place inany or all of the subcycles, not only in one of them. Asmentioned above, there has been a long-standing debate on thereaction mechanism of the third catalytic subcycle, with the

Figure 3. Dispersion corrected relative energies of QM-only model systems. The structures were optimized with the B1 basis set, and single-pointcalculations were carried out with the cc-pVTZ basis set (B1/cc-pVTZ). (a) Energy barriers for 2β-hydrogen abstraction. (b) Energy profile for thefission of the O−O bond in Cpd 0.



formation of a peroxohemiacetal. In that case, the route leadingto enolization could occur in the first and/or second catalyticsubcycle.The contribution of an atom to a normal coordinate

belonging to molecular vibrations can be illustrated by thesum of squares of components of the normal coordinatebelonging to the adequate atom. (The sum of all squares ofcomponents of a normal coordinate is defined as 1 Å2.) InTable S2 Supporting Information, we collected the contribu-tions of 2β-hydrogen and the hydrogen of the proton donormolecule (Hd) to the normal coordinate belonging to theimaginary frequency in the transition state. The contribution of2β-hydrogen is higher than 93% in all cases. Compared to theBQM model, where water is used as acid, in the other models thecontribution of Hd to the normal coordinate is increased (from0.13% to 1.18% in ASDQM, 1.41% in ALKQM, and 0.89% inALDQM), suggesting a stronger interaction with the protondonor molecule. However, the contribution of Hd remains verylow in all systems, suggesting a nonsynchronous process, whichmay be concerted. We did not find any transition states whenwe tried to move the Hd hydrogen to the oxo group in the firststep, which suggests that the shifting of 2β-hydrogen isprerequisite for Hd shifting. These findings are in accordancewith the NBO charges of the atoms (see Figure 4 and theSupporting Information, Table S3). In the reactant complexesboth the substrate (ASDQM, ALCQM, or ALDQM) and theproton donor molecule (water or HOAc) are nearly neutral,but in each transition state the substrate acquires a significantnegative charge. The negative charge on the proton donormolecule remains very low, but it is a bit higher if the donormolecule is acetic acid. We have also compared the NBOnatural charges of the ferrous superoxo complex in thetransition state to the charges of Cpd 0, and these data suggestthat the positive charge resulting from the protonation processis not localized to the OOH− moiety, but covers the full iron−porphine system.Figure 4 and Table S4 Supporting Information contain the

calculated natural spin densities for selected atoms and groups.The data are in accordance with previous results and confirm

the right electronic state of the investigated molecules. In allmodel systems, in the reactant state the spin density is maximalon the O1 and O2 atoms with some spin density located on theiron and negligible on other atoms. In the transition states thespin density is increased on the iron ion and decreased on theoxygen atoms, especially on the O2 atom. In the case of Cpd 0the spin density is maximal on the iron ion, with a smallcontribution on O1 and almost zero on other atoms. This is inaccordance with the electronic conversion described above.After having a strong indication from small model system

calculations that the ferrous superoxo complex may catalyze theenolization reaction while being converted to Cpd 0, wedecided to investigate whether Cpd 0 could catalyze the samereaction. (When we refer to the Cpd 0 model, it is presented inboldface type; however, when it is mentioned just as a state inthe catalytic cycle, it is presented as Cpd 0). However, we werenot able to find a transition state with an imaginary frequencybelonging to the shift of 2β-hydrogen from the ASDQM

molecule to Cpd 0 and could not optimize the geometry ofthe O2-protonated Cpd 0 either, because it converted to N-protonated Cpd 0. This result could be somewhat foreseen,because, as mentioned in the Introduction, the most recentcalculations indicate that Cpd 0 is most likely converted to CpdI by the PCET mechanism, instead of the formation of the O2-protonated Cpd 0.39 Furthermore, our calculations alsocontradict the hypothesis that the substrate could take overthe role of proton donor in the PCET mechanism, instead of athreonine residue such as Thr252 in P450cam, as we were notable to locate any correct transition state for this protontransfer reaction.For comparative purposes with the QM/MM results

presented below, we modeled the O−O fission process ofCpd 0 (denoted FQM model in Figures 3 and 4). Cpd 0 is thereactant state of the FQM model whose structure and electronicconfiguration can be seen in Figure 1. In the course of the O−O fission process we obtained Cpd II and •OH radical inaccordance with other works.30 We note at this point that thereare several theoretical and experimental studies dealing with the

Figure 4. Natural charges (in e) and natural spin densities (in parentheses, in e) for selected atoms or groups at the B3LYP/cc-pVTZ level. (a) CQMmodel system; (b) FQM model system.



relative reactivity of possible oxidizing agents in P450 enzymes(i.e., Cpd 0, Cpd I, Cpd II).29,30

3.2. QM/MM Calculations. In the second part of our workwe used QM/MM calculations to study the enolizationreaction, and we decided to test the reliability of theconclusions based on small model gas phase calculations bystudying various reaction mechanisms in the active site of theenzyme. The model systems are shown in Figure 5, and therelative energies of the stationary points of the energy profileswith the lowest barrier are collected in Figure 6. The mostimportant geometrical parameters of the energy profile with the

lowest barrier are shown in Figure 7. The data for all profilesare given in the Supporting Information (Table S5).In the X-ray structure, and therefore in our original MD

simulation, there were no molecules close to the oxo group ofASD that could serve as a proton donor. Even the spatialposition of Asp309, which has earlier been implicated as apossible proton source, was not appropriate for protondonation, neither directly nor indirectly via water molecules.Therefore, this arrangement (indicated as AQM/MM) wasanalogous to the AQM model system described above, whichdid not contain any proton donor molecule. We performedonly a single energy profile calculation using the AQM/MM

Figure 5. Model systems used in QM/MM calculations.



model, in which the 2β-hydrogen was transferred as a protonfrom the ASD molecule to the O2 atom of the ferrous superoxocomplex. The barrier of the reaction (16.1 kcal/mol using theB1 basis set) was higher than in the gas phase, and the high-energy intermediate formed could convert back to the reactantstate very easily.As it was obvious that for the enolization a proton donor

molecule is needed that could protonate the oxo group of ASD,we have manually modified the AQM/MM model by placing awater molecule between the side chain of Asp309 and the oxogroup (Ooxo) of substrate (BQM/MM model). In this arrangementAsp309 could not protonate the substrate either directly orindirectly, but the position of the water molecule was suitablefor proton donation; therefore, this model was analogous to theBQM model. Although this model contained a proton donormolecule, the enol formation was not formed in the calculations(we carried out a single reaction profile calculation). Weobtained an intermediate state after the abstraction of the 2β-hydrogen as proton, but when we tried to generate the enolform using constraints, the energy of the system increasedcontinually (over 45 kcal/mol), and the constraint-freeoptimization of the last point of the reaction profile

Figure 6. Calculated QM/MM energy profiles for the enolizationreaction. Energy values in all cases are given for the reaction profilewith the lowest barrier using the B1/6-311+G* basis sets. The energyvalues contain empirical dispersion correction.

Figure 7. The most important geometrical parameters (in Å) for the C2QM/MM system, for the lowest barrier profile (III) of 2β-hydrogen abstraction.



transformed back to the intermediate state. These results are incomplete agreement with our conclusions drawn based on QM-only calculations: water molecule is not acidic enough toprotonate the oxo group of ASD molecule. However, thepresence of water had a favorable effect on the energy barrier ofthe reaction, similarly to the QM-only calculations, as it wasreduced to 13.0 kcal/mol compared to 16.1 kcal/mol calculatedin the AQM/MM system.Obviously a stronger proton donor is needed for the

enolization reaction. Unfortunately, there were no structures inthe original MD trajectory in which the side chain of Asp309could protonate the Ooxo atom of the substrate directly.Therefore, we manually rotated the side chain of Asp309 residuearound its Cα−Cβ bond in the X-ray structure, and after a newsystem setup process, an MD simulation was carried out fromthis state. The new position of this side chain was stable in thecourse of the MD simulation, and it was stabilized by ahydrogen bond interaction between the hydroxyl group ofThr310 and the carboxyl group of Asp309. In this newarrangement the side chain of this aspartic acid was able toprotonate the Ooxo atom directly, resembling the CQM model,where the proton donor was an acetic acid molecule. Thissystem will be called the C1

QM/MM model, and we selected threestarting structures for adiabatic mapping. In the tables andfigures in the paper the data of the lowest-barrier profile areshown; data of the other profiles are collected in the SupportingInformation. The Asp309 proved to be acidic enough to catalyzethe enolization with energy barriers for 2β-hydrogenabstraction (13.7, 15.1, and 12.1 kcal/mol using the B1 basisset) for the three profiles. These values were similar to the oneobtained for the BQM/MM model; however, in all cases at the endof the reaction profiles the enolized product was formed in aslightly exothermic, nonsynchronous concerted process. Thecalculated reaction energies show that the endothermicenolization process can be coupled with the exothermicconversion of ferrous superoxo complex to Cpd 0 in anenergetically favored process. Although these QM/MM resultsare in accordance with the QM-only results, namely that acarboxylic acid can serve as a proton source in the enolizationreaction, the obtained barriers are unexpectedly high comparedto the barrier (1.2 kcal/mol) of the pathway suggested for theprotonation of the ferrous superoxo complex in P450cam.

40

These results imply that either (1) in the enzyme the ferroussuperoxo complex cannot catalyze the enolization or (2) ourmodel lacks an important component which reduces the actualbarrier in the enzyme.

It has been shown in several cases that protons may movevery easily, via low barriers, through proton shuttlechannels.81,82 Therefore, in the C2

QM/MM model (which isalso analogous to the CQM model), we have placed a watermolecule between the Asp309 residue and the oxo group of thesubstrate. In this model Asp309 could protonate the oxo groupindirectly through a water molecule. Using this model, thecalculations led to a two-step process with low barriers. Therate-limiting step of the reaction is the 2β-proton abstraction bythe ferrous superoxo complex with barriers of 5.7, 6.8, and 5.2kcal/mol, respectively, for the three profiles, which is followedby the protonation of the oxo group by the aspartic acid via thewater molecule. In this second step the two protons moveconcertedly through barriers of 1.8, 1.8, and 2.8 kcal/mol. Theoverall reaction is quasi thermoneutral or slightly exothermic,and the very low barrier of the reaction makes it likely to bepossible in the enzyme. For comparison, in Table S5 in theSupporting Information, data on the general proton deliverypathway published earlier for P450cam

40 have also beenincluded.In the QM/MM models discussed above there was no

hydrogen bond between Thr310 and the superoxo group,although its important role in the stabilization of ferroussuperoxo complex has been shown previously.30 Therefore, wedecided to study the role of this hydrogen bond for theenolization reaction with the C3

QM/MM model which included ahydrogen bond to the superoxo group from Thr310 in contrastto the C2

QM/MM model. The starting structures were derivedfrom C2

QM/MM by rotating Thr310 manually around the Cα−Cβ

bond with about 10°. The reaction follows a similar, two-stepmechanism as in the C2

QM/MM model, but the barrier of thefirst, 2β-hydrogen abstraction step is increased by about 6 kcal/mol to (10.9, 12.2, and 13.7 kcal/mol using the B1 basis set),while the barrier of the second proton transfer step from Asp309to the oxo group of the substrate remains similar (a smallincrease of about 1 kcal/mol can be observed) to the C2

QM/MMmodel. The considerably increased barrier of the first stepcompared to the C2

QM/MM model is most likely due to the factthat the hydrogen bond to the superoxo group decreases itsproton affinity in agreement with a recent study.83 However,once the proton is transferred to the ferrous superoxo complex,it does not influence significantly the barrier of the secondproton transfer to the oxo group.The calculated NBO charges for selected atoms and groups

are summarized in Table 1. It contains only the profile with thelowest barrier for 2β-hydrogen abstraction (profile IIIbelonging to the C2

QM/MM system). The NBO charges and

Table 1. Natural Charges (in e) and Natural Spin Densities (in Parentheses, in e) for Selected Atoms or Groups of the QMRegiona

RS TS1 IM TS2 PS

ASD −0.04(−0.01) −0.24(−0.01) −0.83(0.00) −0.66(0.00) −0.09(0.00)Porph −1.23(−0.06) −1.05(−0.06) −0.94(−0.03) −0.92(−0.03) −0.91(−0.03)CH3S

− −0.69(0.00) −0.66(0.01) −0.60(0.02) −0.58(0.02) −0.58(0.03)Asp −0.06(0.00) −0.07(0.00) −0.09(0.00) −0.20(0.00) −0.85(0.00)Thr −0.01(0.00) −0.01(0.00) −0.01(0.00) −0.01(0.00) −0.01(0.00)H2O 0.00(0.00) −0.01(0.00) −0.03(0.00) −0.12(0.00) −0.04(0.00)Fe 0.74(0.17) 0.84(0.54) 0.90(0.73) 0.90(0.76) 0.90(0.77)O1 −0.30(0.47) −0.32(0.37) −0.38(0.24) −0.39(0.22) −0.40(0.21)O2 −0.41(0.43) −0.49(0.14) −0.51(0.03) −0.51(0.02) −0.50(0.02)H2β 0.32(0.00) 0.40(0.00) 0.49(0.00) 0.49(0.00) 0.48(0.00)

aData are derived from QM/MM single-point calculations in which the QM region was calculated at the B3LYP/6-311+G* level.



spin densities for all profiles are given in the SupportingInformation (Tables S6 and S7). The changes of geometricalparameters (Figure 7), NBO charges, and spin densities alongthe energy profiles show the same trends as in the case of QM-only calculations, indicating similar changes in the electronicstructure described above.Both the QM and QM/MM calculations suggest that the

enolization of ASD and the protonation of the ferrous superoxocomplex can be coupled in an energetically favorable processwith low barriers, if there is no hydrogen bond between Thr310and the superoxo group coordinating to the heme iron.However, one must also consider whether protonation of theferrous superoxo complex could occur on the pathwayproposed for P450cam, where Asp309 would protonate theferrous superoxo complex indirectly via a water molecule andthe highly conserved Thr310. The calculated barriers for thisreaction in the case of P450cam are rather low (1.2 and 2.5 kcal/mol),40 even lower than the barrier we obtained in theC2

QM/MM model. Therefore, we decided to study thecorresponding pathway in aromatase in the GQM/MM model.We produced three starting structures for adiabatic mappingfrom snapshots taken from the original MD simulation (whichwas used for the C2

QM/MM model, too), by rotating manuallythe side chain of Asp309 around its Cα−Cβ bond by about 40°,placing a water molecule between Asp309 and Thr310 to createthe hydrogen bond chain, and rotating the hydroxyl group ofThr310 to hydrogen bond to the superoxo group of ferroussuperoxo complex. This way a long proton-transfer chain wascreated (see Figure 5) through which, in principle, protonscould shuttle via a low barrier process. Our calculations suggesta concerted reaction similarly to the case of P450cam, but theobtained energy barriers are somewhat higher (10.5, 9.5, and10.2 kcal/mol in contrast to the 1.2 kcal/mol calculated forP450cam). The barrier is also higher than the energy barriers ofthe enolization-coupled process obtained in the C2

QM/MMmodel (5.2 kcal/mol). Somewhat unexpectedly, the reactionturned out to be only slightly exothermic despite the fact that inearlier studies the process was highly exothermic.40 Thedifference between our results for protonation of the ferroussuperoxo complex via the Asp−water−Thr−ferrous superoxopathway and the earlier P450cam studies may in part originatefrom differences in the basis set used, and also different QM/MM methodologies. However, there are differences betweenthe relevant geometrical parameters of the starting structuresbetween the two systems. In human aromatase, the HAsp−Ow,Hw−OThr, and HThr−O2 distances are longer in the startingstructures than in P450cam

40 (see Supporting Information,Table S5), which could also contribute to the significantlyincreased barriers for the proton shift. This phenomenon couldbe due to the presence of the bigger ASD ligand, which has anoxo group close to the water molecule and the side chain ofAsp309. Therefore, on the basis of the results we may concludethat the protonation of the ferrous superoxo complex ispossible on the general pathway, but if the substrate molecule isin the oxo form, the enolization-coupled process will be morefavorable. The higher barriers for protonation of ferroussuperoxo complex in the conventional route are in accordancewith the experimental findings, which suggest the increasedstability of the ferrous superoxo complex in humanaromatase.26

As mentioned above, the consensus catalytic cycle of P450sincludes two protonation steps, and protonation of Cpd 0 toform Cpd I might also take place. In our original research

question we wanted to test the hypothesis that the 2β-hydrogenof ASD could be the proton source of it. However, the QM-only calculations presented above contradicted the hypothesis.Still, we decided to investigate the question using QM/MMcalculations with the FQM/MM model. We tried to apply variousreaction coordinates to force Cpd I formation from Cpd 0 byabstracting the 2β-hydrogen of ASD. First, we tried to make theH2β atom shift to the O2 atom (ξ1 = d(C2−H2β) − d(H2β−O2);ξ2 = d(O1−O2) − d(H2β−O2)), but the energy of the systemincreased continually (over 50 kcal/mol) and cleavage of theO1−O2 bond did not occur. Optimization of the last pointwithout any constraint led back to the reactant state. Next, wetried to cleave the O1−O2 bond in the first step by increasingthis distance in adiabatic mapping and then decrease the O2−H2β distance. In the first step a hydroxyl radical and Cpd IIwere formed based on the charges and spin densities (seeSupporting Information, Tables S6 and S7), but in the secondstep the O1−O2 bond re-formed, so we obtained the reactantstate again. These results suggest that the Cpd I formation isnot possible coupled to the enolization process. The most likelyreason is that the C2−H2β bond is in an unfavorable position: itis quasi perpendicular to the O1−O2 bond and therefore itcannot facilitate the breaking of the O1−O2 bond. In contrast,the OH group of Thr310 is almost in the centerline of the O1−O2 bond, and this position is suitable for promotion of cleavageof the O1−O2 bond in proton-coupled electron transfer(PCET) (see Figure 8). This result is in accordance with theexperiments, which suggest the essential role of Thr310 in theproper functioning of aromatase.43

3.3. Additional Calculations. The aim of these calcu-lations was twofold. On the one hand, they were carried out inorder to check the sensitivity of our results to the usedfunctional (see Table 2). On the other hand, we carried out gtensor calculations in order to compare the properties of ourcomplexes to the experimentally determined g tensor values.The results obtained with various functionals show similar

trends like B3LYP, namely the barriers for the GQM/MM profileare significantly higher in all cases than for the C2

QM/MM system.The OLYP gives anomalous values in the cases of IM, TS2, andPC states of the C2

QM/MM system. The usage of M06 andωB97X-D increases the barriers by about 2−4 kcal/mol, butthese values are still rather low, because these are not zero-point-corrected energies. The TPSSh results are very similar tothe B3LYP relative energies. We checked the spin densities for

Figure 8. Relative positions of H2β and HThr atoms. The C2−H2β bondis in an unfavorable, quasi perpendicular position to the O1−O2 bond,while the OH group of Thr310 is almost in the centerline of the O1−O2bond.



all states and functionals, and observed similar changes in theelectronic structure as discussed previously.In all theoretical studies it is highly desirable to compare the

theoretical findings to the experimentally available data, as onlythose hypotheses could be accepted which are in line with theexperimental findings. As the intermediates of the P450catalytic cycle contain unpaired electrons, EPR is a frequentlyused method in revealing the properties of the intermediates.30

As mentioned in the Introduction, the very short lifetime andthe high reactivity of the ferrous superoxo complex aresuggested by an experimental work dealing with camphorhydroxylase (P450cam),

27 while the increased stability of theoxy−ferrous complex in human aromatase is suggestedexperimentally.26

Here, we calculated the g tensors for two simple gas-phasemodels (ferrous superoxo complex and Cpd 0) and then for thereactant and product states of all profiles of our best model(C2

QM/MM) at the B3LYP/6-311+G* level in the presence ofpoint charges. We tested the effect of the basis set on the gtensor values using the structures from profile III of theC2

QM/MM system, but test calculations with the cc-pVTZ basisgave almost identical values (differences only in the thirddecimal place of g tensor values were observed), indicating thatthe smaller basis set could be reliably used. The results aresummarized in Table 3.The obtained values suggest the high importance of the

inclusion of the protein environment in the calculations. TheQM-only values for the ferrous superoxo complex deviateconsiderably from the experimental values, while the valuesderived from QM/MM calculations show reasonable agreementwith the experiments. In contrast, the calculated g tensors ofCpd 0 show better agreement with the experiment, whichmight be due to the fact that the unpaired electron is located tothe π*O−O orbital in the ferrous superoxo complex and to theπ*yz orbital (mainly to the iron) in Cpd 0, and is thereforemore shadowed and less sensitive for the presence of pointcharges.In the case of the QM/MM results we see slightly different

trends. Unfortunately, several values (including the values forCpd 0) in the case of human aromatase are not known, butbased on the similarity between the g tensor components of theferrous superoxo complexes of P450cam and human aromatase,

we might suppose that those of Cpd 0 would also be verysimilar. The calculated gzz values differ considerably more fromthe measured one in Cpd 0 than in the ferrous superoxocomplex. This difference might originate, for instance, from thefact that the conformation of this state is not ideal, as no MDequilibration of the product state was carried out in ourcalculations. In the product state the hydrogen atom of the Fe−O−O−H moiety is oriented toward its original location, theASD ligand, and this state is a local minimum on the potentialenergy surface. However, the rotated conformer, in which theFe−O−O−H moiety forms a hydrogen bond with one of theporphyrin nitrogens, has been suggested to be more stable, sowe rotated the Fe−O−O−H moiety in the product states ofthe three profiles and performed a QM/MM optimizationfollowed by g tensor calculations. We note that the Fe−O−O−H moiety was also not hydrogen bonded to the heme nitrogenin other QM/MM studies dealing with protonation of ferroussuperoxo complex.40 These values are shown in parentheses inTable 3. This rotation led to slightly different values which arecloser not to the values belonging to the Cpd 0 state, but tothose of the 19-hydroxy-ASD-bound state of the enzyme. Thecalculated g tensor is the closest to the measured reference inthe case of profile III, which has the lowest barrier for 2β-hydrogen abstraction. The values calculated for profile IIdeviate most from the experimental value, due to the fact thatin the rotated structure the conformation of the Fe−O−O−Hmoiety is a bit different from in the other structures.

Table 2. Relative Energies (in kcal/mol) Obtained fromSingle-Point Calculations on the B3LYP/B1 GeometriesUsing Various DFT Functionalsa

model B3LYP M06 TPSSh OLYP ωB97X-D

CQM RC 0.0 0.0 0.0 0.0 0.0TS 3.3 5.9 2.0 3.9 7.4

C2QM/MM (III) RS 0.0 0.0 0.0 0.0 0.0

TS1 5.7 7.6 6.3 9.2 9.7IM −1.8 0.6 1.3 8.7 0.9TS2 1.1 5.4 4.5 12.4 4.5PS −1.7 1.9 2.4 10.7 1.1

GQM/MM (II) RS 0.0 0.0 0.0 0.0 0.0TS 9.4 12.1 9.2 12.2 13.8PS −4.3 −2.5 −2.7 2.1 −2.3

aRC, reactant state; TS, transition state; IM, intermediate state. Thebasis set was cc-pVTZ for the CQM model and 6-311+G* in all QM/MM calculations. The profiles with the lowest energy barriers(indicated by roman numbers) were investigated. B3LYP energiesare dispersion corrected.

Table 3. Comparison of Measured and Calculated g TensorComponentsa

state gzz gyy gxx

P450cam [ref 27] ferrous superoxocomplex

2.246 2.166 1.958

Cpd 0 2.29 2.166 1.958human aromatase[ref 26]

ferrous superoxocomplex

2.254 2.163 −

Cpd 0 − − −19-hydroxy-ASDbound

2.40 2.245 1.925

QM-onlycalculation

ferrous superoxocomplex

2.401 2.025 1.626

Cpd 0 2.365 2.289 1.996C2

QM/MM

I RS (ferrous superoxocomplex)

2.207 2.002 1.968

PS (Cpd 0) 2.336 2.232 1.985(2.364) (2.272) (1.976)

II RS (ferrous superoxocomplex)

2.177 2.022 1.972

PS (Cpd 0) 2.397 2.262 1.966(2.473) (2.327) (1.984)

III RS (ferrous superoxocomplex)

2.226 1.996 1.959

PS (Cpd 0) 2.341 2.234 1.991(2.369) (2.274) (1.977)

III (ext) RS (ferrous superoxocomplex)

2.227 1.995 1.950

PS (Cpd 0) (2.368) (2.273) (1.977)aThe calculated results are derived from single-point calculations atthe B3LYP/cc-pVTZ level in QM-only calculations and the B3LYP/6-311+G* level in QM/MM calculations. Values in parentheses belongto modified structures with rotated Fe−O−O−H moiety. The modelC2

QM/MM III (ext) was extended with the side chains of the hemeresidue.



In the case of the C2QM/MM III (ext) system the QM region

was extended with the side chains of the heme residue. Thecalculated EPR parameters are almost identical to those of thenonextended system, which suggests the sufficiency of theunsubstituted porphyrin ring for QM/MM calculations.The similarity between the calculated g tensor for the Cpd 0

state and the measured g tensor for the 19-hydroxy-ASD-boundstate raises an interesting question. May this mean that in theexperiment no significant accumulation of Cpd 0 was observed,because the components of the g tensors of the two states arevery close to each other, making the distinction difficult? Wecannot answer this question based on the available data, butfurther experimental and computational investigation in thisarea might explain the experimental findings.3.4. Effect of Entropy. The calculations presented above

only aimed at describing a small section of the potential energysurface of the studied systems. However, it should be kept inmind that chemical thermodynamics and kinetics are, inprinciple, governed by Gibbs free energy differences, whichmay be different from the trends suggested by energycalculations due to the contribution of entropy. In order toobtain reliable free energies, it is essential to accurately samplethe conformational space, which becomes unfortunately ratherexpensive with ab initio or DFT QM components, which arenecessary for the investigation of P450 enzymes due to the sizeand complicated electronic structure of the heme center. This iswhy QM/MM free energy studies on P450s are still very rare.Fortunately, the differences between QM/MM energy and freeenergy profiles in enzymatic reactions tend to be small as longas local chemical events are investigated.84 It is explained that insome cases (e.g., hydrogen abstraction in the P450cam) theentropic contribution to the free energy barriers is significantlylower in the enzyme than in gas-phase calculations, since theentropic penalty, due to the loss of translational and rotationaldegrees of freedom upon formation of the transition state, isabsorbed into the substrate binding step, which is driven mainlyby the expulsion of water molecules from the binding pocket.85

We inserted the calculated Gibbs free energy values of the QM-only model systems into Table S1 in the SupportingInformation. These free energy barriers are very similar to orslightly lower than the relative energies (by 1−2 kcal/mol). Onthe basis of the above arguments it can be assumed that theobtained energy profiles can be considered as a reasonableapproximation to the Gibbs free energy changes of the studiedreactions.

4. CONCLUSIONIn this work a key element of the catalytic cycle, the enolizationof the substrate molecule, of human aromatase has been studiedusing QM and QM/MM calculations. It has been shown thatthe energetically unfavored enolization of androstenedione canoccur in a process coupled to the energetically favorableprotonation of the ferrous superoxo complex (traditionallycalled ferric peroxo complex), which is an essential step of thecatalytic cycle of oxidative P450s. According to our results, inthe first step of the reaction, the 2β-hydrogen of androstene-dione is shifted selectively as a proton to the ferrous superoxocomplex in accordance with the experiments,17 which isfollowed by the protonation of the oxo group of the substrateby the highly conserved Asp309 residue via a water molecule.This mechanism cannot serve as a general pathway for theprotonation of ferrous superoxo complex in most P450s, as itrequires the enolization of the substrate molecule. However, in

the case of human aromatase this conversion is essential for thecatalytic process to be completed.19 The higher barriersobtained in this study for the conversion of the ferroussuperoxo complex to Cpd 0, and the relatively low reactionenergy, are in agreement with the experimental findings, whichsuggested in human aromatase the relative stabilization of theferrous superoxo heme and a more hindered proton transferchain26 than in most other P450s. It has also been shown thatthe protonation of the ferrous superoxo complex may followthe general mechanism proposed for other P450 enzymes,where the Asp309 residue protonates the ferrous superoxocomplex indirectly via a water molecule and the highlyconserved Thr310 residue. However, based on our results, thebarrier of the latter process is higher than that of theenolization-coupled process; therefore, if the substrate is inthe oxo form the enolization-coupled process is more likely tooccur in human aromatase.Our QM-only calculations also suggest that the enolization-

coupled protonation of the ferrous superoxo complex can occurin human aromatase independently of the identity of the C19group of the substrate (methyl, hydroxymethyl, or formyl), as itdoes not significantly influence the obtained barrier heights.These results imply that enolization might take place in any orall of the subcycles, not only in one of them. On the basis of theobtained low barrier of the enolization reaction, in the future itmay be worth considering other possibilities for the debatedthird catalytic subcycle besides peroxo hemiacetal formation,e.g., routes involving Cpd I, the traditional oxidizing agent ofP450 enzymes.It has also been shown that the formation of Cpd I from Cpd

0 is not possible in an enolization-coupled process, because theC2−H2β bond of androstenedione is quasi perpendicular to theO1−O2 bond of Cpd 0, which has to be cleaved in thisconversion. The O−H group of Thr310 is quasi in the centerlineof the O1−O2 bond, and this position is suitable for promotionof cleavage of the O1−O2 bond in proton-coupled electrontransfer (PCET).39 Therefore, our results are in agreement withthe experiments that suggest the essential role of Thr310 in thecatalysis.43

■ ASSOCIATED CONTENT*S Supporting InformationSelected geometrical parameters and fragment charges for QM-only optimized structures using various basis sets, and details ofall reactant, TS, and product complexes obtained in QM/MMcalculations. Furthermore, total energies and Cartesiancoordinates of all QM-only structures, geometries of the QMregion of the QM/MM structures, and MM parameters for theASD ligand are also provided together with tables and figuresindicated in the text. This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Tel.: +36-1-463-1286.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Prof. Jeremy Harvey (University of Bristol)for useful discussions and the New Szechenyi Plan (TAMOP-4.2.2/B-10/1-2010-0009) for financial support. J.O. acknowl-



http://pubs.acs.org

mailto:[email protected]

edges receipt of an EU Marie Curie ERG Fellowship (Project“Oestrometab”).

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