steric factors override thermodynamic driving force in regioselectivity of proline hydroxylation by...

Steric Factors Override Thermodynamic Driving Force in Regioselectivity of ProlineHydroxylation by Prolyl-4-hydroxylase Enzymes

Baharan Karamzadeh,† Devesh Kumar,*,‡ G. Narahari Sastry,‡ and Sam P. de Visser*,†

The Manchester Interdisciplinary Biocenter and the School of Chemical Engineering and Analytical Science,The UniVersity of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom, and MolecularModelling Group, Indian Institute of Chemical Technology, Hyderabad 500-607, India

ReceiVed: September 20, 2010; ReVised Manuscript ReceiVed: NoVember 4, 2010

Prolyl-4-hydroxylase is an important nonheme iron-containing dioxygenase in humans involved in theregioselective hydroxylation of a proline residue in a peptide chain on the C4 position. In biosystems thisprocess is important to create collagen cross-linking and cellular responses to hypoxia. We have performeda series of density functional theory (DFT) studies into the origin of the regioselectivity of proline hydroxylationby P4H enzymes using a minimal active site model (where substrate is unhindered in the binding site) anda larger active site model that incorporates steric hindrance of the substrate by several secondary spherearomatic residues. Our studies show that thermodynamically the most favorable hydrogen atom abstractionposition of proline is from the C5 position; hence, the small model gives a low reaction barrier and largeexothermicity for this process. However, stereochemical repulsions of the substrate with aromatic residues ofTyr140 and Trp243 in the second coordination sphere prevent C5 hydroxylation and make C4 hydroxylation thedominant mechanism, despite a lesser driving force for the reaction. These studies explain the remarkableregioselectivity of proline hydroxylation by P4H enzymes and show that the regioselectivity is kineticallycontrolled but not thermodynamically. In addition, we calculated spectroscopic parameters and found goodagreement with experimental data.

Introduction

Mononuclear nonheme iron-containing enzymes are versatileenzymes in nature that catalyze important reaction processesin many biosystems, including the human body.1 These enzymesoften share a common motif, whereby the metal is bound tothe protein via interactions with two histidine and one carboxylicacid group of either an Asp or a Glu amino acid side chain toform a characteristic facial metal binding triad.2 A large groupof mononuclear nonheme iron-containing enzymes utilize R-ke-toglutarate (RKG) as a cofactor and catalyze aliphatic hydroxyl-ation reactions as well as substrate halogenation, desaturation,and ring-closure reactions.1,3 In nature, these enzymes, forinstance, are involved in the biosynthesis of vancomycin andfosfomycin in antibiotics,4 as well as in DNA and RNA baserepair mechanisms in mammals.5 In addition, the R-ketoglut-arate-dependent dioxygenases are also involved in cross-linkingof collagen and responses to hypoxia.6

The most extensively studied R-ketoglutarate-dependent di-oxygenase is taurine/R-ketoglutarate dioxygenase (TauD) foundin Escherichia coli. TauD is involved in the hydroxylation oftaurine and is highly soluble and relatively stable.7 Thanks tothis, it was the first enzyme where a high-valent iron(IV)-oxospecies was characterized with resonance Raman, Mossbauer,and X-ray absorption spectroscopy.8 As a consequence, TauDhas become the template for mononuclear nonheme ironenzymes and has prompted many additional studies. Thecatalytic cycle of TauD enzymes is well-established9,10 and startswith RKG binding to an Fe center and substrate in its vicinity.

Subsequently, dioxygen binds to the last binding site of themetal, which attacks RKG at the R-keto position to form abicyclic ring structure. Subsequently, decarboxylation leads tosuccinate and a high-valent iron(IV)-oxo species, which hasbeen spectroscopically characterized with various methods forTauD.8 The final steps in the catalytic cycle start with a hydrogenatom abstraction from the substrate by the iron(IV)-oxo oxidantfollowed by rebound of the hydroxyl group to form alcoholproducts.

Another R-ketoglutarate-dependent dioxygenase with impor-tance for human health is prolyl-4-hydroxylase (P4H), involvedin the regioselective hydroxylation of proline groups on the C4

position (Scheme 1).11 This is an important reaction in biosys-tems due to cross-linking collagen helices, oxygen sensing, andcellular response to hypoxia through the hypoxia inducible factor(HIF).12,13 Thus, the cross-linking strand in collagen contains aseries of repeated triads, where the first residue usually is proline,the second is often 4-hydroxyproline, while the last residuetypically is a glycine.14 A combination of spectroscopic andkinetic studies on substrate hydroxylation by P4H using a smallpeptide (Pro-Ala-Pro-Lys)3 showed that the proline residues arereadily hydroxylated by the enzyme.15 Studies using 18O2 showedthat one oxygen atom of molecular oxygen is incorporated into

* To whom correspondence should be addressed. E-mail: [email protected] (S.P.d.V.), [email protected] (D.K.).

† The University of Manchester.‡ Indian Institute of Chemical Technology.

SCHEME 1: Proline Hydroxylation by P4H Enzymes

J. Phys. Chem. A 2010, 114, 13234–1324313234

10.1021/jp1089855 2010 American Chemical SocietyPublished on Web 11/29/2010

hydroxo-proline products.16 Further stopped-flow absorptionand Mossbauer experiments provided evidence of an iron(IV)-oxo intermediate with features similar to those detected for theiron(IV)-oxo oxidant of TauD above. Moreover, a large kineticisotope effect was measured for replacement of the 2,3,3,4,4,5,5-positions by deuterium atoms. Mechanistic studies using 5-ox-aproline-containing peptides as a substrate found evidence of aGroves-type radical rebound mechanism.17 By contrast, studiesof the reaction of P4H with fluorinated proline residues gaveno reaction products, due to the strength of the C-F bond.15

On the other hand, with a sulfide group in this position, substratesulfoxidation was observed.

Figure 1 displays the active site structure of P4H enzymesas taken from the 2JIG Protein Databank file.18 The metal isbound to a 2His-1Asp binding motif through interactions withHis143, Asp145, and His227 similar to other nonheme irondioxygenases. Instead of RKG, the PDB contains pyridine-2,4-dicarboxylate, which binds as a bidentate ligand to the metalsimilarly to RKG. Currently no substrate-bound crystal structuresare available. The metal center is closely located to the proteinsurface, and a water channel is seen above the metal, and it hasbeen proposed that the substrate will bind here.19 Directly abovethe metal are located a series of aromatic residues, such as Tyr140

and Trp243, that may restrict substrate approach to the bindingsite. At the moment, the origin of the substrate selectivity ofP4H enzymes is unclear. Thus, the enzyme regioselectivelyhydroxylates a proline residue at the C4 position exclusively,and so far there is no evidence of products from hydroxylationof the C3 and C5 positions. To gain insight into the substratehydroxylation mechanism of this important enzyme for humanhealth and the factors that determine the regioselectivity ofsubstrate hydroxylation, we have performed a density functionaltheory (DFT) study on models of the active site of P4H enzymes.In principle, the origin of this regioselectivity can arise fromthermodynamic factors, i.e., the relative strength of the C4-Hbond with respect to the C3-H and C5-H bonds or, alterna-tively, through electrostatic interactions such as substrateapproach to the active center. In this study, we investigated bothpossibilities and show that stereochemical factors within thesubstrate binding pocket favor C4 hydroxylation over C3 andC5 hydroxylation.

Methods

The mechanism of proline hydroxylation by P4H enzymeswas modeled using density functional theory using methodstested and benchmarked on nonheme iron enzyme models inour group.20 We use the hybrid density functional methodB3LYP21 combined with a double-� quality basis set containingLACVP on iron and 6-31G on the rest of the atoms (H, C, O,N), the basis set BS1.22 Geometry optimizations were done withJaguar 7.6,23 while analytical frequencies were calculated withGaussian 03.24 Energetics were improved through single-point

calculations with a triple-� quality LACV3P+ basis set on ironand 6-311+G* on H, C, O, N atoms, the basis set BS2. Theeffect of the environment was tested through single-pointcalculations in Jaguar using the self-consistent reaction fieldmodel with a dielectric constant of ε ) 5.7 and a probe radiusof 2.72 Å; however, these single-point calculations had littleeffect on the overall energetics of the process; see SupportingInformation, Table S6 and S7. In the past, the methods havebeen extensively benchmarked against experimental data andit was shown that free energies of activation are reproducedwithin 3 kcal mol-1,25 although this is a systematic error andgenerally barrier heights have standard deviations between 1and 2 kcal mol-1.20,26 In addition, extensive calibration studieswere done on related systems in which spectroscopic parametersas well as kinetic isotope effects were calculated.27 These studiesgenerally reproduced experimental data well and confirm thatthe methods are reliable and suitable for these types of systems.

Kinetic isotope effects (KIE) were calculated from thesemiclassical Eyring equations (eq 1) using the free energy ofactivation (∆GH

‡) of the reference structure and those whereone or more hydrogen atoms of the substrate were replaced bydeuterium atoms. In eq 1, R represents the gas constant and Tthe estimated temperature (298 K).

Further corrections to the isotope effect due to tunneling wereapplied by multiplying KIEEyring with the tunneling ratio (QtH/QtD) (eq 2). Two methods to estimate the tunneling ratio wereused due to Wigner and Bell as given in eqs 3 - 6.28 In theseequations h is Planck’s constant, kB is the Boltzmann constant,and ν the imaginary frequency in the transition state.

In addition to replacing hydrogen atoms by deuterium atoms,we also investigated the effects of replacing 16O2 by 18O2 onthe IR frequencies of the iron(IV)-oxo species. The geometrywas optimized as described above and followed by a frequencycalculation at the UB3LYP/BS1 level of theory in the Gaussian03 suits of programs. A single-point calculation was performedon this structure with isotopic substitution of 16O2 by 18O2 andthe obtained frequencies were analyzed. All frequencies werescaled by a factor of 0.9257.29

Figure 1. Active site of P4H enzyme as taken from the 2JIG ProteinDatabank file. Amino acids labeled as in the crystal structure.

KIEEyring ) exp[(∆GD‡ - ∆GH

‡)/RT] (1)

KIEtunneling ) KIEEyring × QtH/QtD (2)

Qt,Wigner ) 1 + ut2/24 with ut ) hν/kBT (3)

Qt,Bell ) Q1 + Q2 (4)

Q1 )ut

2 sin(12

ut)(5)

Q2 ) - ∑n)1

∞

(-1)nexp(Vt∆GH)

Vtwith Vt )

ut - 2nπut

(6)

Regioselectivity of Proline Hydroxylation J. Phys. Chem. A, Vol. 114, No. 50, 2010 13235

Results and Discussion

Our models were based on the crystal structure coordinates(PDB file 2JIG) of P4H.18 We selected strand A of the dimer,since its peptide chain is longer. Subsequently, we addedhydrogen atoms, replaced the metal by iron and pyridine-2,4-dicarboxylate by RKG, and inserted an oxo group trans to His227.Two different models of the active site of P4H enzymes wereset up; see Figure 2. Model A is a minimal model analogous tothe one we used in our earlier studies of TauD33 and comprisesof an iron(IV)-oxo group that is linked to two imidazole groupsthat mimic the His143 and His227 ligands, while the twocarboxylic acid ligands of Asp145 and succinate (Succ) areabbreviated to acetate groups and proline as a saturated five-

membered ring without side chains. A more elaborate model Bwas built by inclusion of the peptide chain between His143 andAsp145, whereby amino acid 144 is modeled by Gly. Further-more, His227 is mimicked as methylimidazole, and two distalaromatic amino acids (Tyr140 and Trp243) are included as p-cresoland indole groups, respectively. Finally, substrate proline isextended with a peptide bond on the N-terminus. To preventthe individual groups from reorienting from the original positionsin the crystal structure, during the geometry optimization weconstrained the position of the carbon atom of the methyl groupof Tyr140 and Trp243 with respect to the metal center as well asthe angle and dihedral of its neighboring carbon atom. However,no constraints were placed on the metal ligands or the substrate.

The active species of P4H enzymes is a high-valentiron(IV)-oxo species, which is one of the few iron(IV)-oxospecies of enzymatic systems that has been spectroscopicallycharacterized with resonance Raman, EPR, and Mossbauerspectroscopic methods.15 In order to benchmark our methodsand procedures, we started our work with a detailed analysis ofthe iron(IV)-oxo species (1) using models A and B as depictedin Figure 2. Here, for each considered species, the spinmultiplicity is given as a superscript and model as a subscriptbefore and after the species label, respectively. We ran a fullgeometry optimization on the lowest lying singlet, triplet,quintet, and septet spin states and subsequently calculated IRspectra as well as EPR and Mossbauer spectroscopic parameters.

The high-lying occupied and low-lying virtual orbitals of theiron(IV)-oxo species of P4H (1) are shown in Figure 3. Forsimplicity, we only show the orbitals for the small model; thosefor the large model are essentially the same. The metal 3d type

Figure 2. Models of P4H studied in this work.

Figure 3. High-lying occupied and virtual orbitals of the iron(IV)-oxo species of P4H.

13236 J. Phys. Chem. A, Vol. 114, No. 50, 2010 Karamzadeh et al.

orbitals split into three lower lying π* orbitals and two higherlying σ* orbitals. The lowest one is the π*xy orbital that isorthogonal to the Fe-O bond and interacts with the His143,Asp145, and succinate ligands. Slightly higher in energy are twoπ*FeO orbitals (labeled as π*xz and π*yz) for the antibondinginteractions of the metal 3dxz,yz with corresponding 2p orbitalson the oxygen atom. Higher in energy are two σ* type orbitalsfor the antibonding interactions along the O-Fe-NHis227 axis(σ*z2) and one in the plane of the His143, Asp145, and succinateligands (σ*x2-y2). This set of orbitals is occupied by four electronsto give the metal oxidation state Fe(IV). DFT calculations onthe iron(IV)-oxo species of TauD predicted a quintet spinground state with π*xy

1 π*xz1 π*yz

1 σ*x2-y21 occupation.30 Inbiomimetic nonheme iron(IV)-oxo species, on the other hand,usually a triplet spin ground state is found with π*xy

2 π*xz1 π*yz

1

occupation.31,32 DFT calculations showed that electron-donatingaxial ligands tend to reduce the energy gap between the π*xy

and σ*x2-y2 orbitals and thereby lower the triplet-quintet energygap in favor of a triplet spin ground state in biomimeticsystems.33

Subsequently, we did a full geometry optimization of thelowest lying spin state structures of models A and B and theresults are shown in Figure 4, which clearly demonstrates thatboth models predict the ground state of 1 to be a quintet spinstate with orbital occupation π*xy

1 π*xz1 π*yz

1 σ*x2-y21. The tripletspin state has π*xy

2 π*xz1 π*yz

1 occupation, whereas the septetspin state has π*xy

1 π*xz1 π*yz

1 σ*x2-y21 σ*z21 lpO1 occupation.

The latter orbital refers to a lone pair on the oxo group. Finally,the singlet spin state has π*xy

2 π*xz1 π*yz

1 occupation with thetwo unpaired electrons having opposing spin direction. Ourassignment supports experimental EPR studies15 that indicateda quintet spin ground state. DFT and quantum mechanics/molecular mechanics (QM/MM) studies on the active speciesof the related nonheme iron enzyme TauD predicted a similarspin state ordering to that obtained for P4H here with a quintetspin state as the molecular ground state.10,34

The quintet spin ground state is well-separated from the septet,triplet, and singlet spin states by 7.4, 13.6, and 25.2 kcal mol-1

for Model A. For the iron(IV)-oxo intermediate in the catalyticcycle of TauD, Siegbahn and co-workers10 found a quintet spinground state that was 6.0 kcal mol-1 lower in energy than the

lowest septet spin state, in perfect agreement with our calcula-tions here. This spin state ordering is similar to previous studiesof nonheme iron(IV)-oxo complexes in enzymatic systems,30,34,35

where also large quintet-triplet and quintet-singlet energy gapswere obtained. In the nonheme iron enzyme isopenicillinN-synthase the iron(IV)-oxo intermediate is in a quintet spinground state with the triplet and septet spin states 5.2 and 10.3kcal mol-1 higher in energy.36 It appears therefore that therelative energy of the quintet and triplet spin states is verysensitive to the ligands bound to the metal, and minor changeslead to major shifts in the quintet-triplet energy gap. This isalso apparent from studies on biomimetic nonheme iron(IV)-oxocomplexes that tend to have a triplet spin ground state with thelowest lying quintet spin state several kcal mol-1 higher inenergy.32,37 Usually in biomimetic nonheme iron(IV)-oxocomplexes the septet spin state is high in energy and does notplay a role of importance. Obviously, this is quite different fromthe situation in enzymatic systems such as TauD and P4H, wherethe septet spin state is significantly lower in energy, sometimeswithin 5 kcal mol-1 of the quintet spin ground state. Clearly, inP4H the triplet and singlet spin states are so high in energy inthe reactants that they will not contribute significantly to thereaction mechanism; therefore, in the remainder of the paper,we will focus on the lowest lying quintet and septet spin statesonly. Extensive DFT studies on nonheme iron-containing enzymessuch as TauD and R-ketoglutarate-dependent halogenase showedthat the singlet and triplet spin state surfaces stay high in energyalong the substrate hydroxylation mechanism.10,30,35,38

Interestingly, the optimized geometries do not seem dramati-cally affected by the size of the model. For instance, aniron-oxo bond length in the quintet spin state of 1.652 and1.649 Å is found for 51A and 51B, respectively. In addition, thecalculated metal-ligand bond distances between Fe and His143,Asp145, His227, and Succ give only small differences betweenthe two models. It appears, therefore, that the optimizedgeometry is very little affected by increasing the size of themodel through addition of mainly secondary sphere amino acids.The calculated iron(IV)-oxo distances are in excellent agree-ment with those calculated for complexes of heme and nonhemesystems before.32,39 An Fe-O distance ranging from 1.62 to1.65 Å was found for heme-based iron(IV)-oxo complexes

Figure 4. Optimized geometries of the iron(IV)-oxo species of P4H models A and B with bond lengths given in angstroms. Also given arerelative energies of the quintet, septet, triplet, and singlet spin states. The inset gives a top view of the metal and its direct ligands for 51B.


dependent on the axial ligand. Our calculated Fe-O stretchvibration of 811 cm-1 for 51B is in line with previouslydetermined values for iron(IV)-oxo complexes.

Resonance Raman experiments of Hausinger et al.8b on TauDsupplied with 16O2 and 18O2 provided a difference spectrum withoxygen sensitive peaks at 583, 821, and 859 cm-1 in the 16O2

spectrum. The latter vibration, however, coincided with a strongsolvent band, so its identification was not unambiguous. To findthe corresponding vibrations for the iron(IV)-oxo species ofP4H, we analyzed the frequencies of 51B and its 18O2 substitutedspectrum. Thus, in the catalytic cycle of P4H and TauD,molecular oxygen donates one oxygen atom to RKG to formsuccinate and an iron(IV)-oxo species. Therefore, we replacedthe oxo group and one of the oxygen atoms of the carboxylicacid group of succinate that binds iron by 18O atoms to calculatethe 18O2 spectrum. Vibrational frequency analysis of 51B[16O2]gives an FedO stretch vibration at νFedO ) 811 cm-1, whilethe stretch vibration along the Fe-O bond between the metaland the succinate group is located at νFe-OSucc ) 857 cm-1. Ourcalculations, therefore, support the experimental assignment ofan FedO stretch vibration at 811 cm-1 and confirm that thereindeed is an oxygen-sensitive band at 857 cm-1, which originatesfrom the first oxygen atom transfer process in the catalytic cyclewhere succinate is formed. Two bending vibrations in thecarboxylic acid group of succinate are identified at 557 cm-1

(out-of-plane) and 583 cm-1 (in-plane). Substitution of 16O2 by18O2 in 51B downshifts νFedO by 33 cm-1 and the νFe-OSucc by18 cm-1. Much smaller frequency shifts are obtained for thetwo bending vibrations of -11 cm-1 for the in-plane vibrationand -4 cm-1 for the out-of-plane vibration. However, thesetwo bending vibrations give sharp differences in the calculatedRaman intensities, whereby the higher vibration is more Ramanactive in the 16O2 spectrum, whereas the lower vibration is moreactive in the 18O2 spectrum. As a consequence of this, theyimplicate a single peak that shifts by about 30 cm-1 but in factrepresent two peaks with different Raman intensities in the 16O2

and 18O2 spectra.In addition, we calculated Mossbauer spectroscopic param-

eters of 1 and focused on the electric field splitting (∆EQ), theasymmetry parameter of the nuclear quadrupole tensor (η), andthe isomer shift (δ) (Supporting Information, Table S8).Although the error bars for these types of calculations are high,a modest agreement between the calculated values of ∆EQ for51 with the experimental data from ref 15 is found. Hence, DFTcalculations on the iron(IV)-oxo species of P4H support thespectroscopic assignments in the literature in favor of a quintetspin as the ground state. The benchmark calculations ofspectroscopic parameters on models A and B give goodagreement with experimental data and support the use of thesemodels for our calculations.

Substrate Hydroxylation by P4H Enzymes. The generalmechanism of substrate hydroxylation by the iron(IV)-oxospecies of P4H is shown in Scheme 2. Thus, the iron(IV)-oxospecies (1) picks up a hydrogen atom from the substrate via ahydrogen atom abstraction barrier (TSHA) to form an iron(III)-hydroxo complex with a nearby prolyl radical (2). A radicalrebound barrier (TSreb) separates this radical intermediate fromthe alcohol product complex (3). This mechanism is similar tothe rebound mechanism proposed for substrate hydroxylationreactions by the iron(IV)-oxo heme(+•) active species ofcytochrome P450 enzymes and confirmed by biomimetic andcomputational studies.40

Theoretically, substrate hydroxylation by P4H enzymesshould lead to products originating from hydroxylation at the

C3, C4, and C5 positions of proline; however, in the enzymeonly the C4 position is activated. To find out what the mostlikely hydroxylation site of a proline molecule is, we did a seriesof DFT calculations to determine the bond dissociation energies(BDECH) of the various C-H bonds of proline; see Figure 5.The C-H bond strength or BDECH was calculated from eq 7:

Experimental studies of hydrogen abstraction reactions bymetal(IV)-oxo oxidants showed that the rate constants andhence free energies of activation correlated with the strengthof the C-H bond that is broken in the process.41 Recent DFTstudies in combination with valence bond modeling rationalizedthese hydrogen abstraction reactions and showed that thecorrelation follows from electron transfer processes fromreactants to radical intermediates.26c In the case of testosteronehydroxylation by the iron(IV)-oxo species of cytochrome P450,the barrier heights of hydrogen atom abstraction for fourdifferent C-H positions of the substrate correlated to thestrength of these C-H bonds.42

Figure 5 displays the calculated BDECH values of the C3-H,C4-H, and C5-H positions of proline. As follows, H-abstractionfrom the C5 position from proline should be the easiest becauseit has the weakest C-H bond strength (BDECH ) 85.4 kcalmol-1). Furthermore, the C4-H bond strength of prolineapparently is the strongest of these C-H bonds and, therefore,the least likely position for hydroxylation with a BDECH ) 92.4

SCHEME 2: General Mechanism of SubstrateHydroxylation by P4H Enzymes

Figure 5. Calculated BDECH values of proline C-H bonds withenergies in kcal mol-1.

Pro-H f Pro · + H · + BDECH (7)


kcal mol-1. Therefore, our BDECH calculations show that prolinehydroxylation on the C4 position is thermodynamically the leastexpected hydroxylation site, despite the fact that this is thetargeted site in the enzyme. Using a small Ala-Pro-Ala peptide,the calculated BDECH values at the C3, C4, and C5 positions ofproline are 93.5, 92.9, and 86.7 kcal mol-1, respectively, whichindicates that these BDECH values give little sensitivity regardingthe size of the substrate. In order to find out why P4H enzymeshydroxylate proline on the C4 position while the driving forcefor the reaction is the smallest at this position, we ran a seriesof DFT calculations to establish environmental (stereochemical)effects on the reaction mechanism and barriers.

Subsequently, we investigated the mechanism of prolinehydroxylation at the C5, C4, and C3 positions using the minimalmodel (model A), and the results are shown in Figure 6. Prolinehydroxylation at the C4 position was calculated on the lowestlying singlet, triplet, quintet, and septet spin states. The singletand triplet reactants, however, are well higher in energy than51A (Figure 4) by 25.2 and 13.6 kcal mol-1, respectively, andso are their corresponding hydrogen atom abstraction barriersleading to an iron-hydroxo intermediate (>28 kcal mol-1).Therefore, we will focus in the main text on the quintet andseptet states only; details of the other spin states can be foundin the Supporting Information. Proline hydroxylation at the C3

and C5 positions was only calculated on the quintet spin statesurface.

Although the reactant state has a quintet spin ground state,after hydrogen abstraction close-lying iron(III)-hydroxo com-plexes (2) in the quintet and septet spin states, 52A,C4 and 72A,C4,are found within 1 kcal mol-1. These two states have the sameorbital occupation (π*xy

1 π*xz1 π*yz

1 σ*z21 σ*x2-y21 πSub1) with a

singly occupied metal d-block coupled to an unpaired electronon the proline rest-group; this interaction is ferromagnetic inthe septet spin state and antiferromagnetic in the quintet spinstate. Even though the intermediate has close-lying quintet and

septet spin states, as a matter of fact, in the H-abstractiontransition states, the quintet state is well-separated from theseptet by more than 10 kcal mol-1 for the C4-hydroxylationmechanism. Consequently, P4H hydroxylation will proceed viasingle-state reactivity on a dominant quintet spin state surfaceonly. The energy gap between 5TSHA and 7TSHA is in contrastto substrate hydroxylation by TauD models, where two-statereactivity patterns on close lying quintet and septet spin stateswere found.26b,30 Nevertheless, the H-abstraction step is rate-determining as the radical rebound barrier is much smaller.

In agreement with the thermodynamic analysis mentionedabove, the relative energies of the H-abstraction transition statesand radical intermediates follow the trend in BDECH with5TSHA,A,C5 < 5TSHA,A,C3 < 5TSHA,A,C4. Hydrogen abstraction of aH-atom from the C5 position (5TSHA,A,C5) is 10.5 kcal mol-1

lower in energy than that for H-atom abstraction from positionC4 (5TSHA,A,C4), whereas the energy difference between the tworadical intermediates (52A,C4 and 52A,C5) is 9.8 kcal mol-1. Thedifference in energy between the BDECH values for H-abstractionat the C5 and C4 positions (Figure 5) is calculated to be 7.0kcal mol-1, which is comparable to the difference obtainedbetween 52A,C5 and 52A,C4. Thermodynamically, the exothermicityof formation of 52A from 51A is the difference in energy betweenBDECH of the C-H bond of the substrate that is broken andthe BDEOH of the FeO-H bond that is formed (eq 8).41a,b

For a small model of TauD that closely resembles model Ahere, a BDEOH of 95.7 kcal mol-1 was calculated.26b Combina-tion of this BDEOH value with the BDECH values from Figure5 gives predicted reaction exothermicities ∆H using eq 8 forformation of intermediate complexes for H-abstraction at theC4 and C5 position of -3.3 and -10.3 kcal mol-1. These values

Figure 6. Potential energy landscape of proline hydroxylation at the C5, C4, and C3 positions using model A. Relative energies are in kcal mol-1,obtained with basis set BS2, while ZPE corrections are calculated with basis set BS1. Optimized geometries contain bond lengths in angstroms andthe imaginary frequency in wavenumbers.

∆H ) BDECH - BDEOH (8)


are a few kcal mol-1 less exothermic than those calculated for52A,C4 and 52A,C5. The reason for this is the close proximity ofthe radical to the iron(III)-hydroxo complex. As a matter offact, the hydrogen atom of the amide group of the radical formsa hydrogen bond with the carboxylic acid ligand of the metaland thereby lowers the energy of this radical intermediate.Nevertheless, the trends in relative energies of 52A,C4 and 52A,C5

in Figure 6, therefore, reflect the differences in BDECH of theC-H bonds of the substrate that are broken. Consequently,thermodynamically and kinetically one would expect P4Henzymes to hydroxylate the C5 position of proline rather thanthe C4 position, which was found in the enzyme. To find outwhether stereochemical interactions change this regioselectivitypreference, we calculated also a larger model of the P4H activesite, vide infra.

Recent studies of hydrogen abstraction trends by iron(IV)-oxospecies of heme and nonheme oxidants showed that the barrierheight from reactants to iron(III)-hydroxo intermediates cor-relates linearly with BDECH, whereas the reverse reaction barriercorrelates with BDEOH instead.20,26 The calculated BDECH valuesin Figure 5, therefore, support the ordering of the hydrogenabstraction barriers calculated for the small P4H model complexwith ordering 5TSHA,A,C5 < 5TSHA,A,C3 < 5TSHA,A,C4. The hydrogenabstraction barrier 5TSHA,A,C4 of 10.6 kcal mol-1 is slightly higherin energy than the barrier obtained before for toluene (7.6 kcalmol-1)26b hydroxylation by the iron(IV)-oxo species of TauD,where a C-H bond of BDECH ) 85.8 kcal mol-1 is broken.The C4-H bond strength in proline, actually, compares to theBDECH value for hydrogen abstraction from the secondarycarbon atom of propane (BDECH ) 93.0 kcal mol-1), whichwould imply that the active oxidant of P4H enzymes is apowerful enough oxidant to hydroxylate very strong C-H bondslike in propane.

Thermodynamically, one would expect a similar barrier heightfor hydrogen atom abstraction from the C3 and C4 positions.The TSHA barrier for abstraction of a hydrogen atom from theC3 position, however, is lowered with respect to that of the C4

position due to more stabilizing hydrogen-bonding interactions.The structure is stabilized by a short hydrogen bond of 2.231Å between the amide proton of the substrate to one of thecarboxylic acid oxygen atoms of the succinate residue, whilethe complementary hydrogen bond in the C4 mechanism is 2.650Å long. This stronger O · · ·H-N interaction in the C3 mechanismlowers the barrier height significantly with respect to thatobtained for the C4 mechanism.

Geometrically, 5TSHA,A,C4 is in a more upright position than5TSHA,A,C3 with an Fe-O-Cproline angle of 150.8° compared to140.8°, respectively. Furthermore, 5TSHA,A,C4 is early with shortC-H and long O-H distances (rCH ) 1.228 Å, rOH ) 1.320Å) in comparison to those found for the 5TSHA,A,C3 (rCH ) 1.301Å, rOH ) 1.207 Å). The magnitude of the imaginary frequencyin the transition state for all H-abstraction barriers are large,which is indicative of narrow and steep barriers, which willresult in a significant amount of tunneling in the H-abstractionprocess. The final H-abstraction transition state, 5TSHA,A,C5, iswell lower than that expected based on the BDECH of the C5-Hbond that is broken. Thus, a BDECH for H-abstraction fromproline at the C5 position of 85.4 kcal mol-1 compares to thevalue obtained for aliphatic hydrogen abstraction from toluene,which gave a H-abstraction barrier of 7.6 kcal mol-1 using asimilar nonheme iron-containing oxidant.26b The substantiallowering of the barrier height is due to the geometric distortionof the oxidant, whereby the imidazole group of His143 bends

upward and donates a hydrogen bond to the amide group ofthe substrate and hence is lowered in energy.

This considerable distortion of the orientation of the histidinegroup may not be possible in the actual enzyme due to stericinteractions. The small model (A), therefore, may not be anappropriate model for the studies of proline hydroxylation byP4H enzymes, and in particular, the C5 hydroxylation mecha-nism is disturbed due to artificial bonding patterns that cannothappen in the actual enzyme configuration. We, therefore,decided to study a larger enzyme model of P4H enzymesinstead.

Figure 7 shows the potential energy profile of prolinehydroxylation using the large active site model of P4H (modelB) as calculated on the quintet spin state surface. Similarly tothe results shown above using the smaller model, the reactionis stepwise via a radical intermediate with a rate-determininghydrogen atom abstraction barrier. Substrate hydroxylation atthe C4 position gives energies in line with those obtained withthe small model above. The H-abstraction barrier (5TSHA,B,C4)is 12.6 kcal mol-1 higher in energy than reactants and is onlyslightly raised with respect to the small model system (10.6kcal mol-1), where the substrate has no stereochemical interac-tions with second sphere amino acids. Hydrogen atom abstrac-tion from the C3 position is raised by 7.3 kcal mol-1 in modelB with respect to model A and is now slightly higher in energyto that obtained for the C4 position: 5TSHA,C,C3 ) 13.5 kcal mol-1.

In addition to the mechanisms for hydrogen abstraction fromproline at the C3 and C4 positions for model B, we also madeefforts to find the C5-H abstraction mechanism. Although wewere able to locate the radical intermediate (52B,C5), we did notmanage to find its corresponding H-abstraction transition state(5TSHA,B,C5). Thus, a geometry scan for the hydrogen transferstep starting from 52B,C5 led to a high-energy pathway (>30 kcalmol-1), which implies that the steric hindrance of the twoaromatic residues (Tyr140 and Trp243) included in the model blockthe hydrogen atom abstraction from the C5 position effectively.Although, the substrate approach leading to H-abstraction fromthe C5 position gave favorable hydrogen-bonding interactionsand lowering of the barrier for the small model, as a matter offact for the larger and more realistic model these hydrogen-bonding interactions are not possible and instead the approachis hampered and the barrier considerably raised in energy. Thisis important for the enzyme due to the weak C5-H bond strengthof proline and prevention of hydroxylation at that position. Itappears, therefore, that the substrate approach to the active centeris aligned in such a way as to favor the C4 hydroxylationmechanism and stereochemically disfavor hydroxylation on theother positions of proline. Furthermore, it may be anticipatedthat mutants whereby the Tyr140 and/or Trp243 amino acids arereplaced by smaller size amino acids should give substantialhydroxylation at the C3 and C5 positions. Recent experimentalstudies on an R-ketoglutarate-dependent halogenase highlightedthe intricate involvement of the active site amino acid residuesby favoring a regioselective halogenation over hydroxylationof the substrate, which could be reversed by active sitemutations.43

Geometrically, 5TSHA,B,C4 is quite different from 5TSHA,A,C4

(compare Figures 6 and 7) with a relatively long C-H distanceof 1.326 Å (compared to 1.228 Å in 5TSHA,A,C4) and short O-Hdistance of 1.194 Å (compared to 1.320 Å in 5TSHA,A,C4). Thus,5TSHA,B,C4 is late with a geometry close to intermediate, whereas5TSHA,A,C4 has a more symmetrical C-H-O orientation.

The hydrogen atom abstraction process leading to radicalintermediates is slightly exothermic and has the largest exother-


micity for H-abstraction from the C5 position. This is in agreementwith the discussion above that the energy difference betweenreactants and radical intermediates equals the BDECH - BDEOH

difference of substrate and oxidant (eq 8). The reaction energyfor formation of the radical intermediates in model A issignificantly larger than that obtained for model B due to theformation of hydrogen-bonding interactions of the amide groupof proline with hydrogen-bonding donors of the metal ligands.Due to stereochemical interactions of the aromatic residues in

model B, the substrate cannot approach the iron center closelyand therefore these hydrogen-bonding interactions are notpossible; hence, those structures are somewhat higher in energy.The reaction energies for formation of 52B,C4 and 52B,C5 are within1 kcal mol-1 of the ∆H values calculated from eq 8 and hencefollow thermodynamic principles.

A comparison of Figures 6 and 7 reveals a further effect ofthe stereochemical interactions of the aromatic residues, namely,on the radical rebound barriers that increase from 1.5 kcal mol-1

Figure 7. Potential energy landscape of proline hydroxylation at the C4 and C3 positions using model B. Relative energies are in kcal mol-1,obtained with basis set BS2, while ZPE corrections are calculated with basis set BS1. Optimized geometries contain bond lengths in angstroms andthe imaginary frequency in wavenumbers.

Figure 8. Kinetic isotope effects (KIE) as calculated with the Eyring, and Wigner and Bell models for the replacement of one or both of thehydrogen atoms at the C4 position of proline with deuterium atoms. Also given are the imaginary frequencies in the transition states. Ha is thetransferring hydrogen atom, and replacement of Hb with a deuterium atom gives the secondary kinetic isotope effect.


in model A (5TSreb,A,C4) to 8.2 kcal mol-1 in model B (5TSreb,B,C4).Similar trends are observed for proline hydroxylation at the C3

position. These enlarged radical rebound barriers will increasethe lifetime of the radical intermediates significantly duringwhich, for instance, rearrangement of the radical can take place,leading to side products.44 Despite the rise in rebound barrier,however, the H-atom abstraction barrier is still the rate-determining step in the reaction mechanism. The reactionexothermicities found for the large models are similar to thosefound for the smaller models (compare Figures 6 and 7). Theselarge values for reaction exothermicities follow thermodynamicprinciples and are comparable to values obtained before forsubstrate hydroxylation by iron(IV)-oxo complexes.45 Inparticular, for substrate dehydrogenation, which also starts withan initial hydrogen atom abstraction step, it was shown that thereaction energy can be written as a sum of bond dissociationenergies of the bonds that are broken and formed during thereaction process.46 That way, reaction energies of 50 kcal mol-1

were rationalized.Subsequently, we calculated the kinetic isotope effect for

replacement of one or two of the hydrogen atoms at the C4

position of substrate proline by deuterium atoms using modelB, and the results are shown in Figure 8. These procedures usethe semiclassical kinetic isotope effect due to Eyring and arebased on the free energies of activation of the hydrogen anddeuterium-substituted reactions (eq 1). Subsequently, twomethods to estimate the difference in tunneling properties of ahydrogen versus a deuterium atom were used, namely, via themethods of Wigner and Bell.28 The Eyring kinetic isotope effectfor replacement of the transferring hydrogen atom by deuteriumis substantial (7.7) and increases to 9.9 when tunnelingcorrections due to Wigner are included and to 16.0 using theBell model. Interestingly, the secondary isotope effect is largerthan 1, although only slightly, which contrast the typical valueof 0.9 obtained before.44 As a consequence, the KIE value forsubstitution of both hydrogen atoms by deuterium atomsincreases to 11.2 and 18.4 with tunneling corrections includeddue to Wigner and Bell, respectively. These values point to asubstantial kinetic isotope effect and considerable tunnelingcorrections, which are as expected for hydrogen atom transferprocesses. Nevertheless, the values obtained here are somewhatsmaller than those observed experimentally for the nativesubstrate, where a KIE ) 60 was obtained.15 This may have todo with the difference in substrate used or due to interactionsof the rest of the protein with the substrate-oxidant complex.

Conclusions

In this work, the regioselectivity of proline hydroxylation byP4H enzymes is investigated using density functional theorymethods. It is shown that thermodynamically substrate hydroxyl-ation at the C5 position should be favorable over that on the C4

position with significantly larger H-abstraction exothermicity.Stereochemical interactions, in particular, of two aromatic aminoacid residues (Tyr140 and Trp243) hamper H-abstraction from theC3 and C5 positions and thereby lead to a regioselectivitypreference of C4 hydroxylation. These stereochemical interac-tions affect the hydrogen abstraction as well as radical reboundbarriers, but the former remains the rate-determining step inthe reaction mechanism. Thus, these stereochemical interactionsare essential for the enzyme activity and make sure that prolineis regioselectively hydroxylated at the unfavorable C4 position.We validated the models by matching the computed spectro-scopic values with experimental ones (IR spectra, Mossbauerand EPR spectroscopy). The iron(IV)-oxo model gives good

agreement between experimental and DFT calculated spectro-scopic parameters.

Acknowledgment. The research was supported by CPU timeprovided by the National Service of Computational ChemistrySoftware (NSCCS). D.K. holds a Ramanujan Fellowship fromthe Department of Science and Technology (DST), New Delhi(India), and acknowledges its financial support (Research GrantsSR/S2/RJN-11/2008 and SR/S1/PC-58/2009).

Supporting Information Available: Cartesian coordinatesof all structures, tables with group spin densities, charges, andabsolute energies of all structures calculated in this work, andfigures with optimized geometries and geometry scans. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

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