Proximal Pocket Controls Alkene Oxidation Selectivity of Cytochrome

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Proximal Pocket Controls Alkene Oxidation Selectivity of Cytochrome P450 and Chloroperoxidase toward Small, Nonpolar Substrates David C. Chatfield, and Alexander N. Morozov J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04279 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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The Journal of Physical Chemistry

TITLE: Proximal Pocket Controls Alkene Oxidation Selectivity of Cytochrome P450 and Chloroperoxidase toward Small, Nonpolar Substrates David C. Chatfield* and Alexander N. Morozov Department of Chemistry and Biochemistry, Florida International University, 11200 8th St., Miami, FL 33199, United States [email protected] ABSTRACT: This paper examines the influence of the proximal pockets of cytochrome P450CAM and chloroperoxidase (CPO) on the relative favorability of catalytic epoxidation and allylic hydroxylation of olefins, a type of alkene oxidation selectivity. The study employs quantum mechanical models of the active site to isolate the proximal pocket’s influence on the barrier for the selectivity-determining step for each reaction, using cyclohexene and cisb-methylstyrene as substrates. The proximal pocket is found to preference epoxidation by 2-5 kcal/mol, the largest value being for CPO, converting the active heme-thiolate moiety from being intrinsically hydroxylation-selective to being intrinsically epoxidation-selective. This theoretical study, the first to correctly predict these enzymes’ preference for epoxidation of allylic substrates, strongly suggests that the proximal pocket is the key determinant of alkene oxidation selectivity. The selectivity for epoxidation can be rationalized in terms of the proximal pocket’s modulation of the thiolate’s electron “push” and consequent influence on the heme redox potential and the basicity of the trans ligand. 1. INTRODUCTION Cytochrome P450 (P450) and chloroperoxidase (CPO), versatile heme-thiolate enzymes with similarities in active-site structure and reactivity, have elicited wide interest due to impact on human health (P450) and biotechnological potential (both enzymes).1-4 By hydroxylating them, P450 renders xenobiotic molecules water-soluble and thus expellable via the liver; P450 also plays a role in steroid synthesis. Chloroperoxidase, produced by the marine fungus C. fumago, halogenates small substrates to create compounds toxic to predators.5-7 In addition to these, their native biological reactions, P450 and CPO manifest

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promiscuous activities, including the dismutation of peroxides and the dehalogenation, dehydrogenation, and epoxidation of small organic molecules.3, 8-9 The epoxidation and allylic hydroxylation of alkenes, competitive monooxygenase activities, are of particular interest because either activity may be desirable or undesirable, depending on the circumstance. For example, both enzymes catalyze the epoxidation of small, olefinic substrates with high enantiospecificity, a potentially useful activity because chiral epoxides are synthons10-11 for the production of drug molecules. On the other hand, epoxides formed via P450 activity can covalently modify or intercolate into DNA, with harmful health effects.3 Numerous experiments aimed at creating a practical, CPO-based, biocatalytic system have been reported, e.g., via immobilization of CPO on nanostructures.12-18 Understanding the selectivity of P450 and CPO for allylic hydroxylation vs epoxidation of alkene substrates is thus topical and important. The catalytic cycle of both enzymes begins with formation of Compound I (Cpd I), a highly reactive ferryl porphyrin pradical cation intermediate common epox

H

H

O

H

H

O

to classical peroxidases, catalases, and

Fe

Fe

monooxygenases, via two-electron

SR

SR

oxidation of the heme center using a

+

suitable peroxide (CPO) or molecular

O

oxygen and cofactors such as

Fe SR

H hydrox

H

H

OH

NAD(P)H (P450).3 A powerful oxidant,

O Fe

Fe

SR

SR

Figure 1. Oxidation reactions of cyclohexene with Compound I.

Cpd I directly catalyzes the wide set of

oxidative reactions mentioned above. The mechanisms of P450- and CPO-

catalyzed hydroxylation and epoxidation from Cpd I, illustrated for cyclohexene in Figure 1, have been elucidated with experiment and theory;19-27 both are oxygen insertion reactions, insertion being into a C-H s-bond (hydroxylation) or a C=C p-bond (epoxidation). The ratio of epoxidation to allylic hydroxylation products [C=C/C-H ratio or alkene oxidation selectivity (AOS)] has been measured for several substrates, including propene, 2-butene, cyclohexene, and cis-b-methylstyrene (CBMS), for catalysis by CPO or P450


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isozymes.10,

28-30

The AOS varies according to substrate and enzyme; on average,

epoxidation is slightly favored.24-25, 30 Theoretical studies of the AOS have been reported for several substrates using small Cpd I models as catalysts.24-26, 31 These studies provided a rationale for trends in the AOS based on properties of substrates.25 The impact of the environment of the proximal sulfur was assessed computationally using ammonia molecules to mimic hydrogen bonds from backbone amide groups.24, 26 The AOS of cytochrome P450CAM (P450) toward cyclohexene and propene has been evaluated with fullenzyme simulations,32-33 but the observed preference29,

34

for epoxidation was not

reproduced. The methodology in these simulations was to sample substrate/enzyme binding poses with molecular dynamics (MD) simulation; calculate reaction barriers for a set of frames selected from the MD trajectory, using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach; and determine the average barrier. Implicitly, though, the uncertainties were very large due to limited sampling, highlighting the challenge that conformational sampling presents for predicting reaction selectivities from molecular simulation.35-36 Our investigation focuses on two structural aspects of the proximal pocket and their influence on the AOS. The first of these is hydrogen bonding to the sulfur atom of the proximal cysteinate, a conserved feature of heme-thiolate enzymes.37 Located in a turn at the end of an a-helix, this sulfur is an acceptor for two (CPO38) or three (P45O39) hydrogen bonds from backbone amides of the helix. In addition to these NH-S hydrogen bonds, we also considered the possible influence of the dipole moment of the proximal a-helix on the AOS. Oriented nearly perpendicular to the heme plane in CPO and parallel to the heme plane in P450 (Figure 2), the helix possesses a dipole moment whose electropositive end points toward the N-terminus, decreasing the anionic character of the proximal thiolate ligand.37, 40-42


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a)

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b)

Figure 2. a) CPO active site with proximal helix, b) P450 active site with proximal helix. The reason for our focus on the NH-S hydrogen bonds and proximal helix’s dipole moment is that these features are known to influence the reactivity of the heme center, via modulation of the electron donating ability (the “push”) of the proximal cysteinate. For example, experimental and theoretical evidence indicates that they increase the heme’s redox potential by significant amounts: 36- 200 mV for an individual NH-S hydrogen bond and 130 or 70 mV for an a-helix in the CPO or P450 orientation, respectively.29, 43-44 We refer the reader to our prior publications for a review of the literature on the influence of the NHS hydrogen bonds and the proximal helix on the electron-donating properties of the proximal thiolate ligand.27, 45 The influence of the NH-S hydrogen bonds on the full reaction profiles for hydroxylation and epoxidation was first studied with DFT calculations on small heme-thiolate models that incorporated ammonia molecules hydrogen bonded to a proximal SH– group.24-25 More recently, our group has used models incorporating most of the proximal helix to study the combined influence of the NH-S hydrogen bonds and the helix dipole moment on the formation of Cpd I,45 the mechanism of epoxidation of CBMS,27 and the enantiospecificity of epoxidation of CBMS.46-47 We have shown that the barrier for epoxidation of CBMS is lowered significantly (4.6 kcal/mol) by the proximal helix, with at


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least one third of this amount being due to the helix dipole moment and the rest due to the NH-S hydrogen bonds.27 Here we expand on this work to understand the source of the AOS. The substrates studied here lack strongly orienting interactions with residues of the distal binding pocket; consequently, the intrinsic reactivity of the heme-thiolate group with these substrates may make a significant contribution, perhaps the dominant one, to the AOS of P450 and CPO toward them. This observation led us to predict the intrinsic AOS of P450 and CPO in a manner that avoids the issue of sampling mentioned above, and in so doing, to identify the structural basis for the AOS. Our approach is based on determining key portions of potential energy surfaces (PES's) for the respective enzymatic reactions. For each substrate/enzyme combination, two active-site models were created, one with a bare hemethiolate and one that also includes the proximal pocket, so that comparison of the PES’s reveals the influence of the proximal pocket on the intrinsic reactivity. The selectivitydetermining barrier is higher for epoxidation than for hydroxylation for all the bare hemethiolate systems. Inclusion of the proximal pocket lowers all the barriers, with those for epoxidation being lowered more, resulting in a preference for epoxidation for all three enzyme/substrate combinations. As this is in accord with experiment, we hypothesize that for small nonpolar substrates that can float freely in the distal pocket, the intrinsic reactivity determines the alkene oxidation selectivity. These results can be rationalized in terms of the proximal pocket’s modulation of the thiolate’s “push,” and consequently of the heme redox potential and of the basicity of the trans ligand (vide infra). 2. COMPUTATIONAL METHODS The reactions proceed on two closely spaced spin surfaces, the doublet and the quartet. We have shown previously that the influence of the proximal pocket on the epoxidation of CBMS by CPO is similar on the two spin surfaces.27, 45 We restricted the investigation of AOS presented here to the doublet spin surface for simplicity, but from our previous work, we expect that the results will be similar on the quartet surface. The doublet spin surfaces for hydroxylation and epoxidation were determined with unrestricted DFT calculations using the B3LYP hybrid density functional

48-49

without symmetry

restrictions, employing NWChem 6.1 software.50 The LANL2DZ effective core potential


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(ECP) double-ζ basis set for Fe51 and the 6-31G** basis set for H, C, N, O52 and S53 atoms (basis set B0) were used for geometry optimization. Geometry-optimizing with polarization functions on hydrogen atoms, represented by the second asterisk, is especially important when, as is the case for hydroxylation, hydrogen atom transfer is involved.54 Our calculations confirmed this, as without these polarization functions, transition state energies were higher by ~2 kcal/mol for hydroxylation were changed by much less than (0.1-0.7 kcal/mol) for epoxidation. Stationary-point energies were refined using the LANL2TZ+ ECP triple-ζ basis set for Fe51 and the 6-311++G** basis set for H, C, N, O55 and S56 atoms (basis set B1). The stability of the density functions obtained was checked with the B0 basis set using Gaussian-09.57 Zero point energy (ZPE) corrections were determined from frequency calculations performed with the B0 basis and are included in the energies of all stationary points. Natural population analyses58 (NPA) of atomic spin densities and charges were carried out using NBO 6.0 software.59 The Cpd I models were based on R-FePor(IV)=O (N4C20H12) species in which, for computational efficiency, the heme’s vinyl and propionate side chains were omitted. The 43-atom, bare heme-thiolate model (HT-I) employs R = (SCH3) and thus lacks the proximal pocket, most importantly the iron’s secondary coordination sphere (hydrogen bonds to the axial sulfur) and the proximal helix. Calculations involving the HT-I model used no geometric constraints; consequently, transition state structures had one imaginary normal mode frequency, and stable structures had only positive, real frequencies. The influence of CPO’s proximal pocket was studied using a model (CPO-I) with R = (CH3-CO-Ala-Pro-Cys-Pro-Ala-Leu-Asn-NH-CH3) (Figure 2a). This peptide fragment comprises CPO residues 27-33, the backbone carbonyl of residue 26, and the NH-C H fragment of residue 34, and includes all atoms within 8 Å of the axial sulfur. Residues 2934 of the proximal α-helix are represented, as is the iron’s secondary coordination sphere (the A31:NH-C29:S and L32:NH-C29:S hydrogen bonds) and the sulfur’s immediate steric environment. The α-helix in this model is truncated; its dipole moment is derived primarily from the two backbone hydrogen bonds closest to the proximal thiolate, C29:CO–N33:NH and P30:CO–A34:NH. Residues 35-38, not included due to computer resource limitations, would add three more backbone hydrogen bonds to the α-helix. Thus, the effect of the


6

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proximal helix dipole in CPO-I represents a lower bound to the actual effect. The proximal peptide fragment of CPO-I was constrained to maintain backbone and side-chain hydrogen bond distance and angle parameters, backbone φ and ψ dihedral angles, and the orientation of the proximal helix relative to the heme moiety as in the crystal structure of CPO (PDB Code 1CPO).38 The influence of P450’s proximal pocket was studied using a model (P450-I) with R = (CHO–Cys–Gly–Gly–Gln–Gly–Gly–Ala–NH–CH3) and additionally including a CHO-Phe-NHCH3 fragment to complete the axial sulfur’s steric environment (Figure 2b). The side chains of residues Leu358, Glu359, His361, and Leu362, which protrude away from the helix core and the immediate environment of the axial sulfur, were replaced with hydrogen atoms, creating Gly residues. Residues 351-356, which join the two fragments, were omitted (with the exception of the terminal NH and CO groups) because they belong to a loop that bends first away from the axial sulfur, and then back to situate Phe350 in close proximity to the axial sulfur. In this way, the most important components of the secondary coordination sphere were included while keeping the model small enough to treat with our computer resources. Just as for CPO-I, constraints were applied to the R group of P450-I to maintain the crystal-structure conformation. A special set of constraints was designed to maintain the orientation of the Phe350 fragment relative to the axial sulfur and the proximal helix. The stationary points of the CBMS/CPO-I, CBMS/P450-I, and cyclohexene/P450-I reactant, product and transition state complexes have extra normal modes with imaginary frequencies caused by the constraints (imaginary frequencies were not included in the ZPE corrections). The NH–S hydrogen bonds were not constrained. Further details regarding the constraints applied are given in the Supporting Information (SI) (Data S1). Structures of the reactant complex and the highest-energy transition state were determined for each reaction type for all five substrate/active-site-model combinations. We have previously published doublet and quartet PES’s for the epoxidation of CBMS by CPO-I and by HT-I.27, 47 Two epoxide enantiomers can be formed, 1S2R and 1R2S, the former being favored by CPO (96% experimental enantiomeric excess10). We modeled only the transition state that leads to the 1S2R product for the epoxidation reactions described here. The reactant and transition-state structures for the epoxidation of CBMS by CPO-I and


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HT-I are the same as those published previously.27 These reactant structures were also used for hydroxylation. The hydroxylation transition-state structures were generated from the reactant structures by using constrained optimization to adjust the position of the reactive hydrogen, followed by full transition-state optimization. For the P450-catalyzed hydroxylation and epoxidation reactions of CBMS and cyclohexene, we began with the crystal structure of a camphor/P450 complex.32 The camphor was replaced with a transition-state-like conformation of the substrate (either CBMS or cyclohexene), using an atomic coordinate set from a previously determined, fullenzyme, QM/MM transition-state structure60 and orienting it to fit within the van der Waals envelope of the deleted camphor. This effectively constituted a transition-state-precursor structure for our system. Transition-state optimization generated the transition state, and from it, intrinsic reaction coordinate (IRC) calculations followed by full optimization led to the reactant. For HT-catalyzed hydroxylation or expoxidation of cyclohexene, the proximal pocket was deleted from the cyclohexene/P450-I reactant structure, except that the SCH2 moiety of Cys357 was retained and mutated to a methyl thiolate. Full optimization then generated the desired reactant. Constrained optimization to reposition the reactive hydrogen, followed by full transition-state optimization, generated the transition state. The Cartesian coordinates of all reactant and transition state structures are given in the SI (Data S2). 3. RESULTS The hydroxylation reaction begins with hydrogen atom transfer from C3 (methyl for CBMS, methylene for cyclohexene; see atom numbering scheme in Figure 3 below) to the ferryl oxygen, following which the resulting methyl and hydroxyl radicals recombine (“rebound” step). Epoxidation begins with formation of the O-C2 bond via ferryl oxygen attack on the olefinic p-bond, followed rapidly by O-C1 bond formation to complete the ring and sever the O-Fe bond. The completion of either reaction and product release returns the heme to the ferric resting state, ready for another round of catalysis.


8

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Figure 3. Atom numbering scheme for CBMS and cyclohexene.

The optimized structures of the key stationary points for these reactions are shown in Figures 4 and 5. Each panel depicts the structures of the reactant and the transition states for the epoxidation and hydroxylation reactions for one enzyme/substrate combination. [Expanded images depicting just the substrate and the OFeN4 portion of the heme are given in the SI (Data 3).] The reactant, substrate-bound Cpd I, is assumed to be the same for both reaction types. Reorientation of the substrate to facilitate approach of C2 to the ferryl oxygen leads to the transition state for epoxidation. Likewise, substrate reorientation and incipient hydrogen atom transfer from C3 to the ferryl oxygen leads to the hydroxylation transition state. The transition state C2-O distances (epoxidation) and H-O distances (hydroxylation) are consistent with previous calculations for similar systems.24-26


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(a)

(b)

(c)

Figure 4. Reactant (left), epoxidation transition state (center), and hydroxylation transition state (right) conformations for the reaction of CBMS with the (a) HT, (b) P450, and (c) CPO models.


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(a)

(b)

Figure 5. Reactant (left), epoxidation transition state (center), and hydroxylation transition state (right) conformations for the reaction of cyclohexene with the (a) HT and (b) P450 models. Selected interatomic distances for the structures are given in Table 3; some are also shown in Figures 4 and 5. The P450 and CPO reactant models have longer S-Fe distances than the HT model, indicating that the NH-S hydrogen bonds decrease the iron-sulfur bond covalency, as observed previously.44 The length of the iron-oxygen bond decreases modestly in response. For the transition state structures, the O-C2 distance (epoxidation) or the O-H distance (hydroxylation) indicates whether the transition state is early (long distance) or late (short distance). In all cases except one, early vs. late tracks the barrier heights, given in Table 2. Thus, for example, for the epoxidation of CBMS, the P450 model has the longest O-C2 distance and the smallest barrier, while the HT model has the shortest


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O-C2 distance and the largest barrier. Similar trends are observed for the epoxidation and hydroxylation of cyclohexene. The one system that does not fit this trend is the hydroxylation of CBMS by the CPO model, which has a smaller OFe-H distance but a larger hydroxylation barrier than the P450 model. This is probably related to the effects of the proximal pocket on the heme’s redox potential, which influence the hydroxylation reaction in compensating ways (vide infra). Table 1. Selected bond lengths (Å). S-Fe

Fe-O

a O-H

H-C

O-C1

O-C2

CBMS HT

R

2.623

1.623

2.019

P450

R

2.719

1.622

2.014

CPO

R

2.777

1.619

2.015

HT

TSep 2.554

1.705

2.020

1.984

2.634

P450

TSep 2.541

1.657

2.021

2.134

2.696

CPO

TSep 2.569

1.658

2.021

2.099

2.671

HT

TShy 2.510

1.714

2.015

1.240

1.307

P450

TShy 2.600

1.710

2.015

1.247

1.298

CPO

TShy 2.624

1.711

2.016

1.263

1.281

HT

R

2.623

1.623

2.019

P450

R

2.722

1.621

2.014

HT

TSep 2.511

1.723

2.020

1.925

2.534

P450

TSep

2.519

1.662

2.015

2.090

2.620

HT

TShy

2.511

1.715

2.020

1.257

1.282

P450

TShy

2.577

1.706

2.015

1.283

1.260

Cyclohexene

aAverage over the four heme nitrogens.

The natural spin densities and charges serve to confirm the identity of species and


12

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provide insight into the reaction. Selected values are shown in Figure 6 and 7 below; more comprehensive listings are given in tabular form in the SI (Data S3 and S4). The values are consistent with previous calculations for the heme thiolate species involved.23-26 Here we describe a few of the salient trends. In the reactant, Cpd I, almost all of the methyl thiolate’s -1 charge is shifted to the porphyrin ring in the HT model, so the delocalized radical resides mainly on the sulfur. In the P450 and CPO models, the NH-S hydrogen bonds shift a portion of this charge back to the sulfur, so the radical is more evenly distributed between the sulfur and the porphyrin. These trends can be observed in the data in Figure 6 and are consistent with previous model-system calculations.23-26 Figure 6. Selected natural group spin densities (left) and charges (right) for the reaction of CBMS with the HT, P450, and CPO models.

The NH-S hydrogen bonds in CPO alter the electronic mechanism of step 1 in the epoxidation reaction, as we have shown previously.27 With the HT model, the first electron transfer reduces the iron from Fe(IV) to Fe(III), following which a second electron transfer


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reduces the porphyrin/sulfur radical cation. With the CPO model, on the other hand, the porphyrin/sulfur radical cation is reduced first. This difference can be traced to stabilization of the a2u+ss orbital by the NH-S hydrogen bonds and can be observed in the spin densities and charges for the epoxidation transition states (TSep). Comparison of the sum of the spins on the S-R and porphyrin groups (S-Por) with the corresponding sum for the Fe and O (Fe-O) is particularly instructive (Figures 6 and 7). For the CBMS reaction, as the reaction progresses from reactants to the transition state, the S-Por spin is nearly unchanged for the HT model (-1.0 à -1.0), while the Fe-O spin changes noticeably (2.0 à 1.7). For the P450 and CPO models, the reverse is true: the S-Por spin changes substantially (-1.1 à -0.5), while the Fe-O spin is only slightly changed (2.1 à 1.9). Meanwhile, the sign of the transition-state spin density on C1 (Figure 7) is different for the HT model compared to the P450 or CPO models because, for the doublet state, the half-filled a2u+ss orbital and p* orbitals (p*xz and p*yz) are anti-ferromagnetically coupled (having different spins, they must receive electrons with different spins transferred from the substrate’s p-orbital). These observations are consistent with the mechanistic difference described above and are nearly identical for the cyclohexene reaction. The charges reflect this mechanistic difference in a more muted way. No such change in electronic mechanism is observed for the hydroxylation reaction, consistent with previous calculations.24, 61 The iron is reduced in the first step in all of the models; this is reflected in the spin densities.


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Figure 7. Natural atom/group spin densities for the reaction of CBMS with the HT, P450, and CPO models.

The barrier heights for epoxidation and hydroxylation are given in Table 2. For the HT model, hydroxylation is favored, in contrast to the experimental results for P450 and CPO,10, 28-29 which favor epoxidation. The HT barriers are consistent with previous calculations on HT models (3-5 kcal/mol differences between the barriers for epoxidation and hydroxylation).32-33 The particularly high barrier for epoxidation of cyclohexene is probably due to the unstable radical intermediate; the CBMS radical intermediate is resonance-stabilized by the adjoining phenyl ring. When the proximal pocket is added to give the P450 or CPO model, epoxidation becomes favored, bringing the model computations and experiment into qualitative agreement. Table 3 shows the stabilization of the transition state (relative to the reactant) by the proximal pocket. The degree of stabilization is greater for epoxidation than for hydroxylation in all cases. P450’s proximal pocket provides greater transition-state


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stabilization than CPO’s for both epoxidation and hydroxylation, judging from the CBMS results. The largest and smallest differences between the epoxidation and hydroxylation barrier heights are for CBMS/CPO and cyclohexene/P450, respectively, consistent with the experimental enantiomeric excesses.10,

29

The agreement between calculation and

experiment is striking, suggesting that the proximal pocket is the key factor in determining the alkene oxidation selectivity. Table 2: Transition state energies (B1//B0, kcal/mol).a

HT

CBMS

Cyclohexene

E‡ep

14.8

16.3

E‡hy

14.3

13.8

8.6

9.2

E‡hy

10.5

11.6

E‡ep

9.5

E‡hy

14.1

‡ P450 E ep

CPO

aRelative to reactant energies.

Table 3: Stabilization of transition state by proximal pocket (kcal/mol).a CBMS CPO

Epox

cyclohexene

5.3

Hydrox 0.2 P450 Epox

6.2

7.1

Hydrox 3.8

2.3

aCalculated as the difference between the transition-state energies (Table 2) for the HT

model and the corresponding CPO or P450 model. 4. DISCUSSION For substrates that tumble freely in the binding pocket, the relative barriers for substrate oxidation determine the AOS (thinking of enzymes like P450 and CPO).62 Molecular dynamics simulations demonstrate that cyclohexene and CBMS, being nonpolar,


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do tumble freely in the binding pockets of P450 and CPO.46, 60, 62 Comparison of the transition state energies for the substrate oxidation steps leading to epoxidation and hydroxylation is therefore appropriate for predicting the AOS. In other cases of oxidation by these enzymes, for substrates that do not tumble freely, the substrate oxidation step does not determine the AOS, as the second electron transfer step is rate determining.62-63 This subtlety has led to differing predictions for the AOS of P450CAM,32-33 as discussed previously.33, 62 In any case, since the substrates considered here do tumble freely, the relative transition state energies are expected to determine the AOS. Comparison of our results with experimental data strongly suggests that the proximal pocket is a key determinant of the AOS. This is demonstrated in Table 4, which compares the differences between the epoxidation and hydroxylation barrier heights calculated for our model systems with barrier height differences calculated from experimental enantiomeric excesses using a simple, Arrhenius relationship. Epoxidation is favored over hydroxylation in all cases, to a degree increasing in the order CBMS/P450, cyclohexene/P450, CBMS/CPO. Experiment and the computations using the P450 and CPO models display the same trend. The agreement is striking, particularly given that the model systems only represent a portion of the enzyme systems. Although there is a modest discrepancy between the experimental and theoretical values, it is probably within the error of the theoretical method,54 particularly given the assumptions in applying an Arrhenius relationship to the experimental data. By contrast, the barrier differences for the HT model favor hydroxylation over epoxidation. The evidence strongly suggests that the proximal pocket is an important determinant of the observed selectivity. Table 4. Effective barrier height differences (epoxidation minus hydroxylation, kcal/mol) from experiment and calculation. Experimenta

Calculationb

substrate/enzyme

Selectivity

DE‡eff

DE‡enz

DE‡HT

CBMS/P450

2:1

+0.4

+1.9

-0.5

cyclohexene/P450 5:1

+1.0

+2.4

-2.5


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CBMS/CPO

∞:1c

--

+4.6

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-0.5

aDetermined from experimental enantiomeric excesses.10, 29 Selectivity is moles epoxidation

product : moles hydroxylation product; DE‡eff is the effective barrier height difference assuming an Arrhenius relationship. bD E‡enz and DE‡HT are the barrier height differences for the enzyme (P450 or CPO) and HT active site models, respectively. cNo hydroxylation product could be detected.10 The data raise the question of why the proximal pocket favors epoxidation over hydroxylation. An early explanation invoked “redox mesomerism,” whereby electronwithdrawing moieties were thought to influence the electrophilic and radical character of the iron-oxo moiety and thereby the favorability of hydroxylation.24 However, models with and without NH-S hydrogen bonds do not show a significant difference in the spin density on the ferryl oxygen in DFT calculations.24 Later, also on the basis of DFT studies, it was shown that the AOS is correlated with the ionization energy and CH bond dissociation energy of the substrate.25 Selectivities for a given oxidant toward different substrates can be predicted on this basis, but not differences between oxidants. More recently, it was discovered that the ferryl oxygen of Cpd I is quite basic in heme-thiolate proteins, and consequently, P450-catalyzed hydroxylation of hydrocarbon substrates depends on the interplay of redox potential and basicity.62, 64-66 We believe this is the key to understanding why the proximal pocket favors epoxidation over hydroxylation. The first and rate-determining step for conversion of the substrate/Cpd I complex to the enzyme-bound hydroxylation product is H atom transfer from the substrate’s carbon to the ferryl oxygen. This can be considered a combination of an electron transfer, favored by a high redox potential, and an H+ transfer, favored by a high basicity. Therefore, either a high redox potential or a high basicity can in principle drive the reaction. The particularly high Cpd I basicity of heme-thiolate enzymes such as P450 enables these enzymes to oxidize substrates while avoiding a redox potential so high as to risk autoxidation of Tyr or Trp residues in the protein matrix.64-65 The epoxidation of alkene substrates, on the other hand, does not involve H+ transfer, so the redox potential of Cpd I is of prime importance, while the basicity is relatively unimportant.


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The strong electron “push” exerted by the proximal cysteinate ligand, thought to be critical for heme-thiolate enzymes’ impressive spectrum of reactivities,25, 65 is fine-tuned by the proximal pocket.27, 43, 45, 47 In P450 and related model compounds, NH-S hydrogen bonding and the helix dipole moment have both been shown to increase the redox potential and would also be expected to reduce the basicity. This would have a mixed influence on hydroxylation, which is promoted by an increased redox potential yet impeded by a reduced basicity, but a strictly favorable influence on epoxidation, which involves electron but not proton transfer. It is understandable, then, that the proximal pocket favors epoxidation over hydroxylation in all three cases that we studied. Several earlier studies are directly pertinent to our findings. Morishima and coworkers29 showed, via studies on the L358P mutant of P450, that loss of one of three NHS hydrogen bonds decreases the C=C/C-H ratio for cyclohexene oxidation. The decrease is small, but then the L358:N-C357:S hydrogen bond is the longest and weakest of the three NH-S hydrogen bonds. Shaik and coworkers67 showed via computations on a heme-thiolate Cpd I model system that an external electric field will modulate the regioselectivity of propene oxidation. In particular, when the field is oriented perpendicular to the heme plane with the electropositive direction pointing toward the proximal sulfur, the C=C/C-H ratio is increased. Both of these reports are consistent with our findings concerning the proximal pockets of P450 and CPO on the intrinsic AOS. On the other hand, computations by Shaik and coworkers24 showed a decrease in the C=C/C-H ratio for the oxidation of propene when NH-S hydrogen bonds were added to a small heme-thiolate Cpd I model system. The calculations employed SH— to represent the proximal cysteinate and ammonia molecules to replace the amino acid residues involved in the NH-S hydrogen bonds. The use of SH— is common in such situations, as it gives a good representation of the orbitals obtained using a full cysteinate ligand.68 Nevertheless, given the inconsistency with other data, it is possible that the model does not lead to an accurate evaluation of the AOS. In any case, the preponderance of evidence is consistent with our finding that the proximal pockets of P450 and CPO increase the C=C/C-H ratio for oxidation of cyclohexene and CBMS, and with our proposal that the influence on the heme redox potential and the basicity of the trans ligand is the root cause.


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5. CONCLUSION Our principal finding is that the barrier for alkene epoxidation is lowered by more than the barrier for allylic hydroxylation by the proximal pockets of P450 and CPO, leading to an intrinsic selectivity for epoxidation for the substrates CBMS and cyclohexene. Because this is consistent with experimental measurements on reactions catalyzed by the whole enzymes, we propose that the proximal pocket of these enzymes is the principal determinant of alkene oxidation selectivity for small, hydrophobic substrates. We propose a simple, conceptual framework for understanding the results. The proximal pocket reduces the electron donating strength (the “push”) of the proximal cysteinate ligand via a secondary coordination sphere effect (hydrogen bonding to the proximal sulfur) combined with the electrostatic influence of the enzyme (principally the dipole moment of the proximal helix). The combined effect is expected to increase the heme redox potential and decrease the basicity of the ferryl oxygen. The increase in the redox potential appears to be the dominant influence on both types of reaction, as both reaction barriers are lowered. However, the net impact is smaller for hydroxylation due to reduction of the ferryl oxygen’s basicity, to which H atom transfer is sensitive. This is the first computational study of which we are aware to calculate alkene oxidation selectivities for allylic oxidation in qualitative agreement with experiment for P450 and CPO. The work has important implications for molecular modeling, as it demonstrates that the proximal helix must be included for models to reproduce the relative favorability of the enzymes’ several reaction types. The work also has practical significance for the rational design of useful enzyme variants, as it suggests that the reaction selectivity may be controlled via the proximal ligand’s secondary coordination sphere and the electric dipole moment of the proximal helix. Not only epoxidation and hydroxylation, but also other reactivities, such as peroxide dismutation, could potentially be influenced with mutations affecting these enzyme properties. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: …. Data S1: Cartesian coordinates of reactant and transition state structures. Data


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S2: Constraints used in geometry optimization. Data S3: Natural group spin densities. Data S4: Natural group charges. Figure S1: Expanded images of stationary structures. AUTHOR INFORMATION Corresponding Author *(D.C.C.) Telephone +1-305-348-3977. Email: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT D.C.C. and A.N.M. thank the Instructional and Research Computing Center at Florida International University for access to the computer cluster, on which all computations reported here were run.


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Proximal Pocket Controls Alkene Oxidation Selectivity of Cytochrome

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