MM

LETTER pubs.acs.org/JPCL

Will P450cam Hydroxylate or Desaturate Alkanes? QM and QM/MM Studies Wenzhen Lai, Hui Chen,† Shimrit Cohen, and Sason Shaik* Institute of Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

bS Supporting Information ABSTRACT: The hydroxylation versus desaturation for the enzyme P450cam is addressed by comparing the reactivity of the active species Por•+Fe(IV)dO (Cpd I) toward cyclohexane (CH), camphor (CAM), and cyclohexene (CHE). The quantum mechanics (QM)-only calculations, which reveal protein-free trends, show mixed and nonselective hydroxylation/ desaturation activities, branching from the PorFe(IV)OH/R• intermediates. By contrast, the quantum mechanics/molecular mechanics (QM/MM) results with CAM and CHE show exclusive alcohol formation. Two distinct modes by which the protein controls the hydroxylation/desaturation selectivity were identified: (a) with the native substrate CAM, the tight binding site of the P450cam protein prevents the second hydrogen abstraction and leads to exclusive C5
H hydroxylation, and (b) with the freely tumbling CHE, the protein stabilizes the polarizable electromers, Por•+Fe(III)OH/R•, which possess intrinsic hydroxylase preference. The latter mechanism is common for substrates that are not tightly bound. It is a unique mechanism to P450 Cpd I, which possesses the Por•+Fe(III)OH electromers that dominate the in-protein reactivity. This is contrasted with nonheme enzymes, which lack such electromers. SECTION: Biophysical Chemistry

I

n its normal activity, cytochrome P450 catalyzes the hydroxylation of alkanes by utilizing two reduction equivalents (2e
), one mole of O2, and two proton equivalents (eq 1). Occasionally, as in eq 2, the enzyme converts all the dioxygen into water, while dehydrogenating the alkane,1,2 and functioning like the nonheme enzymes that catalyze fatty acids metabolism and known as desaturases.3,4

Scheme 1. (a) P450 Cpd I and Substrates (1
3) Used in This Study; (b) The Oxidation Mechanism and the Bifurcation of the FeOH/Sub• Intermediates (2S+1I) to Alcohol by Rebound (reb) and to Olefin by a Second H-Abstraction (2H)a

O2 þ 2e
þ 2Hþ þ HR 2 C
CR 02 H f HR 2 C
CR 02 OH þ H2 O

ð1Þ O2 þ 2e
þ 2Hþ þ HR 2 C
CR 02 H f R 2 C ¼ CR 02 þ 2H2 O

ð2Þ Thus, for example, whereas P450cam hydroxylates camphor (CAM) exclusively,5 some mammalian isozymes give a hybrid hydroxylase/desaturase reactivity with certain substrates.1,6 The seminal study7 of C
H hydroxylation versus desaturation of valproic acid showed that the hydroxylase/desaturase activity branches from the same oxidation mechanism that involves the high-valent active species compound I (Cpd I), shown in Scheme 1a. This and other studies5,8
12 concluded that the mechanism (see Scheme 1b) involves an initial hydrogen abstraction (1H) from the substrate by Cpd I with subsequent branching via rebound (reb) to form alcohol, or via abstraction of one more hydrogen atom (2H; possibly also by sequential loss of electron r 2011 American Chemical Society

a The hydrogen atoms involved in the first and second H-abstraction steps are marked in red and blue, respectively.

and proton), thus producing olefin and water. In fact, a similar dichotomy was recently noted in synthetic models of Cpd I with Received: June 5, 2011 Accepted: August 16, 2011 Published: August 16, 2011 2229

dx.doi.org/10.1021/jz2007534 | J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters certain substrates,13,14 and is commonly observed in nonheme chemistry.14
16 This dichotomy and the lack of predictability of the patterns in this hybrid reactivity2a raise questions of significant current interest: What is the root cause of the switch between the two pathways for P450 Cpd I? Is hybrid reactivity intrinsic to Cpd I, or is it the substrate that matters? Or does it all depend on the protein? It is known that the P450cam leads to exclusive 5-exo hydroxylation of CAM. However, there is no information about the intrinsic reactivity of Cpd I and CAM: Will we still see exclusive hydroxylation, or will we see hybrid reactivity? Why does cyclohexene give only oxygenation products with no desaturation?17,18 To understand how substrates behave intrinsically and when within the protein, we studied reactions of P450cam with three substrates indicated as 1
3 in Scheme 1a: 1, cyclohexane (CH); 2, the native substrate, camphor (CAM); and 3, cyclohexene (CHE). To these ends, we used (i) quantum mechanics (QM(B3LYP))only of bare Cpd I for 1
3, and (ii) in-protein hybrid QM(B3LYP)/MM calculations19
21 for the latter two alkanes. The comparison of the behaviors of 2 and 3 in the protein environment is expected to be revealing since 2 is well bound in P450cam,22
25 and hence its in-protein 2H/reb reactivity may be influenced by this binding, while 3 seems to tumble almost freely,26,27 and thereby its reactivity pattern may be more intrinsic. Since our focus herein is rebound versus desaturation, known results25
27 for the first H-abstraction step are relegated to the Supporting Information (SI) document. Past the first H-abstraction, the generated iron-hydroxo/substrate
radical intermediate (2S+1I) has four low-lying electronic states, which differ in the oxidation states on iron and the porphyrin (Por) macrocycle, as well as in the spin quantum number (S), which leads to doublet and quartet states. These are the 2,4Por•+FeIIIOH/Sub• and 2,4 PorFeIVOH/Sub• states, labeled henceforth as 2,4I(III) and 2,4 I(IV), respectively.28 Gas Phase Behavior. Figure 1 shows the generic energy profile for the 2H/reb bifurcation in the gas phase. Thus, as shown before,28 in the bare system, the lowest intermediate states are 2,4I(IV), with the doublet state being slightly lower by ca. 0
1.2 kcal/mol for 1•
3•. These intermediates are followed by 2I(III) and 4I(III), which lie, ca. 2.5
7.3 kcal/mol higher than the 2,4I(IV) pair, at the B3LYP/B2 and B3LYP-D/B2 levels (where D stands for the employed empirical dispersion correction29). As such, 4I(III) is energetically inaccessible in the gas phase, and even though its rebound barriers are known to be large,28,30 this electromer should not play a role in consideration of the intrinsic reactivity patterns. Among the remaining three intermediate states, both reb and 2H processes are barrier free for 2I(III), but have non-negligible barriers for 4I(IV). Starting, however, from 2I(IV), it is not possible to localize the transition states (TSs) along the scans (due to energy drops caused by the sudden change of electronic configuration that cannot be handled smoothly by density functional theory (DFT); see SI, e.g., Figures S5
7 in the case of CH). The barriers for the respective doublet reb and 2H processes were estimated from the highest energy points of the corresponding one- or, wherever possible, the two-dimensional scans. Table 1 collects the B3LYP/ B2 (B3LYP-D/B2) calculated barriers and reaction energies for the two processes originating from the 2,4I(IV) intermediates for the three substrate-radicals. Before discussing the data in Table 1, we have to consider the kinetic scenario for the two processes in Figure 1. Thus, since 2 I(IV) and 4I(IV) are close in energy and both seem to possess barriers for the two processes, we can think of two scenarios:31

LETTER

Figure 1. Generic QM-only energy profiles for hydroxylation (reb) versus desaturation (2H) of the 2,4I intermediates (FeOH/R•).

(i) If the two spin states are in fast equilibrium, then the reb/2H branching will be determined by the lowest possible barriers, and (ii) if the spin-state equilibrium is slower than the reb/2H process, then the branching will be determined jointly by the two spin states. Since the two spin states differ by a spin flip on the radical center (Scheme 1b), the spin pre-equilibrium may not be too fast, and we need to consider both spin manifolds. Inspection of Table 1 shows that the three substrate radicals exhibit clear intrinsic reactivity patterns, as observed recently for nonheme systems.15 Thus, for example, the quartet state reb versus 2H barriers follow the exothermicities of the respective processes, given by the reaction energy (ΔEreb/ΔE2H) values. As such, the intrinsic preferences of the substrate radicals for reb/2H follow the Bell
Evans
Polanyi principle.32,33 Calculations of the bond dissociation of the C
H bond (BDECH) in the radicals show that the reaction energy trend is dominated by the trend in the BDECH values of the radicals (see SI, Figure S4), being BDECH(3•) > BDECH(2•) > BDECH(1•), such that the larger the BDECH, the less exothermic the 2H process.13 More specifically, the cyclohexyl radical (1•) and camphoryl (2•) radicals have small barriers for the two processes on both doublet and quartet surfaces, with hardly much of a preference for 2H or reb. One may therefore conclude that, intrinsically, these two substrates should produce mixtures of hydroxylation and desaturation, irrespective of whether the spin equilibrium is fast or slow. In the case of cyclohexenyl (3•), reb is significantly preferred over 2H on the quartet surface, wherein the 2H barrier is large (7.5 kcal/mol, after zero-point energy (ZPE) correction). As such, the slow spin-equilibrium scenario predicts that CHE will exhibit a predominant if not exclusive hydroxylation (and epoxidation) due to the two-state reactivity, while the fast spin equilibrium scenario predicts a mixture of hydroxylation and desaturation due to dominance of the doublet-state processes. In Protein Behavior. Since 1 is not metabolized by P450cam, we focus hereafter on 2• and 3•. Figure 2a,b shows the changes in relative energies of the intermediate states as the QM system is transferred from the gas phase to a continuum solvent model (dielectric constant = 5.7) and subsequently to the protein environment. As pointed out before,28,30 and as seen in Figure 2, the protein completely switches the relative stability of the intermediates, preferring the more polarizable species 2,4I(III) to 2,4I(IV). However, with one exception (for I(III) in 3•), the continuum solvent does not achieve this inversion, albeit it lowers the gap between Fe(III) and Fe(IV) states. Importantly, the in-protein inversion in the order of the intermediate stability is obtained in the QM-part of the QM/ MM energy, with little or no contribution from the MM energy 2230

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters

LETTER

Table 1. Barriers and Reaction Energies (kcal/mol)a for Rebound (reb) and Desaturation (2H) Processes Exhibited by the 2,4 I(IV) Intermediates of the Three Substrate Radicals (1•
3•) Formed by the First H-Abstraction from the Corresponding Substrates (1-3) by Cpd I in the Gas Phase quartetb

doubletc

intermediate/substrate radicald

ΔE‡reb/ΔE‡2H

ΔEreb/ΔE2H

ΔE‡reb/ΔE‡2H

I(IV)/1•

0.6/1.2 (2.3/1.4)


57.3/
48.6 (
58.2/
47.3)

1.8/1.0

I(IV)/2•

1.1/2.8 (0.8/2.3)


55.3/
40.9 (
54.9/
38.2)

1.4/1.6

I(IV)/3•

1.7/7.5 (1.9/9.5)


40.2/
33.4 (
40.4/
32.2)

1.4/2.5

a

The energy data are given for the B3LYP/B2 (B3LYP-D/B2) levels in the order reb/2H. b The energies are relative to 4I(IV) and include ZPE correction. c These B3LYP/B2 barriers are estimated from the scans relative to 2I(IV) (see SI, Figures S6
S7 for 1•, Figures S13 and S15 for 2•, and Figures S25
S26 for 3•) and do not include ZPE correction. d See substrates 1
3 in Scheme 1a.

Figure 2. B3LYP/B2 (B3LYP-D/B2) relative energies (in kcal/mol) of the intermediate states, 2,4I(III) and 2,4I(IV) for (a) 2• and (b) 3•. Energy values are shown in gas-phase and in a continuum solvent including ZPE correction, as well as in-protein without ZPE correction. In the in-protein case, we note both the QM/MM and the QM-component relative energies. (c) The electric field vector of the P450cam protein projected on 2I(III) in the case of CHE.

(see also Table S6 and Table S10 in the SI). As such, the inversion reflects only the effect of the point charges of the protein, namely, its oriented electric field,34 which is illustrated in Figure 2c. Separate QM calculations with inclusion of MM charges only for 3• (see Table S12 in the SI) show that as we increase the radius of the included MM charges gradually from 3 Å around the heme and substrate, finally the MM charges contained in the 5 Å sphere reproduce the QM/MM ordering with 2,4I(III) being the lowest energy states. The stability ordering inversion is not associated with any specific residue, but represents a general electric field effect and longer range effects of the protein residues. Thus, an important change brought about by the protein is that 4I(III), which was inaccessible in the gas phase, becomes one of the two lowest energy states in protein, and under the scenario of slow spin equilibrium, this state must be taken into account. In-Protein Reactivity of 2•. The QM/MM energy profiles for reb/2H starting from the various intermediate states of PorFeOH/camphoryl• are shown in Figure 3. The rebound phase behaves in accord with early studies;24 it is essentially barrier free for 2I(III) and 4I(IV), having a small barrier of 1.9 (1.4) kcal/mol for 2I(IV) and a significant barrier, 9.9 (8.5) kcal/mol, for 4I(III) at B3LYP/B2 (B3LYP-D/B2). By contrast, we were unable to find any low-energy direct path that leads to desaturation. Starting from 2,4I(III) gave energy scans

with high-energy summits (see Figure S22, and pages S30
31 in the SI), that are much higher than the corresponding rebound TSs. These species are shown in Figure 3b, along with their QM energies in the gas phase, using their geometry within the protein and the QM-component barriers of the corresponding QM/MM barriers. It is seen that the gas phase energies of these species are high, and so are the QM-component barriers. The QM-component energy shows that the high energy of these species is not due to steric repulsion from the protein residues. It is associated with geometric deformation of the structures and their mismatch with the partial charges of the protein environment. A similar mismatch was recently reported by Mullholland et al. for the aromatic hydroxylation of detromethorphan by P450 (CYP 2D6).35 For both spin states of FeIV, 2,4I(IV), an energy scan along the O
H6exo reaction coordinate (Figure S21 in the SI) goes up in energy and then falls to the hydroxylation product, never to the desaturation product. The only way we were able to connect the FeIV intermediates to the desaturation product is to force the conformation that places the C
H6exo bond over the Fe
OH moiety. As shown in Figure 3c, this conformation, 4I0 (IV), lies 15.5 kcal/mol above the most stable 2I(III) intermediate (12.9 kcal/mol above the relaxed 4I(IV) species wherein the C5 radical center is coordinated to the Fe
OH moiety), and most of this energy rise is due to the QM-part (10.2 kcal/mol) of the QM/MM energy. Clearly, therefore, enforcing the 2231

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters

LETTER

Figure 3. (a) QM/MM calculated energy profiles for hydroxylation (reb) versus desaturation (2H) of camphoryl radical in the various 4,2I intermediates. Energy values (in kcal/mol) correspond to B3LYP/B2(B3LYP-D/B2). (b) The 2,4TS2H(III) structures, the corresponding QM/MM barriers (ΔE‡QM/MM), the QM-component barriers (ΔE‡QM), and the gas phase ‘barriers’ (ΔE‡p,g), at the protein geometries. (c) The high-energy conformation of 4I0 (IV) that leads to desaturation and its relative energy to 2I(III). The relative energy values refer, respectively, to the QM/MM and QM-component energy differences (ΔEQM/MM/ΔEQM) at the B3LYP/B2 level.

C
H6exo 3 3 3 O(H) orientation, as we did in Figure 3c, disfavors this potentially reactive conformation by raising its QM energy due to charge mismatch with the protein residues around it. Again, we verified that there is no specific residue effect here, just a long-range effect of the electric field of the protein. Thus, even if this conformation may be accessed, the energy expense of desaturation will be of the order of 10 kcal/mol, and much larger than the corresponding 2,4I(IV) rebound barriers in Figure 3a. The above behavior of CAM in P450cam reflects the match between the protein and its native substrate. The P450cam protein pocket is not large and it fits CAM tightly. Thus, the native C5 3 3 3 O(H)Fe orientation of the relaxed intermediate is juxtaposed22,23,25 via binding of the carbonyl group of CAM by a hydrogen bond donation from the Tyr96 residue of the protein and by hydrophobic interactions from Phe87, Leu244, Val247, and Val295, and long-range interactions with the electric field of the protein. The calculations for all the intermediates states reveal that the non-native C
H6exo 3 3 3 O(H) trajectory that is necessary for desaturation invariably results in high-energy species (Figure 3), due to deformation of the QM system and to its mismatch with the partial charges of the protein environment. Heuristically expressed, these results reveal that the binding machinery of the P450cam protein applies a “restoring force” that prevents the formation of a low energy pathway for the desaturation of its native substrate, CAM. In this manner, the P450cam protein augments the very small intrinsic propensity of Cpd I to hydroxylate CAM and leads to exclusive hydroxylation at the C5 site, which is held by the substrate-binding machinery above the iron-hydroxo moiety.

In-Protein Reactivity of 3•. CHE (3) is an interesting case since it does not have this tight binding machinery in the P450cam active site. Figure 4 shows the situation for 3•. The rebounds nascent from 2I(III)/2I(IV)/4I(IV) have small barriers of 1.9/ 3.8/3.2 kcal/mol and a significant barrier of 11.9 kcal/mol from 4 I(III). The 2H processes initiated from 2I(III)/2I(IV)/4I(IV) occur via TSs that lie 9.0/6.4/8.7 kcal/mol above the most stable intermediate 2I(III), and lead to the generation of the triplet FeOH2 species and singlet cyclohexadiene (2,4P2H) with high exothermicity. By contrast, starting from 4I(III), the 2H process was found to proceed with a much higher energy barrier, 28.8 kcal/mol, and to generate singlet FeOH2 and triplet cyclohexadiene (4P0 2H) with endothermicity of 17.2 kcal/mol. If we consider only the most stable intermediate states, 2I(III) and 4I(III), both fast and slow spin-equilibrium scenarios will predict exclusive hydroxylation of CHE, in contrast to the hybrid reactivity expected in the gas phase. Thus, in this case, the protein affects the selectivity of this substrate by its electric field (see Figure 2c) that renders accessible the 2I(III) and 4I(III) species that prefer a hydroxylase activity. In summary, in contrast to the gas-phase wherein Cpd I is predicted to have hybrid hydroxylase/desaturase reactivity, via the 2,4I(IV) intermediates, the P450cam protein induces exclusive mono-oxygenation for CAM and CHE, in accord with experiment.17,18,36 The P450cam protein applies two different effects depending on the identity of the substrate. In the case of the native substrate, CAM, the substrate binding by P450cam and the rather small pocket prevent a desaturation trajectory, and leads to exclusive regio- and stereospecific hydroxylation 2232

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters

LETTER

it should be similar to CH and CAM, and its only minor desaturation in P450 reflects the predominance of the Por•+FeOH/R• intermediates in the protein and some freedom in the protein pocket to assume the 2H trajectory.

Figure 4. QM/MM calculated energy profiles for hydroxylation (reb) versus desaturation (2H) of cyclohexenyl radical (3•) in the various 2,4I intermediates. Energies, in kcal/mol, are B3LYP/B2 (B3LYP-D/B2) values.

of C5
H. This exclusive hydroxylation of CAM supports the suggestion of Poulos22 that this specificity is due to properties of the protein pocket, most notably the groups in the helix-poor domain, as well as a hydrogen bond between the carbonyl group of CAM and Tyr96 of the enzyme. In contrast to CAM, CHE is not bound, and it tumbles freely in the P450cam pocket. In this case, the protein acts by stabilizing the polarizable 2I(III) and 4I(III) states that are not available in the gas phase and that prefer hydroxylation to desaturation. Indeed, of all the reactions with CHE, in P450 enzymes17,36 as well as in solution by mimetic Cpd I species,18 the only products are allylic hydroxylation and double bond epoxidation, with no hint we know of for desaturation. Thus, the in-protein reactivity of CHE highlights the role of the electric field of the protein environment on the hydroxylase versus desaturase selectivity of P450s. This scenario is the more general one, and it will fit both substrates that tumble freely in P450cam and promiscuous P450 isozymes that possess large pockets, such as CYP 3A4. Additionally, while the tight substrate binding effect should be generally applicable to both heme and nonheme enzymes with their native substrates, the second effect due to the Por•+FeOH/R• I(III)-type states is unique to P450 Cpd I due to its high oxidation state (effectively V). This effect will not play a role in nonheme enzymes that utilize a lower oxidation state (IV),15 and which may therefore exhibit greater propensity toward desaturase activity.3,4 Given that Por•+FeOH/R• is available to most if not all substrates in P450, we may conclude that P450 with its unique Cpd I was well designed for hydroxylase activity. Many other substrates (e.g., ethyl benzene, cumene, propyl benzene, etc.), which may be considered analogues of CHE, undergo major hydroxylation and minor desaturation in P450cam and P450BM3.12,37 This trend may reflect the interplay of the protein directives in stabilizing the Por•+FeOH/R• intermediates along with the intrinsic hydroxylation preference of the substrate radicals. Interesting cases, such as valproic acid with liver microsome P450 or thujone,38 display some desaturase reactivity despite the presence of CdO groups, which can potentially undergo binding in the protein and behave similar to CAM. Our calculation of the BDECH of the valproic acid radical (page S7 in the SI) shows that, intrinsically in the gas phase,

’ COMPUTATIONAL METHODOLOGY Cpd I was represented as a six-coordinate oxo-ferryl species Fe4+O2
(C20N4H12)
(SH)
. The DFT calculations were carried out with Gaussian 09,39 using B3LYP. The QM/MM computations were preformed using ChemShell40 interfaced with Turbomole41 and DL_POLY.42 The CHARMM2243 force field was used for the MM part. In both QM-only and QM/MM calculations, the geometries were optimized with the double-ζ LACVP(Fe)/6-31G(rest)44,45 basis set (B1), followed by singlepoint energy correction with the larger basis set (B2), LACV3P+ (Fe)/6-311+G*(rest).46 ZPE corrections were included only in the gas-phase calculations. Since a recent work27 showed that dispersion correction of B3LYP (i.e., B3LYP-D) gives improved agreement with experiment, we corrected our B2 results using singlepoint DFT-D229 calculations. Dispersion was found herein to have only small effects on the relative energies. Therefore, the values we report herein are at the B3LYP/B2 level, unless otherwise specified. And ZPE correction was also found to have minor effect (∼1 kcal/ mol for reb and ∼3 kcal/mol for 2H. Single point calculations in a solvent at the B1 level were carried out using CPCM solvation model with chlorobenzene as a solvent (dielectric constant = 5.7). It is noted that the QM/MM barriers are not activation free energies, which require extensive sampling.19 However, a QM/MM study47 of H-abstraction from CAM by Cpd I of P450cam showed that the sampling effect is small (1.2 kcal/mol or less). ’ ASSOCIATED CONTENT

bS

Supporting Information. Complete citations for references 39, 40, and 43, computational details, as well as a full set of computational results. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Present Addresses †

Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China.

’ ACKNOWLEDGMENT This work was supported by the Israel Science Foundation (ISF 53/09 to S.S.). ’ REFERENCES (1) Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism and Biochemisty, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Chapter 8, pp 245
304. (2) (a) Guengerich, F. P. Common and Uncommon Cytochrome P450 Reactions Related to Metabolism and Chemical Toxicity. Chem. Res. Toxicol. 2001, 14, 611–650. (b) Sono, M.; Roach, M. P.; Coutler, E. D.; Dawson, J. H. Heme-Containing Oxygenases. Chem. Rev. 1996, 96, 2841–2887. 2233

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters (3) Behrouzian, B.; Buist, P. H. Fatty Acid Desaturases: Variations on an Oxidative Theme. Curr. Opin. Chem. Biol. 2002, 6, 577–582. (4) Buist, P. H. Fatty Acid Desaturases: Selecting the Dehydrogenation Channel. Nat. Prod. Rep. 2004, 21, 249–262. (5) Mueller, E. J.; Lioda, P. J.; Sligar, S. G. In Cytochrome P450: Structure, Mechanisms and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Vol. 2, Chapter 3. (6) Akhtar, M.; Wright, J. N. A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes. Nat. Prod. Rep. 1991, 8, 527–551. (7) Rettie, A. E.; Boberg, M.; Rettenmeier, A. W.; Baillie, T. A. Cytochrome P-450-Catalyzed Desaturation of Valproic Acid in Vitro. J. Biol. Chem. 1988, 263, 13733–13738. (8) Lee, J. S.; Jacobsen, N. E.; Ortiz de Montellano, P. R. 4-Alkyl Radical Extrusion in the Cytochrome P-450-Catalyzed Oxidation of 4-Alkyl-1,4-dihydropyridines. Biochemistry 1988, 27, 7703–7710. (9) Wand, M. D.; Thompson, J. A. Cytochrome P-450-Catalyzed Rearragement of a Peroxyquinol Derived from Butylated Hydroxytoluene. J. Biol. Chem. 1986, 261, 14049–14056. (10) Aoyama, T.; Korzekwa, K.; Nagata, K.; Gillette, J.; Gelboin, H. V.; Gonzales, F. J. cDNA-Directed Expression of Rat Testosterone 7R-Hydroxylase Using the Modified Vaccinia Virus, T7-RNA-Polymerase System and Evidence for 6R-Hydroxylation and Δ6-Testosterone Formation. Eur. J. Biochem. 1989, 181, 331–336. (11) Guengerich, F. P.; Brian, W. R.; Iwasaki, M.; Sari, M.-A.; B€a€arnhielm, C.; Berntsson, P. Oxidation of Dihydropyridine Calcium Channel Blockers and Analogues by Human Liver Cytochrome P-450 IIIA4. J. Med. Chem. 1991, 34, 1838–1844. (12) Whitehouse, C. J. C.; Bell, S. G.; Wong, L.-L. Desaturation of Alkylbenzenes by Cytochrome P450BM3 (CYP102A1). Chem.—Eur. J. 2008, 14, 10905–10908. (13) Kumar, D.; Tahsini, L.; de Visser, S. P.; Kyang, H. Y.; Kim, S. J.; Nam, W. Effect of Porphyrin Ligands on the Regioselective Dehydrogenation versus Epoxidation of Olefins by Oxoiron(IV) Mimics of Cytochrome P450. J. Phys. Chem. A 2009, 113, 11713–11722. (14) Nam, W. High-Valent Iron(IV)-Oxo Complexes of Heme and Non-Heme Ligands in Oxygenation Reactions. Acc. Chem. Res. 2007, 40, 522–531. (15) Usharani, D.; Janardanan, D.; Shaik, S. Does the TauD Enzyme Always Hydroxylate Alkanes, While an Analogous Synthetic Non-Heme Reagent Always Desaturates Them? J. Am. Chem. Soc. 2011, 133, 176–179. (16) Mukherjee, A.; Martinho, M.; Bominaar, E. L.; M€unck, E.; Que, L., Jr. Shape-Selective Interception by Hydrocarbons of the O2-Derived Oxidant of a Biomimetic Nonheme Iron Complex. Angew. Chem., Int. Ed. 2009, 48, 1780–1783. (17) White, R. E.; Groves, J. T.; McClusky, G. A. Electronic and Steric Factors in Regioselective Hydroxylation Catalyzed by Purified Cytochrome P-450. Acta Biol. Med. Ger. 1979, 38, 473–482. (18) Song, W. J.; Ryu, Y. O.; Song, R.; Nam, W. Oxoiron(IV) Porphyrin π-Cation Radical Complexes with a Chameleon Behavior in Cytochrome P450 Model Reactions. J. Biol. Inorg. Chem. 2005, 10, 294–304. (19) The QM/MM method was pioneered by Warshel and Levit. For a recent review in the centennial issue, see: Kamerlin, S. C. L.; Haranczyk, M.; Warshel, A. Progress in Ab Initio QM/MM Free-Energy Simulations of Electrostatic Energies in Proteins: Accelerated QM/MM Studies of pKa, Redox Reactions and Solvation Free Energies. J. Phys. Chem. B 2009, 113, 1253–1272. (20) Senn, H. M.; Thiel, W. QM/MM Methods for Biomolecular Systems. Angew. Chem., Int. Ed. 2009, 48, 1198–1229. (21) Lin, H.; Truhlar, D. G. QM/MM: What Have We Learned, Where Are We, and Where Do We Go from Here? Theor. Chem. Acc. 2007, 117, 185–199. (22) Poulos, T. L.; Finzel, B. C.; Howard, A. J. High-Resolution Crystal Structure of Cytochrome P450cam. J. Mol. Biol. 1987, 195, 687–700.

LETTER

(23) Deng, T.-J.; Macdonald, I. D. G.; Simianu, M.; Sykora, M.; Kincaid, J. R.; Sligar, S. G. Hydrogen-Bonding Interactions in the Active Sites of Cytochrome P450cam and Its Site-Directed Mutants. J. Am. Chem. Soc. 2001, 123, 269–278. (24) Sch€oneboom, J. C.; Lin, H.; Reuter, N.; Thiel, W.; Cohen, S.; Ogliaro, F.; Shaik, S. The Elusive Oxidant Species of Cytochrome P450 Enzymes: Characterization by Combined Quantum Mechanical/Molecular Mechanical (QM/MM) Calculations. J. Am. Chem. Soc. 2002, 124, 8142–8151. (25) Sch€oneboom, J. C.; Cohen, S.; Lin, H.; Shaik, S.; Thiel, W. Quantum Mechanical/Molecular Mechanical Investigation of the Mechanism of C
H Hydroxylation of Camphor by Cytochrome P450cam: Theory Supports a Two-State Rebound Mechanism. J. Am. Chem. Soc. 2004, 126, 4017–4034. (26) Cohen, S.; Kozuch, S.; Hazan, C.; Shaik, S. Does Substrate Oxidation Determine the Regioselectivity of Cyclohexene and Propene Oxidation by Cytochrome P450? J. Am. Chem. Soc. 2006, 128, 11028–11029. (27) Lonsdale, R.; Harvey, J. N.; Mulholland, A. J. Compound I Reactivity Defines Alkene Oxidation Selectivity in Cytochrome P450cam. J. Phys. Chem. B 2010, 114, 1156–1162. (28) Shaik, S.; Kumar, D.; de Visser, S. P.; Thiel, W. Theoretical Perspective on the Structure and Mechanism of Cytochrome P450 Enzymes. Chem. Rev. 2005, 105, 2279–2328. (29) Grimme, S. Semiempirical GGA-Type Density Functional Constructed with a Long-Range Dispersion Correction. J. Comput. Chem. 2006, 27, 1787–1799. (30) Shaik, S.; Cohen, S.; de Visser, S. P.; Sharma, P. K.; Kumar, D.; Kozuch, S.; Ogliaro, F.; Danovich, D. The “Rebound Controversy”: An Overview and Theoretical Modeling of the Rebound Step in MonoOxygenations by Cytochrome P450. Eur. J. Inorg. Chem. 2004, 110 207–226. (31) Hirao, H.; Kumar, D.; Que, L., Jr; Shaik, S. Two-State Reactivity in Alkane Hydroxylation by Non-Heme Iron-Oxo Complexes. J. Am. Chem. Soc. 2006, 128, 8590–8606. (32) Bell, R. P. The Theory of Reactions Involving Proton Transfers. Proc. R. Soc. London, Ser. A 1936, 154, 414–429. (33) Evans, M. G.; Polanyi, M. Inertia and Driving Force of Chemical Reactions. Trans. Faraday Soc. 1938, 34, 11–24. (34) Cho, K.-B.; Hirao, H.; Chen, H.; Carvajal, M. A.; Cohen, S.; Derat, E.; Thiel, W.; Shaik, S. Compound I in Heme Thiolate Enzymes: A Comparative QM/MM Study. J. Phys. Chem. A. 2008, 112, 13128–13138. (35) Olah, J.; Mulholland, A. J.; Harvey, J. N. Understanding the Determinants of Selectivity in Drug Metabolism through Modeling of Dextromethorphan Oxidation by Cytochrome P450. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6050–6055. (36) For a general review on epoxidation by P450, see: Martinez, C. A.; Stewart, J. D. Cytochrome P450’s: Potential Catalysts for Asymmetric Olefin Epoxidations. Curr. Org. Chem. 2000, 4, 263–282. (37) Ortiz de Montellano, P. R.; De Voss, J. J. In Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer Academic/Plenum Publishers: New York, 2005; pp 208
211. (38) Yang, Y.; He, X.; Ortiz de Montellano, P. R. Biochemistry 2006, 45, 533–542. (39) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A. et al. Gaussian 09, revision A.02; Guassian, Inc.: Wallingford, CT, 2009. (40) Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach, G.; Catlow, C. R. A.; French, S. A.; Sokol, A. A.; Bromley, S. T.; Thiel, W.; Turner, A. J.; et al. QUASI: A General Purpose Implementation of the QM/MM Approach and Its Application to Problems in Catalysis. J. Mol. Struct. (THEOCHEM) 2003, 632, 1–28. (41) Ahlrichs, R.; Bar, M.; Haser, M.; Horn, H.; Kolmel, C. Electronic Strucutre Calculations on Workstation Compouters: The Program System Turbomole. Chem. Phys. Lett. 1989, 162, 165–169. 2234

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235

The Journal of Physical Chemistry Letters

LETTER

(42) Smith, W.; Forester, T. R. DL_POLY_2.0: A General-Purpose Parallel Molecular Dynamics Simulation Package. J. Mol. Graph. 1996, 14, 136–141. (43) MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L., Jr.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; et al. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586–3616. (44) Hay, P. J.; Wadt, W. R. Ab Initio Effective Core Potentials for Molecular Calculations. Potentials for K to Au Including the Outermost Core Orbitals. J. Chem. Phys. 1985, 82, 299–310. (45) Friesner, R. A.; Murphy, R. B.; Beachy, M. D.; Ringnalda, M. N.; Pollard, W. T.; Dunietz, B. D.; Cao, Y. X. Correlated ab Initio Electronic Structure Calculations for Large Molecules. J. Phys. Chem. A 1999, 103, 1913–1928. (46) The LACV3P basis set is a triple-ζ contraction of the LACVP basis set developed and tested at Schr€odinger, Inc. (47) Senn, H. M.; K€astner, J.; Breidung, J.; Thiel, W. FiniteTemperature Effects in Enzymatic Reactions: Insights from QM/MM Free-Energy Simulations. Can. J. Chem. 2009, 87, 1322–1337.

2235

dx.doi.org/10.1021/jz2007534 |J. Phys. Chem. Lett. 2011, 2, 2229–2235