External Electric Field Can Control the Catalytic Cycle of Cytochrome

pubs.acs.org/JPCL

External Electric Field Can Control the Catalytic Cycle of Cytochrome P450cam: A QM/MM Study Wenzhen Lai, Hui Chen, Kyung-Bin Cho,† and Sason Shaik*

Institute of Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, Hebrew University of Jerusalem, Givat Ram Campus, 91904 Jerusalem, Israel

ABSTRACT This Letter presents a systematic study of effects of external electric fields (EEFs) on the key species and steps in the catalytic cycle of cytochrome P450cam. The QM/MM/EEF results demonstrate that an EEF can exert significant effects on the entire catalytic cycle of P450. The EEF will control the initial gating of the cycle, its rate-determining step, its O2 uptake, the geometry of its resting state, and the spin-state ordering and electronic structure of its various active species. Furthermore, the EEF has a potential of controlling the bond activation reactions of the active species as well. These effects are pronounced when the EEF is aligned perpendicular to the porphyrin plane. SECTION Biophysical Chemistry

C

electron transfer, by increasing the driving force, would enhance the turnover of the cycle. Can an EEF affect the spin-state energy gaps? Will the EEF change the geometry and electronic structure of the various intermediates and thereby affect the cycle as a whole? These questions are in the focus of the present Letter. EEFs have been known to affect many features of atoms, molecules, enzymes, and so forth.12-20 Previous theoretical calculations18-20 showed that EEFs can change the electronic properties and the spin-state separation of iron-oxo species18,19 and affect their regioselectivity and reactivity in bond activation reactions.18,20 However, these theoretical studies have been carried out on simple models and on a single species/process which lacked the protein environment. Accounting for the protein requires DFT/MM calculations, which have proved reliable for predicting the properties of all of the species in Scheme 1 with reasonable accuracy.21-28 Therefore, we present here hybrid DFT(B3LYP)/MM/EEF calculations which explore systematically the effects exerted on the intermediates in Scheme 1 by oriented EEFs along the three axes x, y, and z (see center of Scheme 1). The magnitude of EEF varies from 0 to 0.0125 au (1 au = 51.422 V/Å) in positive and negative orientations. P450cam is a soluble bacterial P450 enzyme from Pseudomonus putida and is not membrane-bound. The maximal EEF strength used here is of the same order of magnitude as the intrinsic EEF-free values previously determined by QM/MM calculations29 for the active site of P450cam in an aqueous environment and is ∼5-50 fold larger than the values at membrane interfaces (0.01-0.1 V/Å).14,15,30 Initial tests were done to ascertain that the conclusions are conserved for

ytochrome P450 is an important family of versatile catalysts for reactions like hydroxylation, epoxidation, heteroatom oxidation, and dealkylations.1-5 As such, it is important to ponder how to control or tweak P450 activity. The present Letter describes the intriguing effects of external electric fields (EEFs) on the key species in the catalytic cycle of P450cam (CYP101), which constitutes the most widely studied P450 isozyme. The catalytic cycle of P450cam (Scheme 1) starts with the resting state (1), which is a hexacoordinated low-spin state, 21. The entry of the substrate into the binding pocket expels the water ligand and produces a high-spin pentacoordinated ferric complex (62). This ligand loss and spin change are crucial for the ensuing activity of the enzyme.5 Reduction of 2 yields the high-spin pentacoordinated ferrous complex (53), which binds O2 and generates the low-spin oxyferrous intermediate (14), which is a key intermediate in the cycle. Reduction of 14 results in a low-spin ferric peroxo complex (25), which gets protonated to form the ferric hydroperoxide species (26), so-called compound 0 (Cpd 0). Then Cpd 0 abstracts another proton, and a water molecule is liberated to generate the high-valent iron-oxo species (7), called compound I (Cpd I), which is the active species of P450 and is known to exhibit sensitivity to external perturbations.4,6 Cpd I has a virtually degenerate pair of doublet and quartet spin states (4,27), which gives rise to two-state and to multistate reactivity scenarios.4,7,8 Therefore, the doublet-quartet energy splitting/ordering of the electronic states could influence the activity and catalytic properties of the enzyme. Can an EEF affect the major features in this cycle? One might expect that the processes 21 f 62 and 53 f 14, which involve also spin inversion, will become easier if the energy gap between the spin states can be made smaller.9,11 Furthermore, the second reduction, 14 f 25, is considered to be ratecontrolling,1-5 and the reduction of the barrier for this

r 2010 American Chemical Society

Received Date: May 24, 2010 Accepted Date: June 15, 2010 Published on Web Date: June 21, 2010

2082

DOI: 10.1021/jz100695n |J. Phys. Chem. Lett. 2010, 1, 2082–2087

pubs.acs.org/JPCL

highest computational level, which involves a triple-ζ basis set augmented with polarization and diffuse functions. In agreement with previous QM-only calculations,18 it was found that the x,y-oriented EEFs cause mainly charge polarization in the porphyrin plane (e.g., Figure SG5, SI) and smaller effects otherwise. For example, in the presence of x,y fields, the energy gaps between different spin states are almost unchanged for 2, 3, 6, and 7, while for 1 and 4, the energetic effects are within 1.5 and 3.1 kcal/mol compared with 6.2 and 7.2 kcal/mol for the z field. Therefore, we focus hereafter on the effect of z-oriented EEF (perpendicular to the porphyrin plane) and display the results in Figure 1. As mentioned above, the step that triggers the cycle is the conversion of 21 to 62, which is attended by a water ligand loss and spin change.9,10 The Fz was found to have a dramatic effect on the state energies of 2,4,61 (see Figure 1a) as well as on the geometrical features. Specifically, negative Fz lengthens the Fe-O bond, even leading to dissociation of the water ligand and thereby promoting also the spin inversion. In fact, due to water dissociation from 1, for -0.0125 e Fz e -0.0075 au, it becomes degenerate with 2 (in Figure 1b). Clearly therefore, an EEF along the -z direction will facilitate the displacement of the aqua ligand and the generation of 2 and will thereby gate the cycle more efficiently. By contrast, Fz > 0 shortens the Fe-O bond and lengthens the Fe-S bond, upon Fz increase. As such, the direction of Fz will modulate the Fe-O bond strength of 1 and hence act as a “gate”; in the negative direction, it will turn the cycle on, and in the positive direction, it may turn it off. As shown in Figure 1b, an Fz will change the quartetdoublet and quartet-sextet gaps of 2 significantly by mainly affecting the quartet state, which becomes the ground state for Fz > 0 but lies above the sextet state at Fz < 0. The energy gap of the quartet state to the other spin states reaches maximum values of 9.5 and 3.9 kcal/mol at the positive end of Fz = 0.0125 au. Accordingly, the reduction process, 2 f 3, will start from 42 in the presence of the þz field, while in the presence of a -z field (Fz triplet > quintet (0 > -7.4 > -10.2 kcal/ mol), in line with the results of Thiel et al.22 and with experimental assignment (See also Scheme S1, SI). As shown in Figure 1c, an EEF oriented along the z direction will alter the triplet-singlet energy gap while keeping the quintet-singlet gap unchanged. For Fz < 0, the triplet-quintet gap is increased, whereas for Fz > 0, this gap is decreased, leading to state degeneracy at Fz = þ0.0125 au. As found before,31-33 it is the triplet state 33 that couples with 3O2 to form the oxyheme species 14,11 whereas the quintet state 53 has a totally repulsive interaction with 3O2. Since the FeII-O2 binding requires spin inversion from the ground quintet state 5 3 to 33,32 the EEFs will affect the binding of 3 to 3O2 in a selective manner. For positive Fz, which stabilizes 33, the external field will facilitate the O2 binding, and vice versa for negative fields. Thus, once again, the EEF can act as a gating device for O2 binding by the enzyme. The oxyferrous complex 14 has an open-shell singlet ground state,24,27,31 possessing an FeIII-O2- character, where

Scheme 1. Catalytic Cycle of P450cam

different starting points of the protein without and with EEF. Thus, to test the potential effect of the EEF on the structure of the enzyme, we carried out molecular dynamics (MD) simulations on 7 (whose X-ray structure was used), without and with EEF (of Fz = þ0.0025 au). The 1 ns MD results showed that in the absence of EEF, the average root-mean-square deviation (rmsd) of the backbone atoms relative to the X-ray structure is 1.07 Å. Turning on the EEF and performing the MD again showed a minor incremental deviation of 0.19 Å. This ratio of rmsd's shows that while structural changes are expected,14 the major change caused by the MD is relative to the X-ray structure, while the EEF inflicts smaller changes in the nuclear polarization of the protein. Furthermore, to ascertain the impact of the MD changes on the final conclusions, we carried subsequently DFT(B3LYP)/MM/EEF geometry optimizations of 3 for the X-ray structure and compared them with two snapshots (200 ps and 1 ns) taken from the MD/EEF trajectory. The results for the X-ray structure and the two MD snapshots showed precisely the same trends (for details, see Part III in the Supporting Information (SI)). Therefore, as we are studying here the trends of EEF effects, the use of a common snapshot (the X-ray-derived snapshot referred to as the 0 snapshot) is deemed to be the best strategy since it will treat these effects on equal footing for all of the enzymatic species. Accordingly, different spin states of the various species and their interconversions were studied under the influence of EEF for the 0 snapshot. Specifically, we explored spin inversion processes (1 f 2 and 3 f 4) and one-electron reduction steps (2 f 3 and 4 f 5) and considered three lowest spin states of 1-4, 6-7, and the state 25. The full set of results is collected in the SI. Below, we present the key results at the


2083


pubs.acs.org/JPCL

Figure 1. Change in the relative QM/MM energies under the influence of an EEF along the z axis (Fz).

the odd electrons on FeIII and O2- are coupled to a singlet as in the Weiss model.34 As can be seen from Figure 1d, in the range of Fz > 0, the quintet-singlet gap decreases, and the triplet-singlet gap slightly increases; smaller effects occur for Fz < 0. Furthermore, (Fz was found to change the charges and spin densities and also the Fe-O2 bonding mechanism (see details in Table SD5, SI). For Fz > 0, back-donation from O2- to FeIII intensifies with the increasing field strength, and at Fz = þ0.0125 au, the bonding becomes essentially a McClure-Goddard type,35 in which there is a singlet coupling of triplets FeII and O2. The catalytic cycle involves two reduction steps, 2 þ e- f 3 and 4 þ e- f 5, the latter being the rate-determining step in the P450cam cycle.36 Due to the complexity of these processes, their barriers have not been calculated yet, and hence, a thermochemical analysis might be useful here. The absolute value of the reduction energy (ΔE) for the 2 f 3 process at Fz = 0, which can be evaluated by the energy change Ep - Er (Ep and Er are the energies of reduced and oxidized species, respectively), is shifted relative to the experimental value (-98 kcal/mol) due to artificial long-range electrostatic interactions between the QM and MM charged distant residues.22,23


Since we are interested in the changes of ΔE due to the EEF, the absolute value is less relevant. In z-oriented EEFs, the energy change caused by the transfer of one electron from the donor (e.g., PDX) to the acceptor (heme) needs to be included and can be evaluated by the term e 3 d 3 Fz, where e is the basic electric charge and d is the z-axis projection of electron transfer distance. This term (e 3 d 3 Fz) represents the work done/released during the movement of one electron from the position corresponding to PDX to the position corresponding to the heme, under the EEF influence. Therefore, the driving force for the electron-transfer reaction can be expressed as ΔE =Ep Er þ e 3 d 3 Fz, where the last term, that is, work released during one electron movement, is dominant (see Table S1, SI). As no crystal structures are available for the reductase (PDX, having an Fe2S2 cluster) complex with P450cam, we rely on theoretical modeling,37 in which the Feheme-FePDX distance was estimated in the range of 12-17 Å. We considered a distance of 12 Å (along the z axis) and calculated the EEF-induced difference of the electrostatic potentials between the two moieties; this increment was then added to the energy of the electron acceptance processes and used to generate Figure 2 (for more details, see SI). Using values different from 12 Å only

2084


pubs.acs.org/JPCL

Figure 2. Reaction energy ΔE of (a) 2 þ e- f 3 and (b) 4 þ e- f 5 under the influence of an EEF along the z axis.

while flipping the field induces charge transfer from sulfur to porphyrin, thereby creating a thiolyl radical. These changes are attended by FedO bonding changes. With Fz 0, the FeO bond becomes more covalent, as indicated by the increased spin density on O. Thus, the EEF effect on Cpd I demonstrates the chameleonic character of Cpd I. In the sense that these changes can affect its regioselectivity in bond activation reactions, as suggested by model studies,18 this effect is important. In summary, the present QM/MM/EEF calculations demonstrate that an EEF acts as a gating device, with a significant effect on the catalytic cycle of P450. The EEF will control the initial gating of the cycle, its rate-determining step, its O2 uptake, the geometry of its resting state, and the spin-state ordering and electronic structure of its various active species. Especially, an EEF along the -z direction will facilitate the displacement of the aqua ligand (1 f 2) and two reduction processes (2 f 3 and 4 f 5) but obstruct O2 binding (3 f 4). Since the reduction 4 f 5 is the rate-controlling step of the cycle, a -z-oriented EEF will increase the efficiency of the catalytic cycle, and vice versa for a positive field. Of course, a large enough negatively oriented field that will prevent the Fe-O2 formation may convert the O2 binding to the ratecontrolling event and will thereby have an adverse effect, whereas a positively oriented field may be a bonus. An exact prediction cannot be made since it requires a complete kinetic treatment of the cycle in the presence of EEF. Still, the two extreme situations just described project the intriguing opportunities. Furthermore, the EEF has a potential of controlling the bond activation reactions of the active species as well.18 These effects were shown to be pronounced when the EEF was aligned with the axis perpendicular to the porphyrin plane. When perfect alignment cannot be attained, the Fx,y,z effects will be averaged, depending on the misalignment. While this is a purely theoretical study, it is important to note that experimental studies in the presence of EEFs are known; using a pulse electric field in an enzyme-incorporated nanocontainer of the core-shell type polyion complex (PIC) micelles achieved control of the enzymatic reaction of lysozyme,12 an electric field induced molecular-component exchange in constitutional dynamics liquid crystals,13 an

Figure 3. Group spin densities (FX) of the O, Fe, porphyrin, and S under the influence of an external electric field along the z axis.

will change the slope of the lines but will not change the computed tendency. Thus, it is apparent that Fz < 0 makes ΔE more negative and will hence facilitate these two one-electron reduction steps. However, a positive field will have an inhibitory effect, so much so that at Fz = þ0.0125 au, 5 even loses its electron and changes to 4 plus an anion on the carbonyl group of Leu356 adjacent to the thiolate ligand, Cys357 (Table SE2, SI). It follows therefore that the EEF influence on 4 f 5 can have a dramatic impact on the speed of the entire catalytic cycle and can turn it on and off by simply switching the direction of the field. Cpd 0 (6) is calculated to have a ground doublet state, in accord with EPR studies3 and previous DFT and DFT/MM calculations.4,25 From Figure 1e, it is clear that an EEF along the z axis can alter the energy gaps but will not alter the ground state. For Cpd I (7), the EEF changes the sextet, quartet, and doublet energy gaps by no more than -0.4 to þ0.5 kcal/mol (see Figure 1f). However, since Cpd I is a chameleon species,4,6 an EEF oriented along the z axis exerts a dramatic change on the electronic structure. As may be seen from Figure 3, the species becomes a predominant sulfur radical at Fz < 0, while at Fz > 0, it becomes predominantly a porphyrin cation radical, as found in model systems.18 In the resonance theoretical picture proposed,2,4,6,38,39 the electronic structure oscillates between two structures; |aæ has a thiolyl radical with a closed-shell porphyrin, and |bæ has a thiolate anion and a porphyrin radical cation. In the native state,6,26 Cpd I in P450cam is a resonance hybrid of |aæ and |bæ with a dominant thiolyl radical in the gas phase but a major porphyrin radical cation character in the protein pocket. Using Fz > 0 accentuates the porphyrin cation radical nature,


2085


pubs.acs.org/JPCL

ACKNOWLEDGMENT S.S. is supported by an ISF grant (53/09).

interfacial electric field in membrane bilayer affected the gating of the enzyme Cytochrome c,14-16 and application of an electric field in the STM junction can control the reversible isomerization of specifically designed azobenzene.17 Additionally, the existing capabilities to immobilize molecules and enzymes on modified electrodes/surfaces,40 lipid nanodiscs (where charged lipids can be embedded to create strong local fields),41 or on membrane nanolayers42 or trap molecules within “molecular flasks”43 or other nanocontainers12 imply that, eventually, EEF effects, such as the ones described herein, may be incorporated into and tested in experimental setups.44

REFERENCES (1)

(2) (3)

(4)

COMPUTATIONAL METHODOLOGY All QM/MM computations were preformed using ChemShell45 interfaced with Turbomole46 and DL_POLY47 and followed procedures described in the literature.21-28 The hybrid B3LYP functional48 was used throughout this study for the QM part, and the CHARMM22 force field49 was used for the MM part. QM/MM geometry optimization was preformed using a doubleζ basis set LACVP for all of the atoms. The energy was corrected by single-point calculations with a larger basis set which describes iron by the Wachters all-electron basis set,50 augmented with diffuse d and f polarization functions, while all the other atoms used 6-31þþG(d,p). The effects of uniform EEF were studied by adding appropriate charges on two parallel circular plates flanking the enzyme. For z-directed EEF, these two plates are of radius of 92.4 Å and ensure a uniform EEF in the active site since they are much larger than the size of P450cam. The two charged plates were placed with their centers at distances of 46.8 Å to Fe and their normals determined from Fe-O bond in Cpd I species, which makes the EEF thus generated in the direction to be approximately vertical to the porphyrin ring. In total, two plates contain 7082 point charges with an absolute magnitude of 0.01 e per point, which, with the above parameter settings of the charge plate (92.4 Å radius and 46.8 Å to Fe), produce accordingly a uniform EEF of 0.0025 au in the active site. However, for larger EEF values from 0.0050 to 0.0125 au, we simply increased the charge of all point charges accordingly. For the x,y-direction EEFs, the charge plate settings are similar, except that two Fe-NPor bonds were used to determine the direction of EEF. These charges are treated along with the intrinsic MM charges of the enzyme (see the SI for details).

(5) (6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

SUPPORTING INFORMATION AVAILABLE Complete refs (14)

45 and 49, computational details, and full set of computational results (including energies, Mulliken spin densities and charge, and geometry parameters of all studied species). This material is available free of charge via the Internet at http://pubs.acs.org.

(15)

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. Tel: þ972 (0) 2 658 5909. Fax: þ972 (0)2 658 4033. E-mail: [email protected].

(16)

Present Addresses:

(17)

†

Department of Bioinspired Science, Ewha Womans University, 120-750 Seoul, Korea.


2086

Ortiz de Montellano, P. R., Ed. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.; Kluwer Academic/ Plenum Publisher: New York, 2005. Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. HemeContaining Oxygenases. Chem. Rev. 1996, 96, 2841–2887. Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Structure and Chemistry of Cytochrome P450. Chem. Rev. 2005, 105, 2253–2277. Shaik, S.; Kumar, D.; de Visser, S. P.; Altun, A.; Thiel, W. Theoretical Perspective on the Structure and Mechanism of Cytochrome P450 Enzymes. Chem. Rev. 2005, 105, 2279– 2328. Sligar, S. G. Coupling of Spin, Substrate, and Redox Equilibria in Cytochrome P450. Biochemistry 1976, 15, 5399–5406. Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. Medium Polarization and Hydrogen Bonding Effects on Compound I of Cytochrome P450: What Kind of a Radical Is It Really? J. Am. Chem. Soc. 2000, 122, 12892–12893. Shaik, S.; Hirao, H.; Kumar, D. Reactivity of High-Valent IronOxo Species in Enzymes and Synthetic Reagents: A Tale of Many States. Acc. Chem. Res. 2007, 40, 532–542. Shaik, S.; Filatov, M.; Schr€ oder, D.; Schwarz, H. Electronic Structure Makes a Difference: Cytochrome P450 Mediated Hydroxylations of Hydrocarbons as a Two-State Reactivity Paradigm. Chem.;Eur. J. 1998, 4, 193–199. For factors controlling spin-forbidden processes, see, for example, Danovich, D.; Shaik, S. Spin-Orbit Coupling in the Oxidative Activation of H-H by FeOþ. Selection Rules and Reactivity Effects. J. Am. Chem. Soc. 1997, 119, 1773–1786. The state energy gap affects the spin flip probability since it affects the relative slopes of the states at their intersection points . The 21 f 62 differ by more than one spin flip and have to be mediated by mixing with states having intermediate spin, 4 4 1( 2) for 21 f 62. The process 53 f 14 involves coupling to 3O2 such that 53 þ 3 O2 couple to a total triplet spin, while 33 þ 3O2 couple to a singlet. Harada, A.; Kataoka, K. Switching by Pulse Electric Field of the Elevated Enzymatic Reaction in the Core of Polyion Complex Micelles. J. Am. Chem. Soc. 2003, 125, 15306– 15307. Giuseppone, N.; Lehn, J.-M. Electric-Field Modulation of Component Exchange in Constitutional Dynamic Liquid Crystals. Angew. Chem., Int. Ed. 2006, 45, 4619–4624. de Biase, P. M.; Paggi, D. A.; Doctorovich, F.; Hildebrandt, P.; Estrin, D. A.; Murgida, D. H.; Marti, M. A. Molecular Basis for the Electric Field Modulation of Cytochrome c Structure and Function. J. Am. Chem. Soc. 2009, 131, 16248–16256. Murgida, D. H.; Hildebrandt, P. Electron-Transfer Processes of Cytochrome c at Interfaces. New Insights by SurfaceEnhanced Resonance Raman Spectroscopy. Acc. Chem. Res. 2004, 37, 854–861. Kranich, A.; Ly, H. K.; Hildebrandt, P.; Murgida, D. H. Direct Observation of the Gating Step in Protein Electron Transfer: Electric-Field-Controlled Protein Dynamics. J. Am. Chem. Soc. 2008, 130, 9844–9848. Alemany, M.; Peters, M. V.; Hecht, S.; Rieder, K.-H.; Moresco, F.; Grill, L. Electric Field-Induced Isomerization of Azobenzene by STM. J. Am. Chem. Soc. 2006, 128, 14446–14447.


pubs.acs.org/JPCL

(18)

(19)

(20)

(21) (22)

(23)

(24) (25)

(26)

(27)

(28)

(29)

(30)

(31)

(32) (33)

(34) (35) (36)

(37)

Shaik, S.; de Visser, S. P.; Kumar, D. External Electric Field Will Control the Selectivity of Enzymatic-Like Bond Activations. J. Am. Chem. Soc. 2004, 126, 11746–11749. de Visser, S. P. What External Perturbations Influence the Electronic Properties of Catalase Compound I? Inorg. Chem. 2006, 45, 9551–9557. Hirao, H.; Chen, H.; Carvajal, M. A.; Wang, Y.; Shaik, S. Effect of External Electric Fields on the C-H Bond Activation Reactivity of Nonheme Iron-Oxo Reagents. J. Am. Chem. Soc. 2008, 130, 3319–3327. Sch€ oneboom, J. C.; Thiel, W. The Resting State of P450cam: A QM/MM Study. J. Phys. Chem. B 2004, 108, 7468–7478. Altun, A.; Thiel, W. Combined Quantum Mechanical/Molecular Mechanical Study on the Pentacoordinated Ferric and Ferrous Cytochrome P450cam Complexes. J. Phys. Chem. B 2005, 109, 1268–1280. Zheng, J. J.; Altun, A.; Thiel, W. Common System Setup for the Entire Catalytic Cycle of Cytochrome P450cam in Quantum Mechanical/Molecular Mechanical Studies. J. Comput. Chem. 2007, 28, 2147–2158. Wang, D. Q.; Thiel, W. The Oxyheme Complexes of P450cam: A QM/MM Study. J. Mol. Struct.: THEOCHEM 2009, 898, 90–96. Zheng, J. J.; Wang, D. Q.; Thiel, W.; Shaik, S. QM/MM Study of Mechanisms for Compound I Formation in the Catalytic Cycle of Cytochrome P450cam. J. Am. Chem. Soc. 2006, 128, 13204–13215. 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. Chen, H.; Ikeda-Saito, M.; Shaik, S. The Nature of the Fe-O2 Bonding in Oxy-Myoglobin: The Effect of the Protein. J. Am. Chem. Soc. 2008, 130, 14778–14790. Sch€ oneboom, J. C.; Neese, F.; Thiel, W. Toward Identification of the Compound I Reactive Intermediate in Cytochrome P450 Chemistry: A QM/MM Study of Its EPR and M€ ossbauer Parameters. J. Am. Chem. Soc. 2005, 127, 5840–5853. 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. Clarke, R. J. The Dipole Potential of Phospholipid Membranes and Methods for Its Detection. Adv. Colloid Interface Sci. 2001, 89, 263–281. Rovira, C.; Kunc, K.; Hutter, J.; Ballone, P.; Parrinello, M. Equilibrium Geometries and Electronic Structure of IronPorphyrin Complexes: A Density Functional Study. J. Phys. Chem. A 1997, 101, 8914–8925. Jensen, K. P.; Ryde, U. How O2 Binds to Heme. J. Biol. Chem. 2004, 279, 14561–14569. Strickland, N.; Harvey, J. N. Spin-Forbidden Ligand Binding to the Ferrous-Heme Group: Ab Initio and DFT Studies. J. Phys. Chem. B 2007, 111, 841–852. Weiss, J. J. Nature of the Iron-Oxygen Bond in Oxyhamoglobin. Nature 1964, 202, 83–84. McClure, D. S. Electronic Structure of Transition-Metal Complex Ions. Radiation. Res. Suppl. 1960, 2, 218–242. Benson, D. E.; Suslick, K. S.; Sligar, S. G. Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam. Biochemistry 1997, 36, 5104–5107. Wallrapp, F.; Masone, D.; Guallar, V. Electron Transfer in the P450cam/PDX Complex. The QM/MM e-Pathway. J. Phys. Chem. A 2008, 112, 12989–12994.


(38) (39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48) (49)

(50)

2087

Ullrich, V. Cytochrome P450 and Biological Hydroxylation Reactions. Top. Curr. Chem. 1979, 83, 67–104. Ogliaro, F.; de Visser, S. P.; Groves, J. T.; Shaik, S. Chameleon States: High-Valent Metal-Oxo Species of Cytochrome P450 and Its Ruthenium Analogue. Angew. Chem., Int. Ed. 2001, 40, 2874–2878. See assembly of metalloporphyrins in a controllable orientation to an electrified surface: Ashkenasy, G.; Kalyuzhny, G.; Libman, J.; Rubinstein, I.; Shanzer, A. Functional Monolayers with Coordinatively Embedded Metalloporphyrins. Angew. Chem., Int. Ed. 1999, 38, 1257–1261. Kijac, A. Z.; Li, Y.; Sligar, S. G.; Rienstra, C. M. Magic-Angle Spinning Solid-State NMR Spectroscopy of NanodiscEmbedded Human CYP3A4. Biochemistry 2007, 46, 13696– 13703. Note (as proposed by Sligar) that charged lipids can be embedded into the nanodiscs and generate large local fields. Choi, Y.; Yau, S.-T. Field-Effect Enzymatic Amplifying Detector with Picomolar Detection Limit. Anal. Chem. 2009, 81, 7123– 7126. Murase, T.; Horiuchi, S.; Fujita, M. Naphthalene Diels-Alder in a Self-Assembled Molecular Flask. J. Am. Chem. Soc. 2010, 132, 2866–2867. Attachment of P450cam, for example, to an electrode surface,40 may be achieved by use of self-assembled monolayers (SAMs) of bifunctional alkenthiols with anionic end groups (e.g., PO3-SAM and CO2-SAM), which can link to the surface lysines. At the same time, the reductase will be coordinate to the side of Cys357. For such strategies, see ref 15 herein. 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. Ahlrichs, R.; B€ ar, M.; H€ aser, M.; Horn, H.; K€ olmel, C. Electronic Structure Calculations on Workstation Computers: The Program System Turbomole. Chem. Phys. Lett. 1989, 162, 165–169. Smith, W.; Forester, T. R. DL_POLY_2.0: A General-Purpose Parallel Molecular Dynamics Simulation Package. J. Mol. Graph. 1996, 14, 136–141. Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648–5652. 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. Wachters, A. J. H. Gaussian Basis Set for Molecular Wavefuncitons Containing Third-Row Atoms. J. Chem. Phys. 1970, 52, 1033–1036.


External Electric Field Can Control the Catalytic Cycle of Cytochrome

Recommend Documents