The Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons A

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The Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons A Direct Comparison of the “Somersault” versus the “Consensus” Mechanism Involving Compound I Robert D. Bach* Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716 ReceiVed: May 18, 2010; ReVised Manuscript ReceiVed: July 23, 2010

Model theoretical quantum mechanical (QM) calculations are described for the P-450 hydroxylation of methane, isobutane, and camphor that compare the concerted somersault H-abstraction mechanism with the oxidation step involving Cpd I. Special emphasis has been placed on maintaining a balanced basis set in the oxidation step. QM calculations, employing the 6-311+G(d,p) basis set on the Fe atom and all of the key surrounding atoms involved in the C-H abstraction step, reaffirm a mechanism involving rearrangement of the iron hydroperoxide group (FeO-OH f FeO · · · HO•) in concert with hydrogen abstraction from the C-H bond of the substrate by the incipient bound hydroxyl radical HO•. The barrier for the somersault rearrangement of model Cpd 0 (FeO-OH) is calculated to be 21.4 kcal/mol in the absence of substrate. The overall activation energy for the oxidation of camphor involving the somersault motion of the FeO-OH group of P450 model porphyrin iron(III) hydroperoxide [Por(SH)Fe(III)-OOH-] f [Por(SH)Fe(III)-O · · · · HO-] in concert with hydrogen abstraction is ∆E‡ ) 12.4 kcal/mol. The corresponding abstraction of the hydrogen atom from the C-H bond of camphor by Cpd I has an activation barrier of 17.6 kcal/mol. Arguments are presented that the somersault rearrangement is induced by steric compression at the active site. Kinetic isotope effect data are discussed that provides compelling evidence for a rate-limiting step involving C-H bond cleavage. 1. Introduction Cytochrome P450 enzymes catalyze a diversity of oxidations, including such difficult reactions as the hydroxylation of hydrocarbons.1,2 This enzyme has played an exceptionally large role in biology and medicine, consequently a considerable amount of both experimental and theoretical effort has been expended in discerning the various steps involved in P450 oxidation.1 The generally accepted mechanism for cytochrome P450 hydrocarbon oxidation was proposed by Groves2 over three decades ago (eq 1). This “oxygen rebound” or consensus mechanism is postulated to involve a ferryl species (FeVdO) formed by the activation of molecular oxygen. The active site of cytochrome P450, in its resting state (not shown), consists of an iron porphyrin with the iron(III) coordinated to a water molecule and a cysteinyl axial ligand. In the substrate binding and displacement of a loosely bound water ligand, one-electron reduction of the iron occurs, after which molecular oxygen binds to form the last quasi-stable P450 intermediate (Cpd 0).3 Subsequent formation of the supposed catalytically active ferryl species, compound I (Cpd I), abstracts a hydrogen from the substrate to yield a one-electron reduced ferryl species (FeIV-OH) and a carbon radical intermediate (R•) that recombines in the so-called “oxygen rebound” step2 with the formal equivalent of an iron-bound hydroxyl radical, FeIV-OH, to give an enzyme-product complex, the final alcohol product (eq 1).1c,d

* To whom correspondence should be addressed. E-mail: rbach@ udel.edu.

However, there has been considerable controversy about the formation and actual involvement of Cpd I, and extensive quantum mechanical/molecular mechanical (QM/MM) studies have pointed out many problems with the consensus or “oxygen rebound” mechanism involving a proton relay,4a adding to the controversy. We raised serious questions more than 15 years ago concerning the proposed reaction sequence on the basis of the thermodynamic properties of the oxygen-oxygen bond in the proposed intermediate iron(III)hydroperoxide (Cpd 0).5a,6 We pointed out that the proton affinities of the proximal and distal oxygens of the X-FeO-OH system are such that once the hydroperoxo-heme species (Cpd 0) is formed the distal oxygen is much less favorable to protonation.5a Our studies have benefited from many years of a methodical buildup of theoretical studies that we have made on small molecular systems,7,8 that while not directly mimicking the same heme chemistry has provided essential insight into the new mechanism that we propose. Given our lack of detailed understanding of the positions of waters in enzymatic P450 intermediate states, and the lack of any crystal structures for P450 isozymes in the distal protonated/hydroperoxy heme form, it has been difficult to characterize the energy landscape for the protonation steps involved in the overall PES. We have recently described a new class of hydroperoxides where, in certain cases, homolytic O-O bond cleavage is attended by a unique stabilization of the resulting oxyradical fragment. For example, the simplest cyclic peroxide, dioxirane, has several higher lying structures where the O-O bonds in these singlet minima are elongated above that in ground state (GS) dioxirane.7 The O-O bond in peroxynitrous acid (ONO-OH) also has an atypical O-O σ-bond that exhibits a low bond dissociation energy (G2, 22.0 kcal/mol).8 During the O-O bond elongation process the electron of the developing ONO radical fragment shifts from being localized largely on the oxygen of the O-O bond to the central nitrogen atom of the ONO fragment and the lower energy 2A1 ground state ONO

10.1021/jp1045518  2010 American Chemical Society Published on Web 08/06/2010

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Figure 1. The UB3LYP/6-311+G(d,p) reaction energy diagram for the FeO-OH somersault rearrangement in the presence of the thiolate anion (HS-). The 6-311+G(d,p) basis set was used on all atoms except the peripheral C-H groups of the heme (440 basis functions). The total energy values for HO• radical (-75.76234 au) and Por(SH)FeO triplet anion (-2726.11194 au) were taken for the H-bonding energy estimation.

radical and results in a stabilization of 30.2 kcal/mol.8b Simple vinyl and phenyl hydroperoxides (H2C ) CHO-OH)7,8b also have atypically low O-O BDE (20.2 and 19.6 kcal/mol, G3) due to the delocalization of the O-centered radical to a C-centered radical. In general, O-O bond dissociation in a hydroperoxide, attended by some form of unique stabilization of the incipient oxyradical, can produce an exceptional type of peroxo bond that can undergo a somersault or vaulting motion to a stabilized hydroxyl radical intermediate hydrogen bonded to its open shell oxyradical fragment (MO-OH f MO · · · · HO). We recently reported just such an extraordinary reduction in bond dissociation energy (BDE ) 22.9 kcal/mol) accompanying homolytic O-O bond cleavage in a porphyrin iron(III) hydroperoxide (FeO-OH)!5a On the basis of these data we have proposed a new potential pathway for what transpires in the so-called “black box” in the P450cam oxidation sequence.5a This new mechanism involves the intermediacy of a metastable “inverted” iron(III) hydroperoxide produced by a somersault motion (FeO-OH f FeO · · · · HO) of the hydroperoxide moiety in Cpd 0. The isomerization of the neutral parent heme without the axial SH ligand [PorFe(III)OOH ] f [PorFe(III)O · · · · HO ] had an activation barrier of only 16.3 kcal/mol when an all-electron calculation was used with a 6-311+G(d,p) basis set for iron. When the SH ligand was included, rearrangement of the anionic FeO-OH to produce the isomeric ferryl oxygen hydrogen bonded to an HO• radical [Por(SH)Fe(III)-OOH-] f [Por(SH)Fe(III)-O · · · · HO-] exhibited an activation energy (∆E‡ ) 20.4 kcal/mol) that was considerably lower than the calculated O-O bond dissociation energy (31.7 kcal/mol).5a We proposed a novel mechanism for P450 mediated isobutane oxidation where the somersault rearrangement of the FeO-OH moiety is attended by a concerted abstraction of a hydrogen atom from the C-H bond of isobutane by the hydrogen-bonded hydroxyl radical with an activation energy of ∆E‡ )19.5 kcal/mol (Fe(III)O · · · · HO• +

H-CR3 f Fe(III)O · · · · HO-H + •CR3). The resulting water molecule remains strongly hydrogen bonded to anionic Cpd II, as formally represented in Min-1 (Figure 1), and the hydroxylation step involves a concerted but nonsynchronous transfer of a hydrogen atom from this newly formed, bound, water molecule to the ferryl oxygen with a concomitant rebound of the incipient HO• radical to the tertiary carbon radical of isobutane to produce the C-O bond of the final product, tert-butyl alcohol. We were also able to locate a TS on the potential energy surface (PES) for the oxygen rebound step that follows H-abstraction that is 2 kcal/mol lower in energy than the preceding concerted rate-limiting C-H abstraction step. The overall proposed new mechanism is consistent with much of the ancillary experimental data for this enzymatic hydroxylation reaction as noted below. Thus, the controversy over the protonation of Cpd 0 to produce Cpd I has been partially resolved and the first step in our concerted process involving the somersault rearrangement of Cpd 0 has been adopted by Shaik and Thiel9 as described below. More recently we have reported5b hydrocarbon oxidation by aqueous solvated cationic iron(III) hydroperoxides [(H2O)nFeIIIOOH] that also involves a novel rearrangement of the hydroperoxide group (FeO-OH f FeO · · · · HO) in concert with hydrogen abstraction from a hydrocarbon by the incipient HO• radical with activation barriers ranging from 17-18 kcal/mol. The transition structure for this hydroxylation bears a striking resemblance to our P450 TSs despite the fact that this concerted somersault isomerization-H-atom abstraction can take place with a relatively simple FeO-OH system in the absence of the electronic influence of a porphyrin ring. One of the primary goals of this report is to provide a direct comparison between the ground state complexes and transition structures for C-H bond breaking involving Cpd I with our concerted somersault rearrangement attending H-abstraction involving the bound hydroxyl radical HO•. We describe both ground state (GS) and transition structures (TS) for the P-450 hydroxylation of

Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons

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TABLE 1: Calculated Activation Energies (∆E‡) for H-abstraction by Cpd I and the Somersault Rearrangement in Concert with H-Abstraction

hydroxylation reaction in the absence of its local environment. This is especially true if the geometry of the core reaction is optimized with a much larger basis set than typically applied in QM/MM calculations. In this report we use the 6-311+G(d,p) basis set on all atoms except the C-H groups comprising the outer periphery of the ring comprising the heme. When this more flexible all-electron basis set was used on the iron atom, its four equatorial nitrogen ligands, the axial SH and OOH groups, rearrangement of the FeO-OH group produced the isomeric ferryl oxygen hydrogen bonded to the inverted HO• group [Por(SH)Fe(III)-OOH-] f [Por(SH)Fe(III)-O · · · · HO-] that bears an overall negative charge. The classical activation energy for this isomerization was ∆E‡ ) 21.4 kcal/mol (19.1 kcal/mol with ZPVE). This barrier is in excellent agreement with a thermodynamic analysis by Koppenol11a who estimated the homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical (P450FeOOH2+ ) P450FeO2+ + HO•) to be unfavorable with a ∆G° ) 21.99 kcal/ mol. The transition structure, TS-1, for the somersault rearrangement has an O-O bond that is elongated from 1.425 to 2.221 Å with the O-H hydrogen H-bonded to the ferryl oxygen at a distance of 2.149 Å (Figure 1). Animation of the single imaginary frequency (ν ) 60.97i cm-1) for TS-1 shows largely O-O bond elongation with a pendulum motion of the O-H group with clear evidence of a bonding interaction of the O-H hydrogen atom with the ferryl oxygen in the eigenvectors. The very small magnitude of the imaginary frequency supports heavy atom motion in the TS (OH), and the much larger imaginary frequencies shown below are consistent with only light atom movement (H atom) in the TS.10 The resulting product, with the inverted HO• radical bound to the ferryl oxygen, was 15.6 kcal/mol higher in energy than the ground state minimum Cpd 0 as shown in Figure 1. Min-1 is formally Cpd II, which results from the one-electron reduction of Cpd I, hydrogen-bonded to the HO• radical. This is not a highly reactive free HO• radical because it is strongly hydrogen bonded to the ferryl oxygen (∆E ) -15.9 kcal/mol) in nearly a linear inverted fashion with a relatively short O · · · · HO distance of 1.666 Å reflecting the anionic nature of the donor. The “oxyradical” fragment, resulting from homolytic O-O bond cleavage in hydroperoxide GS-1, correlates with the 3Ag(FeIV) triplet state5a that is 28.3 kcal/mol lower in energy than the singlet 1Ag(FeIV) state and is 38.3 kcal/mol more stable than the corresponding 5A2u(FeIII) quintet state.11b In order for the somersault rearrangement of hydroperoxides to readily occur, the barrier for rearrangement must be considerably lower than the homolytic O-O bond dissociation energy for GS-1 (BDE ) 28.8). It is this unique type of stabilization of the 3Ag(FeIV) triplet state, moving the electron spin from the ferryl oxygen down into the heme, in addition to the H-bonding interaction of the O-OH in the TS, that is responsible for the relatively low barrier for homolytic O-O bond cleavage in GS-1. b. A Direct Comparison of the Somersault and Consensus Mechanisms for Methane Hydroxylation. Having established the QM activation energy of the somersault rearrangement (TS-1) at a credible level of theory, we now determine barriers for the overall concerted FeO-OH rearrangement with attending Habstraction from a complexed hydrocarbon. We have included methane as a substrate in this study because many of the earlier QM/MM studies on P450 have used methane as the hydrocarbon involved in C-H bond breaking. An earlier QM/MM barrier reported by Shaik12a for hydrogen abstraction from methane by Cpd I was 26.5 kcal/mol. In a related QM B3LYP/LACVP3p++** study,12b again using Cpd I as the primary oxidant, they reported

transition structure hydrocarbon TS-2 TS-2 TS-3 TS-4 TS-4 TS-5 TS-6 TS-6 TS-7 TS-7

methane methane methane isobutane isobutane isobutane d-camphor d-camphor d-camphor d-camphor

oxidant Cpd I Cpd I FeO · · · HO Cpd I Cpd I FeO · · · HO Cpd I Cpd I FeO · · · HO FeO · · · HO

barrier, basis seta kcal/molb freq, cm1 all-electron ECP all-electron all-electron ECP all-electron all-electron ECP all-electron ECP

19.5 23.7 23.0 14.4 17.0 17.8 17.6 19.7 12.4 24.1

(15.5)c (19.5)d (18.4)e (10.0)f (12.9)g (14.5)h (14.0)i (16.0)j (7.7)k (19.5)l

-1707.9i -1557.7i -1326.3i -1646.6i -1474.8i -234.3i -989.4i -834.9i -713.8i -704.9i

a All structures in this table have the 6-31G basis set for the peripheral C-H groups of the heme. See also individual figures for a more complete description of the basis set. b The barriers in parentheses are with zero-point correction. c The oxygen and nitrogen atoms including the CH4, SH, and Fe have the 6-311+G(d,p) basis set (473 basis functions). d The oxygen and nitrogen atoms including the CH4 and SH have the 6-311+G(d,p) basis set, but the Fe has the ECP basis described in the Computational Details section (437 basis functions). See reference 6 and Supporting Information for the GS and TS structures with the ECP basis. e The oxygen and nitrogen atoms including the CH4, SH and Fe have the 6-311+G(d,p) basis set (503 basis functions). f The oxygen, nitrogen atoms, SH, and C-H of isobutane, including the Fe, have the 6-311+G(d,p) basis set (494 basis functions). g The oxygen, nitrogen atoms, SH, and C-H of isobutane have the 6-311+G(d,p) basis set; the Fe has the ECP basis (458 basis functions). h The oxygen, nitrogen atoms, SH, and C-H of isobutane, including the Fe, have the 6-311+G(d,p) basis set (524 basis functions). i The oxygen, nitrogen atoms, SH, and CH2 group of camphor, including the Fe, have the 6-311+G(d,p) basis set. All camphor atoms, except the C5 π-CH2 group, have the 6-31G basis (569 basis functions). j The ferryl oxygen and C-H group of camphor have the 6-311+G(d,p) basis set; the Fe has the ECP basis. All camphor atoms, except the C5 π-CH2 group, have the 6-31G basis (533 basis functions). k The oxygen, nitrogen atoms, SH, and π-CH2 group of camphor, including the Fe, have the 6-311+G(d,p) basis set. All camphor atoms, except the C5 π-CH2 group, have the 6-31G basis (605 basis functions). l The oxygen, nitrogen atoms, SH, and π-CH2 group of camphor have the 6-311+G(d,p) basis set; the Fe has the ECP basis. All camphor atoms, except the C5 π-CH2 group, have the 6-31G basis (569 basis functions).

methane, isobutane, and camphor that compare the concerted somersault H-abstraction mechanism with the oxidation step involving Cpd I as presented in Table 1. We have maintained a balanced basis set in all calculations (6-311+G(d,p) as described at the end of the paper in Computational Details. 2. Results and Discussion a. The Somersault Rearrangement of Model Cpd 0. Our proposed mechanism operates under the assumption that the oxidizing capacity of P450cam arises from the participation of a low-lying transient hydroperoxide species resulting from FeO-OH isomerization. In our earlier description5a of the somersault rearrangement in model Cpd 0 we reported activation barriers that varied from 20.3-24.8 kcal/mol depending upon whether we used an ECP basis set for iron and which atoms in the model Cpd 0 included the more flexible 6-311+G(d,p) basis set. It became obvious that maintaining a basis set balance throughout [6-311+G(d,p)] on all of the affected atoms in the FeO-OH rearrangement, especially the Fe atom, was necessary in order to provide uniformly consistent barriers. Although we recognize the significance of having the local residues present in a QM/ MM study, we also think that is important to know the comparable intrinsic activation barrier of just the main core

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Figure 2. The ground state prereaction complex of Cpd I with methane and the TS for hydrogen abstraction by the ferryl oxygen. This is an all-electron calculation where the methane (CH4), Fe, O, N, and SH utilized a 6-311+G(d,p) basis set with a 6-31G basis set on the peripheral heme C-H atoms (473 basis functions). The sum of electronic energies with zero-point corrections are -2766.196 801 and -2766.172 125 au. The values in parentheses are for calculations with an ECP basis on Fe (437 basis functions). For complete xyz coordinates for these structures see Supporting Information.

a ∆E‡ ) 22.31 kcal/mol (with ZPVE correction) for methane and 13.37 kcal/mol for camphor. In that series of calculations it was generally conceded that the doublet was the lowest energy state and that the rate-limiting step involves H-abstraction via a transition state typically labeled 2TSH. We find a QM barrier of 23.7 kcal/mol when we also use an ECP for the Fe atom (22 basis functions) and a 6-311+G(d,p) basis set for all other atoms, including the CH4 group, except for the peripheral C-H groups of the heme. The barrier for TS-2 (Figure 2) is reduced to 19.5 kcal/mol (15.5 kcal/mol with ZVPE) with an all-electron calculation that also uses the 6-311+G(d,p) basis set (58 basis functions) for Fe when measured relative to GS-2. We typically see a reduction in the activation energy for hydrogen abstraction with the larger more balanced basis set on iron as evidenced in Table 1. The O-H (1.169 Å) and C-H (1.348 Å) distances in TS-2 differ from the comparable bond distances reported for the above QM/MM method (1.08 and 1.51 Å).12a By comparison the most recent P450 hydroxylation mechanism suggested by Thiel, Shaik et al.9a invokes the O-O somersault rearrangement as the rate limiting step followed by formation of Cpd I (Mechanism II). They have now adopted the somersault FeO-OH rearrangement5a as the homolytic O-O bond-breaking step in P450 oxidation. Upon reexamination, they found that their mechanism I,9a (the consensus mechanism) requiring distal protonated Cpd 0 (FeOOH2), was more than 20 kcal/mol above Cpd 0 and the barrier for its decay was only 3-4 kcal/mol.

The problem with this step in that mechanism is not a result of the dissociation of distal protonated Cpd 0 into Cpd I and H2O because the ∆G° for this reaction this has been estimated11a to be very close to 0 at pH 7. It is the protonation step that is counter thermodynamic because of the greater proton affinity for the proximal oxygen of FeO-OH that initially led us into this mechanistic study.6a Mechanism I is essentially the “oxygen rebound” or consensus mechanism2 described above in eq 1. Consequently, they opted for mechanism II9a with an initial O-O bond cleavage followed by a concomitant proton and electron transfer yielding Cpd I and a water molecule. Their [Por(SH)Fe(III)O-OH- ] f [Por(SH)Fe(III)O · · · · HO- ] rearranged structures were found to be remarkably close to our earlier GS and TS for the somersault isomerization5a except for minor changes in bond angles resulting from enhanced OH moiety interactions due to inclusion of the protein environment in the larger MM regions surrounding the active site. The ratelimiting step in their suggested mechanism is O-O cleavage with a barrier of about 13-14 kcal/mol. However, at this point they chose to deviate from our proposed concerted mechanism5a (shown below) and suggested that their overall process is the favored low-energy pathway to Cpd I by accepting a proton in the Asp251 channel and an electron from the heme in an essentially barrierless process. It was suggested that Cpd 0 and Cpd I are of similar energies with a slight preference for the latter. More importantly, it was advocated that Cpd I is the primary oxidant involved in C-H bond cleavage of the substrate. This, of course, requires a second TS for hydrogen abstraction from the hydrocarbon substrate by the newly formed Cpd I. In our opinion the proposed rate-limiting step in mechanism II is untenable because the observed experimental kinetic isotope effect (KIE)13,14 data requires that C-H bond breaking be involved in the rate-determining step. Our mechanism, where C-H bond breaking is accomplished by the developing HO• radical attending the concerted somersault rearrangement, is consistent with all of the experimental data including KIE as discussed in detail below.



Figure 3. B3LYP-optimized stationary points for the oxidation of methane to methanol with primary reactant Min-1 (PorFe(SH)O · · · · HO-). All the atoms directly involved in the oxidation step (SH, Fe, N, OOH and CH4) were treated with the 6-311+G(d,p) basis set. The total number of the basis functions in this reactant is 503.

In a more recent disclosure Thiel15a retains the concept that the rate-limiting step at the QM/MM level is O-O cleavage with a barrier of about 13-14 kcal/mol in both the Asp251 and Glu366 channels. In this latest scenario Cpd I also retains its role as the decisive oxidant, but no details of the somersault rearrangement or the H-abstraction step were given. It was proposed in that QM/MM study that the role of Thr252 with respect to Cpd 0 was that of a highly specific proton donor to the distal oxygen atom of Cpd 0, and the preference for the coupling reaction in the wild-type enzyme was due to this specificity. This same basic mechanism was preferred in recent QM/MM studies15b that included the methoxythreonine mutant of P450cam. It was again suggested that formation of Cpd I (coupling) always proceeds through the two-step mechanism involving initial O-O bond cleavage9a (our so-called somersault rearrangement5a). The second step is hydrogen atom transfer to MeO-Thr252, which yields Cpd I and water. We hasten to point out that this entire reaction sequence of O-O bond homolysis and proton relay affording Cpd I,9,15 while in the presence of the local residues at the active site, apparently excluded the camphor substrate and from a steric perspective this is a very important point as delineated below. Examination of the prereaction complex of model Cpd 0 with methane, GS-3 (Figure 3) clearly shows that the more basic of the two oxygen atoms of the hydroperoxy functionality is, as previously reported,5a the proximal oxygen with an O · · · · H-C distance of 2.420 Å. The Fe-O and Fe-S bond distances are quite comparable to those typically expected for Cpd 0 models.9,12 The classical activation barrier for C-H bond breaking, TS-3 (23.0 kcal/mol) is measured from GS-3 and hence represents a somersault rearrangement in concert with H-abstraction by the hydrogen bonded HO• radical. The barrier for TS-3 involving the bound HO• radical is 3.5 kcal/mol higher than hydrogen abstraction initiated by Cpd I, TS-2. This is a very early TS as noted by the O-H distance of only 1.090 Å and a C-H distance of 1.433 Å. It is particularly noteworthy that the Fe-S, Fe-O,

and FeO · · · · HO distances in TS-3 are essentially identical to those in Min-1, the product of the somersault rearrangement of model Cpd 0 (Figure 1). These combined data support a ratelimiting step for methane hydroxylation that involves C-H bond breaking. Methane is a very poor P450 substrate because it produces a methyl radical and hence the barrier for the oxidative step (∆E‡ ) 23.0 kcal/mol) is higher than that for the somersault rearrangement itself (∆E‡ ) 21.4 kcal/mol). c. A Direct Comparison of the Somersault and Consensus Mechanisms for Isobutane Hydroxylation. In our earlier study5a we preferred to use isobutane as the substrate, because H-abstraction provides a relatively stable tertiary carbon radical intermediate that, in principle, could serve as a viable substrate for enzymatic P450 hydroxylation. This appears to be the first example of a calculated activation barrier for C-H bond breaking in isobutane using Cpd I as the oxidant. In GS-4 (Figure 4), the tertiary H atom of isobutane is bonded at a distance of 2.575 Å and forms an angle with the Fe-O bond of 126.7°. The classical activation barrier for C-H bond breaking, at the same all-electron level as that used above for methane, provides a much lower activation barrier reflecting the incipient tertiary radical produce by interaction with Cpd I (∆E‡ ) 14.4 kcal/mol). The large magnitude of the single imaginary frequency (ν ) 1646.6i cm-1)10 for TS-4 is consistent with only light atom (hydrogen) movement in the TS oscillating in a nearly linear fashion (163.9°) between the C and O atoms in a central position (C-H ) 1.289 Å, H-O ) 1.256 Å). Heavy atom motion upon going from ground to transition state is minimal as evidenced by the relatively small change in the Fe-O-H angle in TS-4 that forms an angle with the Fe-O bond of 125.4°. However, the distance from the tertiary carbon atom to the ferryl oxygen is only 2.520 Å, and the distance from the nearest CH3 hydrogen to a heme nitrogen is only 3.1 Å. This could portend steric problems with larger more complex substrates. With an all-electron calculation, when the additional three carbon atoms of the isobutane (560 basis functions) also had the 6-311+G(d,p) basis set, the barrier for TS-4 was

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Figure 4. B3LYP-optimized stationary points for the oxidation of isobutane to t-butyl alcohol with primary reactant Cpd I (PorFe(SH)O). All the atoms directly involved in the oxidation step (SH, Fe, N, OOH and the C-H fragment of isobutane) were treated with the 6-311+G(d,p) basis set with a 6-31G basis set on all other atoms. The sum of electronic energies with zero-point corrections are -2884.018 247 and -2884.002 357 au. The total number of basis functions in this reactant is 494.

essentially unchanged (∆∆E‡ ) 0.2 kcal/mol) supporting our assumption that only the C-H bond in isobutane required the larger basis set. We note that both optimized doublet GS and TS structures involving Cpd I exhibit considerable spin contamination (〈S2〉 ) 1.7) as we have discussed previously.5a In general, with the Cpd 0 prereaction complex the spin contamination is minimal with 〈S2〉 ∼ 0.75 for the doublet. With the ECP basis set for iron we calculated a slightly greater activation energy (∆E‡ ) 17.0 kcal/mol) for TS-4 (Table 1). Our earlier report on the concerted somersault rearrangement-H-abstraction TS for Cpd 0 and isobutane5a was at the ECP level for Fe with the 6-311+G(d,p) basis set being limited to only the C-H and OOH atoms (413 basis functions). We reported an activation barrier of 19.5 kcal/mol. We now employ an internally consistent level of theory with this more flexible basis set on all involved atoms including the Fe and all equatorial (N) and axial ligands (SH, OOH and C-H) with the remaining atoms having the 6-31G basis set (524 basis functions). The ground state prereaction complex with isobutane can be difficult to locate because of the long-range H-bonds of isobutane to the OOH moiety. In GS-5a (Figure 5) the primary bonding interactions involve two CH3 groups, whereas the preferred molecular alignment in GS-5b, which must precede the TS, has only the tertiary hydrogen H-bonded to the proximal oxygen at a distance of 2.355 Å; it is slightly higher in energy (∆E ) 0.70 kcal/mol). The activation energy for C-H bond cleavage, TS-5, in concert with the somersault rearrangement of GS-5b (∆E‡ ) 17.8 kcal/mol) is 3.4 kcal/mol higher than that when Cpd I is the primary oxidant. This is a somewhat later TS than that for methane hydroxylation because the C-H bond is elongated to 1.165 Å and the H · · · · OH distance is 1.468 Å. There is very little heavy atom motion required upon going from the ground state to the TS as evidenced by the relatively small change in the angle that the Fe-O bond makes with the tertiary carbon of isobutane upon going from GS-5B (Fe-O-C

) 141.4°) to TS-5 (Fe-O-C ) 146.2°). Comparison of the geometry of Min-1 with TS-5 leaves no question that this transition structure derives from an initial somersault rearrangement of Cpd 0 (GS-1) while complexed to isobutane in concert with H-abstraction by the HO• radical to produce the tBu• radical and a water molecule.5a The relatively high barrier for H-atom abstraction by the HO• radical in TS-5 (∆E‡ ) 17.8 kcal/mol) is a consequence of the fact that this oxyradical remains strongly H-bonded to the ferryl oxygen throughout the reaction sequence as evidenced by the much lower activation energy for H-atom abstraction from isobutane by a free HO• radical [QCISD/631G(d), ∆E‡ )7.2 kcal/mol].5a By contrast, the activation barrier for H-abstraction by Cpd I, TS-4, is measured from Cpd I complexed to isobutane and no consideration is given to the energetic costs of producing the presumed “culprit” oxidizing agent, Cpd I. In the Shaik, Thiel scenario9a one has to first expend the 13-14 kcal/mol to get from Cpd 0 to Cpd I in order to arrive at the same purported t-butyl carbon radical that follows our TS-5. Recall, it was suggested that Cpd 0 and Cpd I are of similar energies with a slight preference for the latter. More importantly, the suggested rate-limiting step in the QM/MM Thiel, Shaik et al.9a mechanism is O-O cleavage with a barrier that increases to 17.1 kcal/mol in a single point calculation when the basis set was increased [LANL2DZ(Fe) 6-31+G*(rest)]. It is important to point out that this series of QM/MM calculations to produce Cpd I were apparently carried out in the absence of any hydrocarbon substrate. As we point out below, homolytic O-O bond cleavage places the incipient HO• very close to the requisite C-H bond in order to produce the carbon-centered radical. Although one can not make a direct comparison of the reported activation barriers because of the different methodologies, we note that their “rate limiting” O-O bond9a cleavage is very close to the activation energy that we suggest for the oVerall concerted somersault rearrangement-proton abstraction TS, TS-5 (∆E‡ )



Figure 5. B3LYP-optimized stationary points for the oxidation of isobutane to t-butyl alcohol with primary reactant Min-I [PorFe(SH)O · · · · HO]. All the atoms directly involved in the oxidation step (SH, Fe, N, OOH and CH) were treated with the 6-311+G(d,p) basis set. The total number of basis functions in this reactant is 524. TS-5 is rotated with respect to GS-5B to give a better view of the H-abstraction step.

17.8 kcal/mol; 14.5 kcal/mol with ZPVE) starting from Cpd 0. In the present study the ferryl bound hydroxyl radical represented by Min-1 is not quite as effective as Cpd I for C-H bond cleavage step in isobutane when the same basis set is used. However, this comparison does not include the energy requirements to actually form Cpd I, nor does it consider the amount of heavy atom motion required to get Cpd I properly aligned and in position to then sequentially abstract an H-atom from the substrate! The extent of C-H bond stretching in TS-4 (1.289 Å) is considerably greater than that in TS-5 (1.165 Å), suggesting that the KIE for Cpd I should be greater that that for our HO• radical mechanism, which is consistent with the observed experimental KIE data presented below. For the most part in this study we first performed calculations on the GS and TS using the ECP basis set on the iron atom and then expanded to the larger basis set. Unfortunately, in the allelectron calculations the additional flexibility for the Fe atom (58 basis functions) introduces a problem with wave function conversion. In several cases we converged to a geometry with a unique total energy that we felt was simply too high. Since the homolytic O-O barrier (TS-1) is 21.4 kcal/mol, somersault rearrangement in concert with C-H abstraction (with a reasonable substrate) should exhibit an overall barrier very close to or less than that value. This wave function conversion problem can be rectified by a slight change in the initial geometry or by starting out with a less rigorously converged wave function (SCFCON)5) and reading that wave function for the next iteration with a more tightly converged wave function (SCF-

CON)6 or 7). One can then arrive at a final geometry with a wave function that can be up to 10 kcal/mol lower in total energy. This does not seem to be a problem with the ECP calculations where we arrive at a converged geometry without too much difficulty. We were guided to look for a lower energy solution simply by the calculated activation energies for C-H abstraction. d. A Direct Comparison of the Somersault and Consensus Mechanisms for Camphor Hydroxylation. Earlier DFT results by Yoshizawa16 on the hydroxylation of camphor by Cpd I suggested an activation barrier of 23.8 kcal/mol for rate-limiting H-abstraction for the doublet state. Friesner et al.4b reported a much lower activation energy (11.7 kcal/mol) in QM-model studies. The lower activation energy was attributed to stabilization of the TS by interaction of positively charged residues in the active site with carboxylate groups on the heme periphery. Prior results by Shaik and Thiel9b for Cpd I initiated C5-exo-H-abstraction from camphor suggests a barrier of 21.1 kcal/mol in the gas phase at the protein geometry and 19.5 kcal/mol by optimization in the gas phase. These values are quite close to their reported QM/ MM barriers in the range of 20-22 kcal/mol for H-abstraction from camphor by Cpd I depending on the QM region and the basis set used. More recent calculations by Thiel17 suggest a QM contribution to the QM/MM H-abstraction barrier of 16-17 kcal/mol in all snapshots considered. The full QM/ MM barriers were reported in the 14-18 kcal/mol range that dominate the QM region. Thus, we see quite a range for

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Bach

Figure 6. B3LYP-optimized stationary points for the oxidation of camphor to 5-exo-hydroxycamphor with primary reactant Cpd-I. All the atoms directly involved in the oxidation step (SH, Fe, N, OOH, and CH2) were treated with the 6-311+G(d,p) basis set. The total number of the basis functions in this reactant is 569.

activation barriers, but in the final analysis the magnitude of the Cpd I barrier for camphor with (QM/MM) and without (QM) the local residues at the active site do not appear to differ significantly. In a recent P450 BM3 study Friesner also found that activation barriers with and without vacuum optimization gave nearly identical barriers (21.4 and 21.5 kcal/mol) for H-abstraction by Cpd I suggesting that the protein pocket does not facilitate the reaction by conformational restraints or imposed strain.4c In the present study our QM barrier in the absence of local residues is in excellent accord with the above QM/MM results in the 14-18 kcal/mol range. The GS and TS for Cpd I induced C-H bond breaking in camphor is shown in Figure 6. The basis set on the Cpd I residue (GS-6) is maintained the same as that for TS-1 in order to ensure internal consistency while the 6-311+G(d,p) basis set for camphor was only on the π-CH2 group at the key reaction site, C5. The ground state complex between Cpd I and camphor (GS6) is rather complex in that the ferryl oxygen is H-bonded to several C-H bonds in camphor with the longest distance (2.896 Å) being that to the exo-hydrogen at C5 (Figure 6). The exo-TS for H-abstraction from C5 by Cpd I exhibits a barrier of 17.6 kcal/mol (14.0 with ZPVE) when measured relative to model Cpd I complexed to camphor (GS-6). In TS-6 the C5-H-O angle is nearly linear as anticipated (174.8°) and the C-H and C-O distances are comparable in this“ central” TS (C-H ) 1.237 and H-O ) 1.293 Å). This is a rather compact TS, and we note that the distance from the ferryl oxygen to C5 of camphor is only 2.567 Å, whereas the distance from the endo-C5 hydrogen to the nearest heme nitrogen is only 2.769 Å, and we will expand upon this fact below. There is a marked difference between the Cpd I TS for camphor (TS-6) and the concerted somersault-H-abstraction TS (TS-7). In the GS connected to this TS, GS-7, the key

C5- bonding interaction with the more basic proximal oxygen (2.370 Å) (Figure 7) is now the shortest of several bonding interactions. We particularly note the relatively short distance (2.720 Å) to the C9 methyl group because if the wrong or opposite enantiomer, (1S)-camphor, is used as the substrate in P450cam hydroxylation,18 the substrate does not fit as well and in some cases the major product is C9 hydroxylation. The TS for combined somersault rearrangement and exoC5-H hydrogen abstraction, TS-7, exhibits a barrier of only 12.4 kcal/mol (7.7 with ZPVE) Fe-S, Fe-O and FeO · · · · HO distances of the inverted hydroperoxide fragment are essentially identical to that in the product of somersault rearrangement, Min-1 [PorFe(SH)O · · · · HO]. Recall, that the barrier for somersault rearrangement of the FeO-OH moiety in the absence of substrate (TS-1) was 21.4 kcal/mol at this exact level of theory. Note also that the geometry and the extent of C-H bond cleavage in TS-7 is indicative of a central TS with less C-H bond breaking (rC_H ) 1.199 Å) that should exhibit a smaller KIE than TS-6 (1.237 Å) as noted below. The activation energy calculated with the ECP basis set on Fe appears to be to high (∆E‡ ) 24.1 kcal/mol), but several attempts to reoptimize this TS always resulted in the same total energy. A single point energy refinement with the 6-311+G(d,p) basis set on the Fe at the above ECP geometry still gave an activation energy of 21.0 kcal/mol, suggesting a geometry problem with the ECP basis set optimization and not a basis set problem. These data specifically address a key part of the dogma associated with P45cam hydroxylation in that the substrate must be present at the active site or hydroxylation is thwarted and uncoupling (H2O2) results.1-3 Our calculations suggest that TS-7 has an proximal oxygen-C5 distance of 3.574 Å, whereas that distance in the X-ray structure was only 3.09 Å. Consequently, expansion of the active site is required in



Figure 7. B3LYP-optimized stationary points for the oxidation of camphor to the corresponding alcohol, 5-exohydroxycamphor. All the atoms directly involved in the oxidation step (SH, Fe, N, OOH, and the C5 π-CH2) were treated with the 6-311+G(d,p) basis set. The total number of basis functions in this reactant is 605. The rotation of TS-7 relative to the ground state provides a better view of the H-abstraction step.

order to accommodate a concerted somersault rearrangement placing the bound HO• in an approximately linear arrangement with the H-C5 bond to effect H-abstraction. However, upon going from the ground (GS-7) to the transition state (TS-7) in our QM surface the distance (Figure 7) of the ferryl oxygen to C5 does not change significantly (∆r) 0.156 Å). Even with this extended geometrical arrangement of the rearranged FeO-OH group, the H-atoms of the substrate come fairly close to the plane of the heme (2.941 Å), suggesting to us that this purports steric problems with Cpd I as the oxidant because it is even closer to the heme plane. We find a surprisingly lower activation energy than that for TS-6 [(∆∆E‡ ) 5.2 kcal/mol], and this does not include the 13-14 kcal/mol9,15 required to first arrive at Cpd I. The bond angle that Fe-O bond makes with C5 in GS-7 (∠Fe-O-C5 ) 126.7°) and TS-7 (∠Fe-O-C5 ) 127.4°) is essentially unchanged, suggesting that the somersault motion of the FeO-OH moiety can take place albeit heavy atom movement is required to move the camphor molecule further out from the plane of the heme so that the resulting HO• radical is in the correct approximately linear orientation to abstract the exo-hydrogen. The H-O-H angle in the developing water molecule in TS-7 is 96°, and the H2O molecule remains strongly H-bonded to the ferryl oxygen throughout the continuing rebound step producing the C-O bond of the product alcohol. Our mechanism is a concerted nonsynchronous pathway involving rate-limiting H-abstraction. It is also evident that the incipient water molecule formed on H abstraction must rotate downward after TS-7 in order to transfer a H atom from water to the ferryl oxygen (FeO• + H-OHfFeO-H + HO•) and present the open shell species of the developing C-OH bond in their proper approach angles for C-O bond formation as discussed previously (eq 2).5a As noted in our description of the overall mechanism, the subsequent hydroxylation involving the combination of

the t-butyl radical with the HO• radical derived from the H-bonded water molecule was suggested to proceed without a barrier.

The so-called “rebound” step in our mechanism involves formation of the C-O bond in the alcohol product by a rapid transfer of HO• from the H-bonded water molecule to the nearly

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stationary higher molecular weight carbon radical with simultaneous transfer of its H-atom to the ferryl oxygen to complete FeO-H bond formation (eq 2). Reduced mass considerations require dynamic motion of the lighter (OH) atoms in the TS for such a process consistent with little movement of the heavier camphor molecule. Consequently, what we actually need in this direct comparison of the two mechanisms is to compare our overall hydroxylation barrier (Figure 7) from the complexed FeO-OH ground state (Cpd 0) to produce the alcohol product (12.4 kcal/mol) with the sum of the two barriers for homolytic O-O bond cleavage (12-14 kcal/mol) and H-abstraction via Cpd I (17.6 kcal/mol). In this comparison our TS-7 has a lower barrier than any of the above hydrogen abstractions involving Cpd I and this does not even have to include the O-O homolytic barrier preceding Cpd I formation. A brief description of what is presumed to be the active site with the Fe-O bond intact based upon earlier X-ray data (1DZ9)19a is instructive. This X-ray shows a distance from the ferryl oxygen to the camphor C5 atom of only 3.045 Å. Although this may prove to be a good model geometry for Cpd I oxidation where the FeO- C5 distance in TS-6 is only 2.527 Å, it is too crowded for our extended HO• induced hydroxylation. There is insufficient room to allow a second oxygen for formation of the ferrous dioxygen complex (Fe(II)-O2) of wild type P450cam, and it is known from X-ray data19 that when the dioxygen analogue is formed the camphor must move away from the plane of the heme. A more recent X-ray study by Poulos (PDB ID 2A1M)20 on the dioxygen complex of wild type P450cam provides several unique clues to support our mechanism. It was suggested in that study that the role of the highly conserved Thr252 was to accept a hydrogen bond from the hydroperoxy Fe(III)OOH intermediate but not to donate a proton as recently proposed by Thiel.15 However, this suggestion was based upon the premise that the rebound mechanism (eq 1) was operating. It is quite evident that at the Fe(II)-O2 stage of the reaction sequence the active site is very crowded. As shown in figure 8a, based entirely upon X-ray xyz coordinate data,20 the dioxygen moiety is pointed toward the Thr252 residue with the O-O dioxygen bond forming an angle of 149° with the Thr252 oxygen and the distal oxygen being only 2.8 Å from this 2° alcohol oxygen, This suggests a very strong hydrogen bond with a distance of only 1.87 Å. We feel that one of the primary functions of Thr252 must be to stabilize the formation of this dioxygen intermediate, Fe(II)-O2, and to keep the O-O bond oriented and preventing its free rotation about the Fe-O bond. The distance from the proximal oxygen to C5 atom of camphor in this intermediate is only 3.09 Å. Consequently, there must be restricted rotation about the Fe- O2 bond because rotation of the O-O bond toward the C5 of camphor places the distal oxygen within less than 1 Å of the exo-C5-hydrogen. The Fe-O-O bond angle in Fe(II)-O2 is 128.6° and its Fe-O and O-O distances are 1.85 and 1.28 Å. After compulsory addition of an electron and a proton to this dioxygen complex, providing the hydroperoxy Fe(III)OOH intermediate (Cpd 0), restricted rotation of the FeO-OH group within the active site is even more evident. The new geometry in Figure 8b based upon the X-ray coordinates, with calculated QM variables of only the FeO-OH portion of the structure, as shown in Figure 8. The S-FeO-OH geometry is adjusted to be the same as that in GS-7 (Figure 7). The Fe-O-O angle is contracted to 116.4 and the O-O bond is elongated to 1.455 Å. This change in geometry results in an FeO-OH moiety that is even more compressed, and the H-bond

Bach

Figure 8. (a) X-ray coordinates for the Fe(II)-O2 dioxygen intermediate, Fe(II)-O2 (PDB ID 2A1M)20 showing only the heme and residues Thr252 and Tyr96. (b) X-ray coordinates with the S-FeO-OH group modified as in GS-7 (Figure 7). (c) X-ray coordinates with the TS portion modified as in TS-7.

distance of the Thr252 OH to the distal oxygen is reduced to 1.607 Å. This steric interaction should induce attempted rotation about the Fe-O bond, but that would place the distal oxygen within 0.9 Å of the exo-C5-hydrogen as it rotates toward camphor. The proximal FeO-OH oxygen is only 2.015 Å from exo-C5-hydrogen providing a weak bonding interaction that holds it in place. It appears that the camphor substrate is blocked on one side by the proximal oxygen and that the opposite direction is fixed by a strong H-bond between the carbonyl oxygen and Tyr96. Thus, the OOH group is essentially frozen in place at the same N-Fe-O-O dihedral angle as in intermediate Fe(II)-O2, 8a. However, we point out that the distance from the proximal oxygen in our calculated ground state (GS-7) is larger (∆r ) 0.33 Å)) than that in the X-ray ground state (8b) reflecting the influence of local residues at the active site.

Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons At this juncture we raise two pertinent questions that heretofore have not been addressed. Why does the camphor substrate have to be present at the P450cam active site in order to initiate hydroxylation and prevent uncoupling (H2O2)? This is highly unusual, and substrate presence is not a requirement in related oxidative enzymes such as flavin monooxygenases (FMO).21 Second, what induces a perfectly stable FeO-OH to spontaneously undergo homolytic O-O bond cleavage in a somersault rearrangement? Although the dioxygen complex intermediate, Fe(II)-O2, can be successfully stabilized by strong H-bonding to the neighboring Thr252 residue, the electronic event in this reaction sequence, involving the transfer of an electron and protonation affording the FeO-OH intermediate (Cpd 0) can occur rapidly and without much heavy atom motion. As noted above, this results in a highly hindered FeO-OH group, and we suggest that it is this resulting steric congestion that induces the atom motion of the somersault rearrangement. In Figure 8c we have maintained the basic X-ray coordinates of the heme, Thr252 and the relationship between Tyr96 and the camphor CdO, but we have modified the FeO-OH geometry to that of the calculated QM geometry in TS-7 (Figure 7). Rearrangement of the FeO-OH group causes the HO• radical to move toward a structure resembling Min-1, simultaneously pushing the camphor molecule away from the plane of the heme. This places the HO• radical in a nearly linear alignment with the exo-C5 H-atom with an O-H-C5 angle of 167.2° in the TS as noted in Figure 8c. The C-H and O-H distances in the TS (1.199 and 1.350 Å) are quite consistent with the kinetic isotope data presented below, and it is evident that rearranged Fe-OH group fits quite nicely into the active site. As the HO• radical reaches outwardly and upwardly during the somersault exercise, it can reach both the exo- and endo-hydrogens at carbon 5 of camphor, but the during the rebound step (eq 2) only the exposed Re face has the proper orientation with respect to the developing C-O-H angle of the alcohol product. The C-O-H angle in the TS for the rebound step (102°) must approximate that of the final angle in the alcohol product (109°)6 in order to add stereospecifically to give 5-exo-hydroxycamphor.22 This is clearly demonstrated in Figure 8c, where the only major movement of the camphor is to increase the distance from the ferryl oxygen to the camphor C5 from 3.09 Å in the GS (Figure 8b) to 3.565 Å in the TS. For example the Fe-O-C5 angle in the GS (Figure 8b) in slightly increased from 126.1 to 127.6° in TS-7. The angle between the three oxygen atoms (FeO-O-O ) C) from the ferryl oxygen to the camphor carbonyl oxygen changes from 98.1 to 94.2°. We emphasize again that the reactivity of the HO• is tempered by its strong H-bond to the ferryl oxygen (15.6 kcal/mol), and its selectivity for the exo-C5 hydrogen is a consequence of the tightness of the pocket holding the camphor substrate. A free HO• could be expected to abstract a hydrogen from the nearest C-H bond that it encountered. This appears to be precisely what happened in the QM/MM studies,9,15 where the camphor substrate was absent and the HO• abstracted an H-atom from a local residue on the way to forming Cpd I. It is evident in Figure 8c that the Tyr96 group can maintain its H-bond (1.621 Å) to the camphor carbonyl oxygen and still be away from the heme carboxylate groups. The Thr252 residue is now poised to interact more strongly with the proximal oxygen, but we found in unrelated studies that this H-bonding interaction has no apparent impact upon the rate of somersault rearrangement. Note, in this exercise, we modified only the atoms directly involved in the TS and the remaining X-ray coordinates remained unchanged.

J. Phys. Chem. A, Vol. 114, No. 34, 2010 9329 Although we have shown that Cpd I is a very reactive oxidizing agent, this remains a moot point if it is never actually involved in this enzymatic oxidation. Although Taraphder and Hummer23 have identified several potential proton sources in the vicinity of the heme in P450cam, there is still no evidence that this part of the initially proposed reaction coordinate is actually operating. The overall calculated activation energy for C-H abstraction by the metastable inverted FeO-OH species, TS-7, is considerably lower than that involving the formation of Cpd I following a proton relay.9,15 The same basis sets were used in both experiments. There are numerous synthetic P450 models that use iodosylbenzene, hydrogen peroxide, or related oxidants to generate an iron(IV)-oxo species that does not contain a ferric-hydroperoxo intermediate and bypasses the somersault mechanism. These synthetic Cpd I -like oxidants are highly reactive and readily affect hydroxylation and epoxidation reactions.24 Indeed, in several instances above we have shown that Cpd I is more reactive than our H-bonded HO• radical, which should discourage comments that HO• radicals might react indiscriminately with other C-H bonds in the local environment. e. Experimental KIE Data Consistent with the Somersault Mechanism. It has been commonly agreed upon that the P450cam catalytic cycle starts from the displacement of a water molecule in the resting state by the hydrocarbon substrate. Thus, in order for the hydroxylation step to be initiated by the iron(III) hydroperoxide (Cpd 0), the hydrocarbon substrate must be present, and the proposed catalytic cycle for the P450cam enzyme1 is initiated by a high-spin to a low-spin transition that triggers an electron transfer process. Following the formation of Cpd 0, the first major step in our mechanism in this enzymatic hydroxylation reaction is the sterically induced somersault motion of the FeO-OH group that moves the camphor substrate up ≈0.5 Å in TS-7 relative to ground state 8B (based upon X-ray data) and places the bound HO• in position and aligned with the C-H bond (∠O-H-C ) 167.2°) for abstraction of the C5 exo-hydrogen atom from the substrate by the hydrogenbonded hydroxyl radical (TS-7, Figure 7). Earlier kinetic isotope effect (KIE) studies using P450cam were typically assumed to be consistent with C-H bond cleavage in the rate determining step1-3 until the recent reversal by Shaik and Thiel.9,15 This controversy is based in part upon a longstanding concern about KIE masking or reduction in intermolecular KIE in enzymatic reactions. Kinetic oxidation involving P450 is often carried out under enzymatic turnover conditions where the rate-limiting step can be difficult to determine, and the isotopically sensitive step may not be the only step that contributes to the overall rate of the reaction. In a particularly relevant study, Guengerich25 has suggested that the overall ratelimiting step involves the second electron transfer step and O-O bond breaking, but the C-H bond breaking step clearly contributes to the rate in the P450 sequence as manifested in the observed kinetic isotope data. These studies lead to a “paradigm of P450 2D6 catalysis in which the rate of C-H bond breaking is a major contributor but not the only contributor” and “C-H bond breaking, or an equivalent step involving the Fe3+O entity (or another high Valent oxidant) is a ratelimiting step in many P450 reactions”.25 However, the present study is not principally concerned with the overall rate but only with the distinguishing between the C-H bond breaking step involving either Cpd I or our HO• radical. Even if slowest step were to be followed by the oxidation step and intermolecular KIE measurements can potentially give rise to atypically low KIE values, such masking or reduction of the intrinsic isotope

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effect can be minimized by the use of intramolecular isotope effects involving the measurement of the ratios of labeled to unlabeled products to arrive at KIEs. Consequently, we will consider only intramolecular KIE in support of our argument that C-H bond breaking is involved in the rate-determining step in P450cam hydroxylation. This KIE masking problem has been recognized for some time and in 1982 Sligar et al.13a unambiguously demonstrated that P450cam can effect hydrogen abstraction from either the exoor -endo-position and yet yield only one stereospecific product, 5-exo-hydroxycamphor. He noted a very small intermolecular isotope effect on the overall reaction but documented a significant intramolecular isotope effect with a series of oxidants including m-CPBA. Subsequently, Jones reported13b that the P450cam hydroxylation of octane was isozyme independent. He showed that the intrinsic isotope effect could be calculated to be 12.3 as compared to the observed isotope effect of 11.77. In 1993 Hanzlik reported13c the cytochrome p450 hydroxylation of xylene where “benzylic hydroxylation is subject to a large intrinsic (intramolecular) deuterium isotope effect (CH3/CD3 ) 7.5-7.9) comprised of a large primary component (5.3-7.8) and a large normal R-secondary component (1.09-1.19).”13c The Hanzlik KIE data13c are consistent with TS-5 for hydrogen abstraction from isobutane showing a nearly linear C-H-OH array in the TS (. 2009, 131, 10620. (c) Newcomb., M.; Zhang, R.; Esala, P.; Chandrasena, J. A.; Halgrimson, J. H.; Makris, T. M.;

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JP1045518

The Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons A

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