Reactivity patterns of cytochrome P450 enzymes: multifunctionality of the active species, and the...

REVIEW www.rsc.org/npr | Natural Product Reports

Reactivity patterns of cytochrome P450 enzymes: multifunctionality of theactive species, and the two states–two oxidants conundrum†

Sason Shaik,* Hajime Hirao and Devesh Kumar

Received (in Cambridge, UK) 16th March 2007First published as an Advance Article on the web 23rd April 2007DOI: 10.1039/b604192m

Covering: up to 2006 but not exhaustively

This focused review discusses mechanisms of oxygenation of organic compounds by cytochrome P450,based on density functional theory (DFT) and hybrid DFT and molecular mechanics (DFT/MM). Thereactivity of the active species, Compound I, generally involves two-state reactivity (TSR) andsometimes multi-state reactivity (MSR). The reactivity of the ferric-hydroperoxide species(Compound 0) is reviewed too. According to DFT calculations, Compound 0 must be silent in thepresence of Compound I. Much of the experimental mechanistic data is shown to beaccounted for by the TSR/MSR concept.

1 Introduction2 The catalytic cycle of the enzyme3 Unresolved issues in the chemistry of P450 enzymes3.1 How many reactive oxidant species does the P450 have?3.2 Unresolved issues in the mechanism of hydroxylation

and epoxidation4 Properties of Cpd I of P4504.1 Electronic structure and geometries of low-lying states

of Cpd I4.2 Origins of two- and multi-state reactivity5 Reactivity of Cpd I towards alkanes and olefins5.1 The mechanism of C–H hydroxylation5.2 The mechanism of double bond epoxidation5.3 The mechanism of desaturation6 Gauging the reactivity of Cpd 06.1 A computational assessment of the electrophilic

reactivity of Cpd 06.2 A computational assessment of the reactivity of Cpd 0

via initial O–O homolysis6.3 An assessment of the reactivity of the two oxidant of

P4507 Conclusions8 Acknowledgements9 References

1 Introduction

Cytochromes P450 (P450s) are heme enzymes that are presentin all aerobic species and carry out vital oxidative processes,namely, detoxification of foreign compounds and biosynthesisof hormones and other essential molecules.1–22 As such, P450sare extremely versatile enzymes which perform a great variety

Department of Organic Chemistry and The Lise Meitner-Minerva Center forComputational Quantum Chemistry, The Hebrew University of Jerusalem,Givat Ram, 91904 Jerusalem, Israel. E-mail: [email protected];Fax: +972-2-6584680; Tel: +972-2-6585909† This paper was published as a part of a special issue on the chemistryand biochemistry of heme proteins.

of stereoselective transformations that are otherwise difficult toachieve, e.g., hydroxylation of strong C–H bonds, epoxidationof C=C bonds, heteroatom oxidation (sulfoxidation), and de-saturation reactions. With all these features, it is no wonderthat P450s have become the focus of intense research thathas led to important insights and generated lively debates thatmay sometimes remind one of the intellectually-laden heyday ofphysical organic chemistry.

Many of the experimental, mechanistic and structural aspectshave been covered in two monographs,1,2 and in a variety ofother reviews.3–22 Three recent articles in Chemical Reviews,16,17,20

one micro-review in European Journal of Inorganic Chemistry,18 achapter in the P450 Monograph19 and three shorter reviews,15,21,22

provide exhaustive coverage of density functional theoretical(DFT) studies and hybrid DFT/molecular mechanical (MM)investigations of the structure and reactivity of P450cam. Therefore,the present manuscript is an updated focused summary of theinsights obtained from theory regarding controversial electronicstructure and mechanistic aspects associated with the activespecies of the enzyme.

2 The catalytic cycle of the enzyme

The consensus catalytic cycle of the enzyme, based on experimentalspectroscopic and structural data, is illustrated in Fig. 1.5,9,23,24 Allof the species have also been computed by DFT and DFT/MMmethods, as outlined in extensive reviews of the topic.16,19 The cyclebegins with the resting state (1), in which a heme is bound to awater molecule on the distal side and to the thiolate side chainof a cysteine residue (abbreviated as CysS) on the proximal side.The entrance of the substrate (e.g., an alkane, AlkH) displacesthe water molecule, causes a change of the spin state, and triggersthe transfer of an electron from a reductase protein. The resultingferrous complex (3) binds molecular oxygen, which gets activatedby an additional electron transfer from the reductase followedby protonation from a suitable proton-relay system on the distalside of the protein, thereby generating the ferric-hydroperoxidespecies (6), also called Compound 0 (Cpd 0). The resulting Cpd 0

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 533–552 | 533

Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online / Journal Homepage / Table of Contents for this issue

http://dx.doi.org/10.1039/b604192m

http://pubs.rsc.org/en/journals/journal/NP

http://pubs.rsc.org/en/journals/journal/NP?issueid=NP024003

Sason Shaik was born in 1948 in Iraq. His family immigrated to Israel in the Exodus of the Iraqi Jewry. He received his BSc and MScin chemistry from Bar-Ilan University, and his PhD from the University of Washington under Nicholaos D. Epiotis. In 1978/9 he spent apostdoctoral year with Roald Hoffmann at Cornell University. In 1980 he started his first academic position as a Lecturer at Ben-GurionUniversity where he became Professor in 1988. He subsequently moved in 1992 to the Hebrew University, where he is currently the director ofThe Lise Meitner-Minerva Center for Computational Quantum Chemistry. Among the awards he received are the Lise-Meitner-Alexandervon Humboldt Senior Award in 1996–1999, the 2001 Israel Chemical Society Prize, the 2001 Kolthoff Award and he is the recipient of theSchrodinger Medal of WATOC for 2007. He is an AAAS Fellow. His research interests are in the use of quantum chemistry, and in particularof molecular orbital and valence bond theories, to develop paradigms which can pattern data and lead to the generation and solution of newproblems. In 1994, he entered the field of oxidation and bond activation by metal oxo catalysts and enzymes. The P450 research started in1998, and has been fascinating ever since!

Hajime Hirao was born in Hyogo, Japan in 1975. He received his BSc (1998) and MSc (2000) degrees in chemistry from Kyoto University.In 2004, he obtained his PhD from The University of Tokyo with Professor T. Ohwada. After three years of work on computer-assistedmolecular modeling (CAMM) at the Novartis Tsukuba Research Institute, he moved in 2005 to The Hebrew University of Jerusalem,where he has since worked with Professor S. Shaik as a postdoctoral fellow, and as a JSPS fellow since April 2007. His research interestsinclude application of computer chemistry to reactions and interactions of chemical and biochemical molecules, as well as development ofnew methods for analyzing chemical reactivity based on molecular orbital and valence bond theories.

Devesh Kumar was born in India in 1965. He received his BSc from the L. N. Mithla University and his MSc and PhD in Physics from theD. D. U. Gorakhpur University, India. He worked at the Centre for Liquid Crystal Research and Education, Nagarjuna University, India asa research associate (July 2001–March 2002) before joining the group of Professor Sason Shaik at the Hebrew University of Jerusalem as apostdoc in May 2002. He is currently pursuing postdoctoral research in the laboratory of Professor Walter Thiel in the Max Planck Institutein Mulheim. His main research interests are theoretical studies of conformations and interactions of mesogens and enzyme catalysis.

Sason Shaik Hajime Hirao Devesh Kumar

accepts an additional proton and splits off a water molecule toform 7, the so-called Compound I (Cpd I) species. Cpd I possessesa high-valent ferryl moiety, FeIV=O, and a radical cationic statein the porphyrin, i.e., Por+•FeIV=O. Cpd I is known from studiesof synthetic models and from the related chloroperoxidase (CPO)enzyme, to be a competent oxidant3,5,25,26 and as such, it is thoughtthat P450 Cpd I is the ultimate oxidant species that transfers anoxygen atom to the substrate leading to the product (e.g., ferric-alcohol) complex, 8. The cycle is restored by release of the oxidizedsubstrate (e.g., the alcohol, Alk-OH) and the binding of a watermolecule to the heme to regenerate the resting state.

As already mentioned, all of the species in the cycle havebeen computed,16,19,20,21 and there is a general agreement betweenexperiment and theory on most aspects of the cycle. Two featureshave, however, remained controversial to this day. Firstly, Cpd I ofP450 is still not fully characterized by experiment, and secondly,the reactivity patterns of P450 suggest that in addition to Cpd I,the enzyme utilizes “other species” in the cycle to oxidize organicmolecules.6 This is an intellectually challenging problem for theory.

And it is this challenge that has been the main trigger for generatingnew mechanistic ideas, such as two-state reactivity (TSR) andmulti-state reactivity (MSR), which may shed light on theseunresolved issues.15,18 Let us first follow with a more detaileddescription of the major problems.

3 Unresolved issues in the chemistry of P450 enzymes

3.1 How many reactive oxidant species does the P450 have?

Cpd I appears to be the consensus primary oxidant, but asit has never been directly characterized, under native turnoverconditions, the fuzziness in its status has left room for speculationson the nature of the active species of the enzyme. A recenttentative characterization of Cpd I under conditions of cryogenicX-ray crystallography,24 has since been cast in doubt.23 Even theconsensus technique of generating Cpd I species using the oxygensurrogate iodosylbenzene has been questioned recently.27 Attemptsto generate Cpd I using other oxygen surrogates such as peracids

534 | Nat. Prod. Rep., 2007, 24, 533–552 This journal is © The Royal Society of Chemistry 2007

Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 1 Schematic representation of the catalytic cycle of P450 (adaptedfrom Fig. 2.2 in ref. 19, with permission from Springer Science and BusinessMedia).

(see Fig. 1) led to the one-electron reduced species, PorFeIV=O,so-called Compound II (Cpd II), and a Tyr radical.28 While this re-peated generation of Cpd II has usually been taken as an alarmingsign that Cpd I may not exist, more recent experiments29 showedhow one could optimize the formation of Cpd I before it convertsto Cpd II or before the heme bleaches due to destruction by theperacid; these findings may make it possible to observe some of thecharacteristic chemistry of Cpd I. Strong but indirect support forthe participation of Cpd I has been provided by determining thereactivity of synthetic Cpd I species and P450 towards the samesubstrates, where both sets of reactions led to similar product dis-tributions and stereochemical scrambling information.5 Similarly,a recent mechanistic study30 used the N-oxo derivative of a seriesof substituted N,N-dimethylanilines to generate the Cpd I speciesof P450cam and P4502E1 in situ, and to measure the resultingkinetic isotope effect (KIE) due to the subsequent reactions ofthese Cpd I species with the N,N-dimethylaniline substrates. Theobtained KIE values were virtually the same as those measuredfor the reaction of the P450 enzymes when the same series of N,N-dimethylanilines were oxygenated via the normal route of catalysis(reduction, addition of O2, and protonation, see Fig. 1). This result

provided compelling evidence for the reactivity of P450 Cpd I inthe C–H hydroxylation of N,N-dimethylanilines. Similarly, theaction of Cpd I was also inferred for camphor hydroxylation,where the product alcohol was found to be formed with its oxygenatom coordinated to the iron center and with the substrate-derivedproton attached to the product alcohol,23 as shown by the asteriskson the Alk-OH moiety, 8, in Fig. 1.

While Cpd I has never been observed in a native turnoversystem, its existence was detected by the use of exogenous oxygensurrogates in P450cam (CYP101)29 and in the thermostable isozyme,CYP119,31 from Sulfolobus solfataricus. Ballou et al.,29 usingtransient electronic spectroscopy and decay kinetics methods,also confirmed the high reactivity of Cpd I and Cpd II/tyrosylradical species (Cpd ES), with several reductants. Further supportfor this assignment of the active species came from theoreticalcalculations,32 which reproduced the split Soret band that wasblue shifted relative to the resting state. There are also closeanalogies between P450 Cpd I and the Cpd I species of the enzymechloroperoxidase (CPO) in spectroscopic features,25 and in thepatterns of reactivity towards a variety of organic molecules.26

Recent EXAFS33 and ENDOR34 studies further refined thestructural and electronic features of CPO Cpd I, and affirmed closecorrespondence between the experimental data for CPO Cpd Iand the DFT/MM calculated values35,36 for CYP101 Cpd I. Thus,despite the lingering uncertainty, the inferences that Cpd I existsand is responsible for the native oxidation processes of the enzymein the cycle are quite strong. Accordingly, in the following sectionswe shall review the computed reactivity patterns of Cpd I.

The evidence for a second oxidant species in the cycle of P450 isindirect; it derives from reactivity studies, lifetime measurementsof putative intermediates, and product distributions of P450sand their mutants. Thus, mutant enzymes, in which the protonrelay that converts Cpd 0 to Cpd I is disrupted by site-directedmutagenesis,6,37 exhibit reactivity patterns different from thoseof the wild type (WT) enzyme. For example, with cis and trans2-butene, the mutant enzyme yields more epoxidation productscompared with C–H hydroxylation products.37 Similarly, theT252A mutant of CYP101,38 in which Thr252, which plays a keyrole in the conversion of Cpd 0 to Cpd I, is replaced by Ala, doesnot hydroxylate camphor, but is capable of epoxidation of thedouble bond of camphene, albeit more sluggishly than the WTenzyme. Since during catalysis, the T252A mutant is thought tobe trapped at Cpd 0, this and the reactivity of the mutant towardcamphene derivatives constituted evidence that Cpd 0 is involvedin the epoxidation of an activated double bond. Curiously,however, the double mutant of P450cam, T252A/D251N, in whichthe protonation machinery has been disrupted by mutations ofboth Thr252 and Asp251, is able to hydroxylate camphor,39 despitethe T252A mutation that should have hypothetically blockedthe formation of Cpd I. This implies either that Cpd 0 has avariable reactivity in different mutants (that include the T-to-Amutation) or that variable quantities of Cpd I may be presenteven in the case of the different T → A mutant. Thus, whilethe participation of Cpd 0 in oxidation is still a clouded issue,the data behave as though more than one oxidant is available toP450. This pattern confronts both theory and experiment with atantalizing mechanistic enigma. We shall review here some of theDFT calculations associated with the reactivity of Cpd 0 towardssubstrates.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Another species that has been invoked as the reactive oxidant ofP450 is the perferryl, 9 (Fig. 1), in which the porphyrin has a closed-shell and iron is in oxidation state +5, hence, PorFeV=O. Thisspecies, that was postulated by Murakami et al. some timeago in reactions of some model systems,40 has recently beenhypothetically invoked as the species responsible for the highreactivity of the enzyme itself.41 There is however no concreteevidence for the actual presence of PorFeV=O in P450, other thanthe following conjectures. Thus, based on a new technique, Cpd IIhas been reported to photo-oxidatively convert to Cpd I, andthe decay of the latter species can be observed in the presenceof substrates.42 It was thus observed that the so-generated Cpd Ispecies of model iron-porphyrin systems are orders of magnitudeless reactive43 than with the corresponding PorMnV=O species.44

When the technique was applied to CYP119,41 the so-generatedspecies, thought to be Cpd I, was rather inactive in the presenceof laurate, a native substrate of the enzyme. Since CYP101hydroxylates camphor with a rate constant exceeding 1000 s−1 atambient temperature,41 it was conjectured that the reactive speciesin CYP119 and CYP101 must be the PorFeV=O species 9 (Fig. 1).In contrast to this result, the use of meta-chloroperbenzoic acid togenerate Cpd I for CYP119 led to the formation of Cpd I, whichreacted with laurate to give hydroxylaurate within the dead-time ofthe experiment.31 Apparently, the two experiments are at odds witheach other, and one may wonder if the photo-oxidative experimentindeed gave rise to Cpd I. Furthermore, close inspection of thespectrum of the photo-oxidatively produced species in CYP119,shows it to be more compatible with Cpd ES, which is Cpd IIand a protein radical that was reported for CYP101.29,45 Cpd II isknown to be less reactive than Cpd I in hydroxylation and epox-idation reactions from both experimental46,47 and computationalresults.48

A process of reasoning that is often used in mechanistic researchis based on the comparison of reactivities in model bimolecularreactions to those of P450. This however requires some carefulscrutiny as we shall now argue. Thus, with a rate constant k ≥ 200–1000 s−1 (at ambient temperatures) for the reaction of CYP101with camphor,41,49 the free energy barrier at room temperatureis ca. 13.4–14.3 kcal mol−1. If we take even a very high rate of106 s−1 the barrier would be ca. 9.3 kcal mol−1. By comparison,for the bimolecular reactions of the model systems,43 the freeenergy barriers are 14.1–17.5 kcal mol−1 (rate constants of 1–300 M−1 s−1). Thus, with this comparison of the barriers, one maybe led to deduce that the reactive species of the enzyme is notCpd I, since the model Cpd I species have higher barriers than theenzymatic species. However, this deduction neglects to consider thedifferent entropic factors for bimolecular and enzymatic reactions.In bimolecular reactions, the transition state is established with aloss of rotational and translational degrees of freedom comparedwith the reactants. The loss of these degrees of freedom in theTS raises the free energy barrier by ca. 6–12 kcal mol−1 (ref. 50assumes a loss of 40 eu,50 which translates to 12 kcal mol−1, butthe actual number depends on the strength of the interaction).By contrast, in the enzyme, this loss of degrees of freedom iscompensated by a gain of degrees of freedom when molecules ofwater leave the pocket as the substrate goes in and binds e.g., ascamphor does in CYP101 due to hydrogen bonding with Tyr96 andhydrophobic interactions with Val295.16,19 The enzymatic reactionthen starts from a “complex” that has lost already the rotational

and translational degrees of freedom, and hence the free-energybarrier for the reaction from this complex should be lower thanthe bimolecular models by 6–12 kcal mol−1. This effect by itselfaccounts for a large part, if not all, of the observed differencesbetween the bimolecular and the P450 enzymatic process. As such,it is not really necessary to invoke PorFeV=O as the species that isresponsible for the high reactivity of P450s; this can be reasonablyextrapolated from the reactivity of model Cpd I systems.

The energetics of the PorFeV=O species were computed recentlyby DFT,51 and all of its possible electromeric states were foundto be higher in energy, by 17–26 kcal mol−1, than the normalCpd I species. It is therefore doubtful that this species can begenerated in the enzyme under turnover conditions, consideringthe fact that the barrier to the formation of Cpd I in CYP101 wascalculated recently to be 12–15 kcal mol−1,52 and in CYP119 theexperimentally determined barrier was 14.1 kcal mol−1.31 Thus, thecombined energy increase needed to reach the PorFeV=O speciesin CYP119 could well be 31–40 kcal mol−1. Indeed, DFT/MMcalculations in our group designed to find transition states thatare associated with FeV=O show that these species lie much higherthan the lowest transition states. Therefore, it is not very likely thatPorFeV=O is the species that accounts for the reactivity of P450s,unless there is some yet unknown basis for that, e.g., crossing of thepotential energy profile for generating the species on the excitedstate surface (starting from PorFeV=O) below the transition stateseparating Cpd 0 and Cpd I.

We are well aware of the fact that FeV species are acessible innonheme systems, e.g., among the Que reagents,53,54 the recentWieghardt complexes,55 and the oxo-iron of polyoxometalate.56

However, the FeV species exist in these cases because they all lackthe easily oxidizable porphyrin. Furthermore, DFT calculationsshow51,57 that the Cpd I analog with ruthenium replacing iron,has a PorRuV=O ground state. Here the reason is more subtle;as discussed in the original literature,51,57 it is associated with theorbital energy levels and exchange interactions that facilitate thetransfer of an electron to the porphyrin cation radical to createPorRuV=O much more easily than with PorFeV=O. To avoidfurther speculation, the PorFeV=O species will not be consideredin this review.

3.2 Unresolved issues in the mechanism of hydroxylation andepoxidation

The above uncertainties in the identity of the oxidant, led tocontroversies regarding the mechanism of C–H hydroxylationby P450.5,6,11,37 The consensus mechanism of C–H hydroxylationby Cpd I is the “rebound” mechanism proposed by Groves andMcClusky58 and depicted in Fig. 2a. The first step in the mecha-nism involves an initial hydrogen abstraction from the alkane byCpd I. Subsequently the alkyl radical (Alk•) is partitioned betweentwo competing processes. It can either instantly undergo reboundto form an alcohol complex, where the alcohol is unrearranged(U), keeping the original stereochemical information possessed bythe alkane, or it can first undergo skeletal rearrangement and thenrebound to give a rearranged (R) alcohol product. The reboundmechanism accounts for the key experimental data; the partialloss of stereochemistry and geometrical rearrangement data, aswell as the observed large kinetic isotope effect (KIE)3,5,10,11 whenthe activated C–H bond is replaced by C–D.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 2 (a) The rebound mechanism58 and the apparent lifetime (sapp) ofthe radical. (b) U and R products of ultrafast clock substrates.

Based on Fig. 2a, the apparent radical lifetime (sapp) isapproximately given by the ratio of rearranged (R) productyield to unrearranged (U) product yield, divided by the rateconstant for rearrangement of the free radical clock (kR). Usingthis approach, an initial study of the lifetime of the radicalintermediate59 indicated a short but finite lifetime, of the orderof 50 ps. Other data gave a range of lifetimes from nanosecondsto picoseconds.5,60 However, studies6 using ultrafast radical clockssuch as the ones depicted in Fig. 2b, questioned the presenceof the radical intermediate and the validity of the reboundmechanism. Determination of [R/U] for these probe substratesled to the conclusion that if one assumes that there is a radicalduring P450 oxidation, then the lifetime of this species wouldbe the order of 100 fs or less, which is too short to supportthe existence of a real intermediate species. Such unrealisticallyshort lifetimes question the validity of the rebound mechanism.Furthermore, in mutant P450s, in which Cpd I was presumablyabsent, the ratio [R/U] was generally larger in comparison to theWT enzyme.6 This in turn suggested that the rearranged productsarose from non-radical intermediates that were generated due tothe presence of another oxidant species that became prominentin the mutants. Probe substrates, which can distinguish betweenradical and carbocationic rearrangement patterns, were used tosupport this hypothesis;6 the corresponding results suggested thatthe major reaction intermediate is in fact a carbocation andnot a radical. Therefore, Newcomb et al.6,61 proposed that C–H hydroxylation proceeds via multiple and competing oxidantspecies (Scheme 1); Cpd I that leads to concerted oxygen insertioninto the C–H bond, and Cpd 0 or ferric hydrogen peroxide thattransfers an OH+ species and generates a protonated alcohol thatsubsequently undergoes rearrangements typical of a carbocationicspecies. In the mutant enzymes (e.g., T252A CYP101) the amountof Cpd 0 presumably grew at the expense of Cpd I and thereforeled to more carbocationic rearrangment, as well as to more ofthe marker products of, e.g., double bond epoxidation37 andphenyl hydroxylation.6 Interestingly, similar Cpd 0 species in heme

Scheme 1 Postulated multiple oxidants in P450.

oxygenase (HO) and in mutants of e.g., myoglobin (Mb) are knownto insert a hydroxyl group into the meso position of the heme.62,63

The key question in our eyes is not whether Cpd 0 can ever effectC–H hydroxylation and epoxidation, but rather does it ever act incompetition with Cpd I during these oxidation processes? Theorycan provide some answers to this question.

In summary: the evidence on P450 reactivity has so far beenindirect; the primary oxidant species is still not fully characterized,its identity is cast into doubt, and the reactivity patterns behaveas though more than one oxidant is responsible for the productdistribution. The identity of this second oxidant is howeverstill shrouded. This is an intellectually intriguing scenario thatcalls for the involvement of theory. Theoretical analyses since199864 offered a possible reconciliation of the controversy basedon the two-state reactivity (TSR) paradigm. Subsequently, theJerusalem group used DFT to study the mechanism of C–Hhydroxylation reactions between Cpd I and a variety of alkanes, bynow more than 10.16,65 In collaboration with the Mulheim group,the two groups explored the mechanism of camphor hydroxy-lation by Cpd I of P450cam using hybrid quantum mechanical(QM)/molecular mechanical (MM) calculations.66 The reactivityof Cpd 0 was explored in C–H hydroxylation,67,68 double-bondepoxidation,48,69,70 and sulfoxidation reactions, without and withacid catalysis.71 The possibility of C–H hydroxylation by the ferrichydrogen peroxide complex, PorFeIII(H2O2), was also studied.67

These extensive computational studies16 have revealed that:(a) Cpd 0 is much less reactive than Cpd I, and cannot be invokedas an oxidant in the presence of Cpd I, unless the barriers tooxidation by Cpd 0 are smaller than the one to the conversion ofCpd 0 to Cpd I,52 (b) ferric-hydrogen peroxide is less reactivethan Cpd I, (c) the reactivity of Cpd I involves at least twoproductive states, and can be described as TSR, or more accuratelyas multi-state reactivity (MSR), and (d) TSR and MSR reconcilethe major mechanistic questions raised above. These features arebriefly reviewed herein in an updated form.

4 Properties of Cpd I of P450

4.1 Electronic structure and geometries of low-lying states ofCpd I

Before discussing the electronic structure of Cpd I, it is importantto assess the quality of the calculations of Cpd I against some


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


experimental data. Fig. 3 compares the DFT(B3LYP)/MMgeometries and spin densities (q) calculated for CYP101Cpd I16,18,19,35,36,74 alongside those obtained experimentally for thecharacterized species of CPO.33,34 The computational studies35,36

employed different models of the thiolate ligand, starting from thesimple representation (L = HS−) through L = CH3S− and all theway to the more extended cysteinate representation (L = CysS−).Since all the models gave similar DFT(B3LYP)/MM results,35 forthe sake of economy we present only the simplest model in thefigure. It is seen (Fig. 3a) that the calculated bond lengths35 forthe two lowest spin states of CYP101 Cpd I are in reasonableaccord with experimental EXAFS values for Cpd I of CPO.33

Similarly, the spin density distribution (for the quartet state) onsulfur and porphyrin, Fig. 3b, matches the results obtained fromrecent EPR/ENDOR experiments.34 Quite a few other spectro-scopic features were shown to match the experimental data, andthe correspondence between DFT(B3LYP)/MM and high-levelconfiguration interaction calculations was noted.36 AdditionalDFT(B3LYP)/MM calculations for Cpd I of human P450s72,73

further reinforce this match between theory and experiment.

Fig. 3 DFT(B3LYP/B3)/MM computed properties of CYP101 Cpd I(for snapshot 29 fs) vs. experimental data for CPO Cpd I; B3 isLACVP(Fe)/6–31 + G* on the immediate coordination sphere/6–31G onall other atoms: bond lengths in (a) and spin densities (q) in (b); computedvalues correspond to quartet/doublet. (c) DFT(B3LYP/LACVP) data ofmodel systems; on the left is the gas phase species (e = 1) and on the rightis the species with two NH–S interactions and bulk polarity (e = 5.7).

As was argued originally by the Jerusalem group,74 Cpd Iis a “chameleon species”; its geometric and electronic featuresare shaped by the polarity in the protein pocket and hydrogen

bonding (H-bonding) of the protein residues to the thiolate ligand.Simple DFT calculations with two NH–S H-bonds and bulkpolarity corresponding to a dielectric constant (e) of 5.7 couldin fact reproduce both the DFT/MM and EPR/ENDOR results,as shown in Fig. 3c; it is apparent that the two H-bonds andbulk polarity effects (evaluated by single point calculations onthe optimized Cpd I•(2NH3) structures), incorporated into themodel, cause a shortening of the Fe–S bond and a shift ofspin density from the thiolate to the porphyrin. These externalperturbations gauge the donor ability of the thiolate ligand vis-a-vis the porphyrin cation-radical center, and thereby control thespin density on the sulfur/porphyrin and the Fe–S bond lengths(see an orbital-based explanation below).

The data in Fig. 3 show that Cpd I is a tri-radicaloid specieswith three unpaired electrons, this and other features as well can beunderstood by a simple orbital picture. Fig. 4 shows the six higher-lying orbitals of P450 Cpd I. Five orbitals (left hand) constitute thed-block; four of these orbitals contain antibonding interactionsbetween iron atomic d orbitals and the appropriate orbitals ofthe ligands, while the fifth orbital, d, is a pure d orbital on iron.The sixth orbital, labelled as a2u, is a porphyrin-based orbital thatalso involves an antibonding interaction between the atomic lobeson the nitrogen atoms of the porphyrin and the hybrid orbitalon thiolate. The effects of polarity and H-bonding are expressedthrough the nature of the latter orbital, as shall be immediatelyanalyzed.16,35,74,75

Based on DFT74,75 and DFT/MM calculations (for CYP101and human P450s)16,35,36,72,73 using the B3LYP functional and avariety of basis sets, the ground state of Cpd I is a pair of doubletand quartet spin-states, which are labeled in Fig. 4 as 4,2W0 andwhich possess three unpaired electrons in the p* and a2u orbitalsthat are coupled ferromagnetically (to generate a quartet spin) orantiferromagnetically (to generate a doublet spin); the superscriptto the left of the state symbol signifies the spin multiplicity, 2S +1. Since the p* and a2u orbitals are disjointed, the coupling is weakand the quartet and doublet states are virtually degenerate, thedoublet lies ca. 10–21 cm−1 below the quartet.35,36

The effects of polarity and H-bonding capability of the proteinare expressed through the nature of the singly occupied a2u

orbital, as shown in Scheme 2, where we construct the orbitalof Cpd I from the fragment orbitals of oxo-iron porphyrin andthe thiolate fragments. In the gas phase, the CysS− center is agood electron donor; its hybrid orbital hs(g) lies closer to theporphyrin’s a2u(Por) orbital, and therefore the orbitals of the twofragments mix strongly. Consequently, the CysS− center releasesmore electron density towards the porphyrin cation radical, andhence, the sulfur becomes more radical-like, it acquires a higherspin density, and the Fe–S bond gets longer and weaker.16,51,74

Changing the environment from the gas phase to the proteinpocket, the H-bonding and polarity effects in the protein pocketreduce the donation power of the thiolate ligand and its hybridorbital hs(p) gets stabilized. Consequently, the orbital gap and themixing of the sulfur hybrid with the porphyrin a2u(Por) fragmentorbital will diminish compared with the situation in the gas phase.The result is that within the protein environment, the orbitalof Cpd I becomes more porphyrin centered, the thiolate has asmaller radical character and the Fe–S bond gets shorter andstronger compared with the gas phase.16,35,74,75 Since the state ofCpd I depends so critically on the donor ability of the thiolate, the


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 4 Key orbitals of Cpd I and occupation diagrams in the low-lying states.

Scheme 2 Construction of the mixed S-a2u orbital from fragment orbitalsof the porphyrin and the thiolate ligand (S) in the gas phase and in theprotein.

simplest ligand representation HS− in the gas phase is the closestto the actual state of the cysteinate ligand (CysS−) in the protein;the gas-phase CH3S− representation gives a wrong electronic stateand geometry of Cpd I.75

Another pair of states, labelled as 6,4W1 in Fig. 4, can begenerated from the d1p*2r*xy

1a2u1 configuration that has five

unpaired electrons coupled to generate sextet and quartet spinstates. Although the d-r*xy orbital gap is quite large, these statesenjoy significant stabilization due to the multiple exchangeinteractions of the d-block electrons, and as a result, they areexpected to be not much higher than the pair of ground states.Both DFT51,76 and DFT(B3LYP)/MM calculations36 show thatthe 6,4W1 states lie only ca. 12 kcal mol−1 above the doublet andquartet states with the d2p*2a2u

1 configuration. With such a small

energy gap to the 6,4W1 states, it is not surprising that DFT76 andDFT/MM77 calculations show that all the four states in Fig. 4potentially contribute to the reactivity of the enzyme.

4.2 Origins of two- and multi-state reactivity

Based on the above discussion, Cpd I is an oxidant that has adegenerate pair of ground states and two more easily accessiblestates. However, even more states become accessible along theoxidation pathways, as the bonds of substrates are activated, andas Cpd I, which has Fe(IV) and a porphyrin cation radical, getsreduced by the substrate and finally becomes a ferric (Fe(III))porphyrin state. Thus, any reaction of Cpd I will ultimately involvetwo formal “electron transfer” events from the substrate beingoxidized to the heme (or a single event with a “double electrontransfer”). To facilitate the discussion we show in Fig. 5 thechanges in orbital occupancy along the reaction paths for C–Hhydroxylation and C=C epoxidation by the degenerate state, 4,2W0,of Cpd I. The Cpd I species is represented by the d-block and thea2u orbitals, while the substrate is represented by a single orbital;i.e. pCC is the orbital for the p-bond, activated during epoxidation,and rCH is the orbital for the C–H bond that is broken duringhydroxylation. In the triradicaloid states in panels (a) and (b),the doublet spin state is indicated using an inversed electron spinwithin parentheses.

Starting from the first set of panels, 4,2W0, the initial bondactivation step (a → b) involves an electron “transfer” event fromthe substrate into Cpd I. Two of the possibilities for this “transfer”are: (1) into the porphyrin hole (a2u-orbital) to generate in(b) the 4,2Irad(IV) doublet and quartet FeIV-type intermediates,with a closed-shell porphyrin, i.e., the PorFe(IV) electromers, and(2) into the p*xz-orbital to generate 4,2Irad(III), the Fe(III)-typeintermediates with a radical cationic situation on the porphyrin


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 5 Orbital occupancy evolution during C–H hydroxylation and C=C epoxidation. (a)–(c) constitute the radical pathways, while the transition from(b) to (d) shows the cationic path.

ring, i.e. the Por+•Fe(III) electromers. In both cases, the substrateretains a radical center with a singly occupied orbital labelled �C.During the second bond formation (b → c), an additional electronis “transferred” from the substrate to fill, either the a2u orbital ofPor+•FeIII or one of the d-orbitals of PorFe(IV). Filling the p*xz

orbital generates the doublet-spin ferric product 2P(III), whereasthe filling of the r*z2 orbital leads to the quartet-spin ferric product4P(III).

In addition to the above intermediate states, there are otherpossible species where the organic moiety becomes carbocationic,as shown in the set of panels in Fig. 5(d). Thus, shifting electronsfrom the �C orbital of the radical intermediate center (in b) into oneof the d-orbitals or into a2u generate 2,4Icat, namely, carbocationicspecies coordinated to a heme complex in the doublet or thequartet spin state. For simplicity we do not show the states thatare nascent from 6,4W1. The latter states are close in energy to theother intermediate states in Fig. 5, and the interested reader canconsult the original literature.76 Clearly therefore, the reactivity ofCpd I will involve at least two spin states, and possibly also a fewelectromeric states where the iron atom and the porphyrin ring arein different oxidation states. This multitude of states typifies TSRand MSR. In the following section we show two examples, one forC–H hydroxylation where TSR accounts for the salient features,and the other C=C epoxidation where MSR is required to accountfor the reactivity patterns.

5 Reactivity of Cpd I towards alkanes and olefins

5.1 The mechanism of C–H hydroxylation

More than 10 cases of C–H hydroxylation have been studied bynow,16,65 and some of the substrates are depicted in Scheme 3. A few

Scheme 3 Substrates studied for their C–H hydroxylation by Cpd Iusing DFT(B3LYP) and/or DFT(B3LYP)/MM. The arrows indicate thereaction sites.

of these were studied by DFT(B3LYP) only, while others with bothDFT(B3LYP) and DFT(B3LYP)/MM.66,77,78,79 The DFT(B3LYP)and DFT(B3LYP)/MM results revealed differences, which arerelatively small as long as the DFT(B3LYP) calculations includepolarity and NH–S H-bonding effects. All of these cases werefound to exhibit a TSR scenario, with two energy profiles nascentfrom the degenerate ground state of Cpd I. The mechanism isbiphasic; the initial phase involves C–H bond activation and thesecond phase a rebound. The C–H bond activation is the rate-determining step of the mechanism and the averaged barriers


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


(over the two spin states) correlate well with the correspondingbond energies (DCH), according to the expression in eqn 1:65

DE (kcal mol−1) = 0.5887DCH − 39.418 (1)

Thus, while a correlation between two variables may notnecessarily also mean a causal relationship, still, the correlation,together with the structures of the TSs, which all virtually possess alinear O–H–C triad of atoms, and the large computed KIEs meanthat the first step involves H-abstraction. However, the interestingstory begins in the rebound phase as shown below.

The computed TSR scenario is displayed schematically in Fig. 6,where the quartet state surface nascent from 4W0 is designatedas the high-spin (HS) state, while the doublet surface nascentfrom 2W0 is labeled as the low-spin (LS) state. The two reactionprofiles remain close throughout the C-H bond activation phase,up to the iron-hydroxo/radical complexes (4,2Irad), where theradical moieties are bound to iron-hydroxo by a weak OH–Alkinteraction. After snapping out of the weak OH–Alk interaction,the alkyl radical assumes a rebound position (4,2Ireb, which arenot real intermediates), and thereafter the two states bifurcate.On the HS manifold, there is a significant barrier (3–10 kcalmol−1 or so16,18,66,77) for rebound en route to the HS ferric-alcoholcomplex (4P). By contrast, the rebound on the LS surface is barrierfree, leading spontaneously to the LS ferric-alcohol complex (2P).Thus, the DFT calculations support the rebound mechanism,58

but modify it by introducing into the mechanistic picture the TSRphenomenon and the multitude of intermediates that give rise todifferent products.

Fig. 6 presents only two generic intermediates, 4,2Irad, but, as weargued above, there are quite a few more. Fig. 7 shows a typicalsituation exemplified by the case of camphor hydroxylation, withfour radical intermediates of the Por+•FeIII and PorFeIV electromervarieties.16,18,66,77 It is seen that the four states are condensed within2 kcal mol−1 or less, and hence, all the four species are accessibleand will generate products. For each electromer type, the LSintermediate collapses to the product complexes without a barrier,

Fig. 7 Intermediates during camphor hydroxylation in the gas phase andin the protein of CYP101 (adapted from Fig. 22 in ref. 16, with permissionfrom the American Chemical Society).

while the HS intermediate has a significant barrier to rebound.Therefore, generally speaking, C–H hydroxylation proceeds viatwo competing processes on different spin-state manifolds; one onthe LS manifold is effectively concerted with an ultrashort radicallifetime, the other on the HS manifold is stepwise with a significantlifetime for the intermediate.

It is this feature of TSR that creates the likeness of two differentoxidants and, in our view, is the cause of the controversialconclusions which were derived from the clock experiments.6 Ina typical clock experiment (Fig. 2) the apparent lifetime (sapp) ofthe radical intermediate, during P450 hydroxylation, is determinedfrom the extent of skeletal rearrangement observed in the alcoholproduct [R/U] and the known speed of rearrangement of the freeradical (kR). This expression for the apparent lifetime is based onthe assumption that there is a single intermediate that is branchingout to give the two products. However, using the TSR scheme,it is apparent that R and U do not originate from the sameintermediate; R arising only from the HS manifold and U mostly,if not only, from the LS manifold. As such, the apparent lifetimeof the clock experiments, e.g., in Fig. 2, is not the real lifetime,since it reflects the relative importance of the HS and LS pathways.Furthermore, since the LS H-abstraction barrier is generally lowerthan the corresponding HS quantity16 (see Fig. 6), the measured[R/U] quantity will generally lead to apparent lifetimes that are

Fig. 6 A two-state reactivity (TSR) scenario in the mechanism of C–H hydroxylation by the quartet (HS) and doublet (LS) states of Cpd I (4,2W0)(adapted from Fig. 2 in ref. 18, with permission from Wiley-VCH GmbH & Co KG).


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


too short. In the cases of C–H hydroxylation where the yield of theHS process is particularly small, the great majority of the productwill be the unrearranged variety, and the apparent lifetime will beartificially ultrashort. In reality, however, radicals exist on the HSmanifold and have normal lifetimes, while the ultrashort apparentlifetimes originate from the assumption that the two products arisefrom a single state of the radical complex.

Analysis of the barrier to rebound on the HS manifold,18 interms of a general model of barrier formation,80 creates some orderin the experimental data coming from the clocks. Fig. 8 shows theformation of the barrier to the HS rebound for the Alk•/FeIVOHspecies due to the states that interchange along the reactioncoordinate and mix to give the rebound transition state, 4TSreb. Asshown already in Fig. 5(b → c), in order to establish the Alk–OHbond, in the quartet state, the Alk• radical has to shift an electronto the high-lying r*z2 orbital. If we shift the electron withoutchanging the geometry of the 4Irad(IV) ground state, we get as inFig. 8 a vertical excited state 4Icat*(III) that contains an Alk+ cationand an excited ferric-hydroxo anion complex. The total energygap, G, of this state relative to the ground state 4Irad(IV) is given bythe ionization potential, IPAlk• , of the Alk• radical (within the gasphase or protein environments of the process), minus the electronattachment energy, EAFeOH, of the Fe(IV)-hydroxo complex, andplus the excitation energy of the attached electron from the low-lying p* orbital to the higher-lying r*z2 orbital, EFeOH(p* → r*).Along the rebound coordinate (shortening the C–O distance fromI to P) this 4Icat*(III) excited state will be stabilized by Alk–O

Fig. 8 A valence bond curve-crossing model for the barrier formationduring the HS rebound process. The state mixing is shown by the dashes(adapted from Fig. 9 in ref. 18, with permission from Wiley-VCH GmbH& Co KG).

bond-making and become the ground state of the ferric-alcoholproduct, 4P(III); at the same time the 4Irad(IV) state correlates to anexcited state of the product, 4P*(IV). The barrier that results fromthe mixing of these two electronic states, will depend on G. Forthe doublet-state process, the corresponding G is smaller since theelectron is shifted to the low-lying p* orbital, and since EFeOH(p* →r*) makes for a major fraction of G,18 the barrier to LS reboundwill be either very small or nil, as indeed it is. The calculationsshow a barrier-free LS rebound (in camphor hydroxylation66 thecalculated LS barrier was 0.29 kcal mol−1),16,18,66 and what remainsis to consider the HS rebound.

Looking at a series of substrates with the common iron-hydroxocomplex of P450, leads to the simple prediction that as the radicalbecomes a better electron donor (lower ionization potential, IP, oroxidation potential), so should the rebound barrier decrease untilit would altogether disappear. This prediction is manifested in theseries of probe substrates used by Newcomb and Toy,6 as shown inScheme 4. Thus, probes 1a and 1b lead to significant amounts ofthe rearranged product, while probe 1e that produces the radicalwith the lowest IPAlk exhibits virtually no rearrangement (belowthe detection limit). Recent DFT calculations,81 show that indeed1b should yield significant amount of the rearranged product,estimated as 20–30% based on the relative HS and LS barriers,while 1e should produce no rearranged products, since its LS andHS pathways are both effectively concerted.

Scheme 4 Probe substrates and their relative unrearranged/rearranged([U/R]) alcohol product yields as a function of the ionization potential ofthe corresponding radical.

In addition, the TSR model predicts the dependence of [R/U]on the strength of the Fe–S bond and the polarity of the pocket;the stronger the bond and the more polar the pocket, in a givenP450 isozyme, the more rearranged products are expected.18 Tothe best of our knowledge, different P450 isozymes give rise todifferent [R/U] values for a given clock. Another prediction ofthe TSR model is that since the KIE(H/D) reflects the HS andLS transition states for H-abstraction, and these transition statesare different, then the [R/U] value would be subject to productisotope effect (PIE), namely different KIE values are expectedfor U and R. Our most current results,77,78 show that the proteinchanges the TS geometries, compared with gas phase calculations,and therefore in cases where the TS structures for the HS and LSstates are close, the derivation of exact PIE values from modelgas-phase DFT calculations, as we proposed in the past,81 is notreliable. Nevertheless, the reason why PIE should be different than1 in TSR is quite evident. Moreover, an observation of PIE �= 1cannot arise from a single intermediate that branches into U and R.Therefore, until a better explanation is found for the observation


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


of PIE, PIE values different than unity indicate the operation ofTSR.

5.2 The mechanism of double bond epoxidation

As shown in Scheme 5, double bond epoxidation is normallyattended by formation of aldehydes and suicidal complexes, inwhich the olefin alkylates the heme through one of its nitrogen

Scheme 5 C=C epoxidation- and by-products formed during styreneepoxidation (adapted from Scheme 1 in ref. 82, with permission fromWiley-VCH GmbH & Co KG).

atoms.1–3 In addition, if one starts with pure cis or trans olefinsone observes a mixture of cis and trans epoxides, althoughthe scrambling of this isomeric information is not large.5 Themechanistic details of C=C activation by P450 have been thesubject of debate, albeit not as intense as in the case of C–H hydroxylation. Our studies of five different olefins (ethene,propene, cyclohexene, camphene and styrene)15,16,48,77,79,82 revealeda multi-state reactivity (MSR) scenario, where different spin statesand electromers participate in the product formation in a state-specific manner.

A representative example is the epoxidation of styrene in Fig. 9.82

It is seen that here, in addition to the usual pair of LS and HStransition states, 2,4TSrad, which lead to the radical intermediates,2,4Irad, there is also a LS cationic transition state 2TScat,yz that leads tothe cationic intermediate, 2Icat,yz, having a d2p*xz

2 p*yz1 configuration

on the porphyrin iron-oxo moiety and a carbocationic center onthe benzylic carbon of styrene. The bond activation here leadsto five low-lying intermediate states (during the self-consistent-field procedure 2Irad(III) became one of the cationic states), of theradical and cationic varieties. All the LS intermediates undergoring closure to give the LS epoxide product, 2P, without a barrierand hence, give rise to effectively concerted LS processes.

In contrast to the LS species, the HS intermediates encounterbarriers to ring closure, and therefore give rise to a stepwise mecha-nism with sufficiently long-lived intermediates that can participatein a variety of side product formations and in scrambling of

Fig. 9 A multi-state reactivity (MSR) scenario during styrene epoxidation by Cpd I (adapted from Fig. 6 in ref. 82, with permission from Wiley-VCHGmbH & Co KG). More states are shown in Fig. 10.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 10 The MSR scenario and the state origins of the various products obtained during the oxidation of trans-2-deuterio-styrene (adapted from Fig. 9in ref. 82, with permission from Wiley-VCH GmbH & Co KG).

stereochemistry (e.g., if one starts with trans-2-deuterio-styrene)to yield a mixture of cis and trans epoxide products.

Fig. 10 shows the MSR scheme for the reaction of Cpd I withtrans-2-deuterio-styrene (two hydrogen atoms are labeled in white,while the deuterium atom is in black). This is a state-specificscheme, where the spin states and the electromers participatein specific reactions. The LS intermediates undergo ring closureto form trans-epoxides and conserve the initial stereochemistry.By contrast, the HS intermediates participate in stereochemicalscrambling and in side-product formation. The rotational barrieraround the C–C bond of the radical intermediate is only 1.3kcal mol−1, which is smaller than the barriers to ring closure forboth HS intermediates. Therefore, both 4Irad(IV) and 4Irad(III) willhave sufficient lifetimes to lose stereochemistry and undergo ringclosure to produce a mixture of cis and trans epoxide products.

The suicidal and aldehyde complexes, which are side productsin the reaction, were found to originate only from the HS cationicstates; one state, 4Icat,z2, with configuration a2u

2d2p*xz1p*yz

1 r*z21

correlates in a barrier-free fashion to the aldehyde complex,and the other, 4Icat,xy, with the configuration a2u

2d2p*xz1p*yz

1 r*xy1

correlates similarly to the suicidal complex. The formation ofthese side-product complexes requires crossover from the HSradical states to the HS cationic states. The barriers to thesecrossovers are of the order of 10 kcal mol−1. Consequently, 4Irad(IV),which possesses a much smaller barrier to ring closure will givea scrambled epoxide but will not produce a suicidal complexor aldehyde. By contrast, the 4Irad (III) intermediate possessesa sufficiently large barrier to ring closure to also be able to

participate in the production of the suicidal complex and aldehydeside products.82

5.3 The mechanism of desaturation

Scheme 6 shows the P450 mechanisms studied by the Jerusalemgroup. It is apparent that not all the reactions involve TSR and/orMSR. There are a few reactions that proceed on a single spin-statesurface. For example, benzene hydroxylation,83 as well as a varietyof substituted benzenes,84 occurs preferentially on the LS surface,and mostly via a cationic Meisenheimer complex. Sulfur oxidationwas originally found to prefer a HS oxygenation.71 But recently,our collaborators85 identified an additional low-energy pathwayfor the LS states, and therefore we label the character of thisprocess, in Scheme 6, with a question mark until we satisfactorilyresolve the identities of different pathways in this reaction.

As can be seen in the scheme, desaturation is another processthat proceeds on the LS via a cationic intermediate state.86 Sincethis reaction bears a relationship to the mechanism whereby P450aromatase, CYP19, converts androgens to estrogen products,87 wediscuss below the mechanism of desaturation to demonstrate theversatility of Cpd I as an MSR reagent.

Thus, in its normal activity, P450 catalyzes the “insertion” of anoxygen atom into substrates by utilizing two reduction equivalents(2e−), one mole of O2, and two proton equivalents; the other[O] equivalent is converted to water, eqn 2. Occasionally, the O2

consumption follows eqn 3, where the enzyme converts all the


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Scheme 6 Mechanisms of P450 oxidation studied by DFT(B3LYP)and/or DFT(B3LYP)/MM, and their characterization in terms of TSR,MSR or a single-state reactivity.

dioxygen into water, thereby acting as an oxidase, but at the sametime it dehydrogenates the substrate,12,88 creating a double bond.

O2 + 2e− + 2H+ + R3C–CR′2H → R3C–CR′

2OH + H2O (2)

O2 + 2e− + 2H+ + HR2C–CR′2H → R2C=CR′

2 + 2H2O (3)

A seminal study88 of C–H hydroxylation of valproic acid (ananti-epileptic drug) showed that the desaturation activity andthe normal monooxygenation function branch from the sameoxidation mechanism that involves Cpd I. Since mechanisticalternatives exist for the desaturation reaction, involving eitherradicals or cations,12 theoretical calculations by the Jerusalemgroup86 were used to establish the preferred mechanism. The modelalkane (Alk–H) chosen for the calculations is trans-2-phenyl-1-iso-propylcyclopropane (probe substrate 1e in Scheme 4). Despitethe fact that there are no clear experimental data that showoxidase/dehydrogenase reactivity for this substrate, it was selectedbecause it leads to both radical and cationic species en routeto rebound; these species were postulated to be involved in thedesaturation/oxygenation dichotomy.12,88,89

Fig. 11a shows the radical, 2Irad and the cation, 2Icat; it is seenthat the C–O distance in 2Icat is longer than in 2Irad (2.587 A vs.2.142 A). But in 2Icat the hydrogens of the methyl groups of theisopropyl moiety are close to the iron-hydroxo anion. As shownby the arrows in Fig. 11a, 2Irad undergoes C–O bond making(barrier-free, as in Fig. 6) to yield the alcohol Alk-OH, while 2Icat

spontaneously loses a proton, from the [(CH3)2C–H]+ moiety tothe ferric-hydroxo anion, to form the corresponding alkene andthe ferric-water complex.

In fact, the 2Icat species is on the downhill slope en route tothe alkene product complex; once the electron shifts from theAlk• radical to the iron(IV)-hydroxo complex, the latter becomesa strong base and abstracts a proton from the carbocation togive rise to an olefin. Thus, theory reveals that the dehydrogenaseactivity is associated not with radicals of the substrates butrather with the corresponding carbocations. This is indeed oneof the possibilities postulated in the experimental literature.12,89

Fig. 11 The DFT(B3LYP) computed mechanism of desaturation occur-ring in competition with C–H hydroxylation (adapted from Fig. 35 in ref.16, with permission from the American Chemical Society).

Furthermore, as shown in Fig. 11b,86 the electron transfer fromthe alkyl radical to the iron-hydroxo complex occurs by surfacecrossing, between the radical and cationic manifolds, during theinitial C–H abstraction phase, wherefrom the reaction proceedsfurther to the alkene. A very similar mechanism was demonstrated,using DFT(B3LYP) and molecular dynamics, to be responsible forthe third “mysterious” step during aromatase catalysis that resultsin androgen aromatization.87

What is the reason for the oxidase-dehydrogenase mode of2Icat? Inspection of the geometry of the species in Fig. 11a showsthat the C–O distance is 2.59 A compared with 2.14 A forthe corresponding radical 2Irad. The reason for the longish C–Odistance is rooted in the steric bulk of the isopropyl group and thecharges of the two moieties of 2Icat. Thus, on the one hand, thesteric bulk of the isopropyl prevents the necessary close approach


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


for instantaneous C–O bond making and alcohol formation. Thisdistance is further augmented by electrostatic preference: in thecationic species, 2Icat, the iron-hydroxo is negatively charged, and itprefers to maintain O−–H+ interaction with the close-by positivelycharged hydrogen ends of the CH3 groups of the Alk cation moiety;this facilitates proton abstraction. Thus, the electro-steric factorcauses a spontaneous proton transfer from the radical to the iron-hydroxo complex, and results in the dehydrogenase activity.

The electro-steric factor links the theoretically studied processto the “pure” oxidase activity of P450, which converts the twooxygen atoms in O2 to water, by using an extra mole of reductasein the course of the reaction (i.e., O2 + 4e− + 4H+ → 2H2O).As has been shown,90 the “decoupling” of oxygen consumptionfrom substrate oxidation occurs when Cpd I accepts two electronsfrom the reductase. Mueller et al.91 have further demonstratedthat whenever the protein pocket is sufficiently encumbered toprevent an approach of the substrate C–H bond to the FeOmoiety of Cpd I, one also observes increased “decoupling” andheightened “pure” oxidase activities. The theoretical study86 showssimilarly that, whenever the substrate is sterically encumbered,thus hindering the C–O approach, the cationic intermediatewill lead to a mixed oxidase–dehydrogenase reaction. Thus, theoxidase–dehydrogenase function requires substrates that combinesteric inhibition of rebound and stable carbocationic complexes.Such conditions are met in the third step of aromatization ofandrogens, where the ferric-hydroxo anion deprotonates a gem-diol, thereby causing deformylation and aromatization of the Aring of androgens.87 It is further reasonable to speculate that inthe protein environment, release of the carbocation, away fromthe iron-hydroxo anion, may compete with dehydrogenation. Insuch an event, the carbocation will either be hydroxylated (e.g., byrebound from a long C–O distance [Fig. 11b], or simply by water),or mediate “pure” oxidase activity by accepting electrons from thereductase, while restoring the substrate by hydrogen abstraction.

6 Gauging the reactivity of Cpd 0

The reactivity of the T252A mutant of CYP101 towards thecamphene analogs of camphor38 has created a tantalizing problemfor both experiment and theory: Must we invoke Cpd 0 as asecondary oxidant in P450, or could the data still be interpretedby Cpd I as the sole oxidant? Let us see how theory attempted totackle this question.

6.1 A computational assessment of the electrophilic reactivity ofCpd 0

Since C=C epoxidation is thought to be a marker of Cpd 0reactivity, this reaction was the first to be studied by DFT(B3LYP)calculations, performed independently by two groups.69,70 The twostudies concluded that the barriers to electrophilic epoxidation byCpd 0 are exceedingly high. Furthermore, the computed barriers toepoxidation by Cpd 0, through either one of the two oxygen atomsof the OOH moiety, were much higher than the correspondingbarriers to ethene epoxidation by Cpd I, for example, >36 kcalmol−1 vs. 14–15 kcal mol−1.69

It is well known from model compounds that Cpd I is capableof oxygenating sulfur.92 Nonheme FeIVO type complexes can alsoachieve this transformation, whereas the corresponding nonhemeFeIIIOOH species fail to produce any reaction under the sameconditions where FeIVO was reactive.93 To gauge the sulfoxidationreactivity of Cpd 0 vs. Cpd I, the Jerusalem group studied thesulfoxidation of Me2S, dimethyl sulfide (DMS), by these twospecies.71,85 The reaction of Cpd I in Fig. 12a (energies includecorrections due to the bulk polarity effect) involves a concertedoxo transfer from Cpd I to the sulfur atom; the lowest barrier is16.0 kcal mol−1 via 4TSI (unpublished results85 show an even lowerbarrier on the LS surface, ca. 12.5 kcal mol−1, and assign the LSpath in Fig. 12a to a porphyrin oxygenation reaction). Fig. 12b

Fig. 12 DFT(B3LYP) results for the sulfoxidation of (CH3)2S by (a) Cpd I, and (b) Cpd 0 (see ref. 71). Work in progress (ref. 85) shows the existence ofan additional 2TS1 species 6.8 kcal mol−1 lower than the one shown in (a).


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


shows the monooxygenation reaction of Me2S via the proximaloxygen of Cpd 0. With a bulk polarity correction, but withoutacid catalysis, the barrier is exceedingly high. Hydrogen bondingby a molecule of water lowers this barrier by ca. 5 kcal mol−1,while assistance from a potent proton source, H3O+(H2O) furtherlowers the barrier. If we take the H3O+(H2O) species to representa limiting acidity, this would mean that even under these limitingconditions, the barrier to sulfur oxygenation by Cpd 0 remains atthe very least 10 kcal mol−1 higher compared to sulfoxidation byCpd I.

The lowering of the barrier to oxygen transfer by Cpd 0 dueto acid catalysis is in line with the experimental observation thatoxo-transfer reactions by MOOHn+ species (M-transition metal) todialkyl sulfides and triarylphosphines require acid catalysis, evenfor positively charged metallo-hydroperoxide reagents.94 It shouldbe kept in mind though that Cpd 0 of P450 carries a negativecharge and this does not favor electrophilic reactivity. While theeffect of the negative charge of Cpd 0 is partially offset by the acidcatalysis, the sulfoxidation study shows that this does not makeCpd 0 competitive with Cpd I. Furthermore, since the barrier31,52

to conversion of Cpd 0 to Cpd I is smaller than the barrier tosulfur oxygenation by Cpd 0, theory does not favor participationof Cpd 0 in monooxygenation in the WT enzyme.

In conclusion, based on the present computational picture,Cpd 0 is a sluggish electrophilic oxidant that cannot compete, assuch, with Cpd I. This conclusion is similar to the one drawnrecently in the nonheme catalysts where the LFeIIIOOH2+ specieswere shown to be silent under conditions where the correspondingferryl species LFeIV=O2+ were reactive.93

6.2 A computational assessment of the reactivity of Cpd 0 viainitial O–O homolysis

The interplay of homolytic and heterolytic O–O bond cleavagesin Cpd 0 species is well known.95–97 For example, the peroxidedependent P450 hydroxylation of cyclohexane,95 gave a smallKIE value ([C6H12/C6D12]) of ∼2, which was diagnostic ofthe HO• radical reactivity due to the homolysis of the O–Obond of the ferric-hydroperoxide. Indeed, the Jerusalem group98

showed recently that Cpd 0 undergoes relatively facile O–Obond homolysis and generates a bound OH• radical, which thenperforms hydroxylation of the meso position of the porphyrin,in a manner analogous to the reaction of heme-oxygenase (HO)enzymes.99 As such, in the presence of an oxidizable substrate,one may expect a competition between substrate oxidation andmeso heme-hydroxylation by the bound OH• radical, as shown inScheme 7. The Jerusalem group has studied this competition forthe two substrates 1 and 2 shown in the scheme;48,67 1 is a probesubstrate (see Scheme 7) that undergoes C–H hydroxylation givingrise to results that were interpreted6 in terms of the “two oxidanthypothesis” in Scheme 1, above, while substrate 2 was shown toundergo expoxidation by the T252A mutant of CYP101, implyingCpd 0 as an oxidant.38

C–H hydroxylation by Cpd 0: Fig. 13 shows the DFT(B3LYP)computed mechanism67 of hydroxylation of 1 by Cpd 0. It is seenthat the first step involves O–O bond homolysis, leading to anintermediate 2IO–O in which the OH• radical is bound to a Cpd IImoiety. Subsequently, the OH• radical abstracts a hydrogen atomfrom 1, and then the alkyl radical rebounds on the ferryl to give rise

Scheme 7 Homolytic cleavage of the O–O bond in Cpd 0, the ensuingchemical reactions induced by the bound OH• radical, and the twosubstrates studied by DFT(B3LYP) to explore the hydroxylation andoxidation capabilities of Cpd 0 via this mechanism.

to a ferric-alkoxy complex, 2PAC, of the alkoxide derived from 1. Analternative process whereby the radical derived from 1 reboundson the water molecule, while the ferryl abstracts a hydrogen atomfrom the water, has a much higher barrier (40 kcal mol−1) and istherefore not shown in Fig. 13.67

In the insets of Fig. 13, we show two other events: inset (a) showsthe meso hydroxylation process nascent from 2IO–O, while inset (b)shows the rearrangement barrier of the free radical derived from1, and is labelled as 2IU in the figure. Two conclusions can bedrawn: (i) the bound OH• radical will carry out meso hydroxylationof the heme in preference to H-abstraction from 1, since the H-abstraction barrier is larger than the barrier to meso attack, and(ii) the radical derived from 1 will rearrange faster than it canrebound to give the hydroxylation product (the rebound barrier is5.2 kcal mol−1; see Fig. 13 insets).

Since the hydroxylation of 1 by Cpd I was studied before atthe same computational level,81 it is possible to compare thebarriers for the rate-controlling steps in the two mechanisms.The data shown in Table 1 involve corrections due to the zeropoint energy (ZPE), bulk solvation (a dielectic constant of 5.7)and NH–S hydrogen-bonding effects. It is seen that the C–Hhydroxylation barrier by Cpd 0 is ca. 9.9 kcal mol−1 higher thanthe corresponding barrier of Cpd I. Clearly, therefore, Cpd 0 isa sluggish hydroxylation reagent that will be silent in the presenceof Cpd I. If Cpd I is not present, Cpd 0 may lead to a limitedamount of hydroxylation due to the competition with meso-hemehydroxylation.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 13 DFT(B3LYP/LACV3P+*) energies during hydroxylation of 1 by Cpd 0 (see ref. 67). Shown in the insets are (a) the barrier to heme hydroxylationstarting from 2IO–O, (data taken from ref. 48) and (b) the barrier to rearrangement of the radical derived from 2IU. (from ref. 81).

Table 1 Energy barriers (in kcal mol−1) for C–H hydroxylation of 1 byCpd I and Cpd 0

DE‡, kcal mol−1 a

Computational level Cpd Ib Cpd 0c

(1) LACVP 19.4 22.9(2) LACVP + ZPE 15.2 18.3(3) LACV3P+(d) 17.0 25.1(4) LACVP + ZPE + 2NH · · · S + e = 5.7 18.3 23.8(5) LACV3P+(d)+ZPE + 2NH · · · S + e = 5.7 16.0 25.9

a Barriers relative to the clusters of the substrate with Cpd I or Cpd 0. b Seerefs. 67 and 81. c See ref. 67.

Another DFT (B3LYP) study of the reactivity of Cpd 0 inC–H hydroxylations68 reached the conclusion that all the P450chemistry can be understood in terms of the chemistry of Cpd 0,without a need to invoke Cpd I. These conclusions were based onthe computational results of the hydroxylation reactions of CH4

and isobutane by Cpd 0. The case of CH4 is of course academicallyinteresting; P450 does not hydroxylate methane. In the case ofisobutane, the authors obtained a barrier of 19.5 kcal mol−1.However, using analogous substrates with secondary and tertiaryC–H bonds, shows that Cpd I is more reactive.16,65 Thus, the barrierto H-abstraction of the isopropylic C-H of propane is 16.9 kcalmol−1 (12.9 kcal mol−1 after ZPE correction), while for a tertiaryC–H of the probe substrate 1e in Scheme 4, the barrier is 16.6(12.7) kcal mol−1.81 Therefore, the barriers for Cpd I are at least 3kcal mol−1 lower than the value obtained for Cpd 0 (if ZPE wereincluded, most likely the difference would be significantly larger,but no ZPE was reported in ref. 68). Furthermore, considering

the fact that the barrier of Cpd 0 is raised by 2.5 kcal mol−1 morethan the barrier of Cpd I, when the effects of polarity and NH–Shydrogen bonding are taken into account (see entries 2 vs. 4 inTable 1), the difference in the barrier becomes at least 5.5 kcalmol−1 in favor of Cpd I.

Other inconsistencies of the C–H hydroxylation by Cpd 0 aremechanistic. Thus, suppose that Cpd 0 was indeed the ultimateoxidant also in the wild type (WT) enzyme, then one would nothave expected to find the original hydrogen of the C–H bond inthe alcohol product as was reported to be the case in the cryogenicEPR/ENDOR study of camphor hydroxylation by CYP101.23

In addition, H-abstraction by OH• radical is expected to lead tosmall KIE values,95 whereas the experimental ones for Cpd I are8–12.5,11,67 Furthermore, if it were true that Cpd 0 was the onlyoxidant of P450, this should have rendered the T252A mutant ofCYP101 considerably more reactive than the WT enzyme, which isobviously not the case. The minimally drawn conclusion at thispoint in time is that Cpd 0 may lead to C–H hydroxylation only inthe absence of Cpd I. In the case of T252A reacting with camphor,no C–H hydroxylation was observed. So it remains to be proventhat P450 Cpd 0 can indeed participate in C–H hydroxylation.

C=C epoxidation by Cpd 0 will proceed by TSR: Fig. 14shows the DFT(B3LYP) energy profile for the oxidation of 5-methylenylcamphor, 2, by Cpd 0 in its two lowest spin states.48

The relative energies include corrections due to ZPE, NH–S H-bonds, and bulk polarity (using a dielectric constant e = 5.7). Thus,as described above, the initial step involves O–O bond homolysisto give rise to the IO–O intermediate, which involves Cpd II anda bound OH• radical. The OH• radical then attacks the doublebond and in a barrier-free fashion gives rise to the hydroxylatedintermediate Id. Being barrier free, the attack on the double bond


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Fig. 14 DFT(UB3LYP/LACV3P+* + NH–S + e = 5.7 + ZPE) results(adapted from ref. 48, with permission from Elsevier) for the mechanismof the epoxidation of 2 by Cpd 0 via the mechanism starting with O–Obond homolysis. Please note the competition between the epoxidation andglycol pathways.

will compete favorably with heme degradation via meso attack (seeFig. 13 inset (a)).

Subsequent to the attack on the double bond, the Id intermediatecan undergo a radical coupling reaction, whereby the radical,derived from the hydroxyl attack on 2, couples with the oxo groupof Cpd II (recall Cpd II is a diradical like 3O2), and leads to aferric-glycol complex, Pg. Being a radical coupling process, thisreaction has a small barrier ca. 6 kcal mol−1.

Alternatively, Cpd II can abstract a hydrogen atom from thehydroxyl group of the radical intermediate in Id, and cause ringclosure to form the epoxide product. This latter step involvessignificant electronic reorganization (C–H bond breaking, Fe=Obond breaking, C–O bond formation, and electron shift from theorganic moiety to convert the Fe(IV) to Fe(III)), and therefore thecorresponding barrier is large, ca. 20 kcal mol−1. As such, it is clearthat Cpd 0 will not lead to epoxidation of 2, but would rathergenerate the corresponding glycol (1,2-diol). Nevertheless, theglycol path may be less plausible within the enzymatic environmentdue to the steric restraints of the substrate in the active site ofP450cam, where Tyr96 can hold the carbonyl by a hydrogen bondand restricts its movements.48 We can tentatively deduce that wereCpd 0 the reactive species in the T252A mutant of CYP101,one would have expected to find alongside the epoxide of 2 alsosome of the corresponding 1,2-diol. Finding the glycol product isimportant because its formation is a clearer signature of Cpd 0reactivity than having merely an epoxide product, especially sincethe latter can be easily formed with Cpd I.

Indeed, as shown in Fig. 15, epoxidation of 2 by Cpd I,48 leadsto a barrier of 12.7 kcal mol−1 for the LS state; 6.2 kcal mol−1 lowerthan the barrier to the rate-controlling step for Cpd 0 (Fig. 14).Once again we conclude that in the presence of Cpd I , Cpd 0 willbe silent towards the epoxidation of 2. However, in the absence ofCpd I , Cpd 0 may become a by-default oxidant via the mechanismthat starts with O–O bond homolysis. Without the observation ofthe glycol, and having no definitive proof that Cpd 0 is the reactive

Fig. 15 DFT(UB3LYP/LACV3P+* + NH–S + e = 5.7 + ZPE) results(adapted from ref. 48, with permission from Elsevier) for the mechanismof the epoxidation of 2 by Cpd I.

species of the mutant, there will remain a lingering question: canCpd I still be present in the mutant but be formed slowly and withtoo poor a juxtaposition to perform hydroxylation, but still in afavorable position to allow some epoxidation?

Interestingly, like Cpd I, one can see from Fig. 14 that Cpd 0is predicted to react by the two-state reactivity (TSR) scenarioand to lead to some loss of stereochemistry in cases where thedouble bond carries some stereochemical cis or trans information.However, a much greater loss is predicted for Cpd 0, since thequartet transition state 4TSe which is almost at the same energy asthe doublet species, leads to the triplet diradical substrate in 4Pe

′.48

Sulfoxidation by Cpd 0: We tried to apply the same homolyticmechanism to the sulfoxidation of DMS. All our attempts showedthat the OH• radical cannot transfer an oxygen atom to sulfur, theenergy expense is simply too high (>25 kcal mol−1). It may well bethat the sulfoxidation can proceed via the Cpd II moiety after O–Obond homolysis, but this was not explored further since it seemedto be the same process that was already studied in Fig. 12b, wherethe proximal oxygen of Cpd 0 is transferred to DMS.

Acid catalysis of the oxidation reactions by Cpd 0: All attemptsto study the effect of acid catalysis on the reactivity of Cpd 0,resulted in the FeIII(H2O2) complex,67,48 once the proton was givenfreedom and was not constrained to remain in the proton source(e.g., NH4

+(H2O)2). We therefore studied also the reactivity offerric-hydrogen peroxide in C–H hydroxylation,67 and performedpreliminary calculations for C=C epoxidation.48 Indeed, thepresence of a second proton on the proximal oxygen loweredthe barriers (but the overall barrier for the processes were stillhigher than those of Cpd I). However, the binding energy ofH2O2 to the ferric porphyrin is rather small (ca. 10 kcal mol−1)and smaller than the barrier to C–H hydroxylation. This inturn means that the FeIII(H2O2) complex will lead to uncoupling(H2O2 release) in preference to substrate oxidation.67 The ratioof uncoupling to substrate oxidation will depend greatly on the


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


substrate; reactive ones will exhibit higher substrate oxidationyields. Thus, establishing the role of the FeIII(H2O2) in substrateoxidation merits future attention.

6.3 An assessment of the reactivity of the two oxidant of P450

Theory shows that in the presence of Cpd I, Cpd 0 would not be acompetent oxidant, unless the barrier separating Cpd 0 and Cpd Iis larger than the barrier to oxidation by Cpd 0, which does notseem to be the case.31,52 This statement is in line with experimentalobservations that the FeIII–OOH species in the T252A mutant ofCYP101 is incapable of camphor hydroxylation.38 This is also inline with the demonstration that in nonheme systems the FeIII–OOH species is silent under the conditions where the FeIV=Ospecies is reactive.93 As such, at present, the reactivity patterns ofC–H hydroxylation by the WT enzymes are best understood interms of TSR (MSR15,76,82) of Cpd I in its various spin states. Theinterpretations that the reactivity of the WT enzyme arises froma competition of Cpd I and Cpd 0,6,37,61 or that Cpd 0 is the onlyoxidant,68 do not seem tenable.

In mutant enzymes, it is conceivable that if indeed Cpd I isabsent, then Cpd 0 can do the job, albeit more sluggishly.38,48

What is missing in P450 research is a direct characterization of thereactivity of Cpd 0 in the mutant: Which oxygen in the Fe–OOHmoiety is transferred to the substrate? What are the KIE patternsfor e.g., C–H hydroxylation? These are some of the questions thatneed to be addressed before a fruitful discussion can go on, onthis topic. An experimental study like the one recently conductedfor nonheme systems93 is sorely missing in the P450 field. Anothernagging question is whether Cpd I really does not exist in themutant? What is the precise mechanism of the conversion of Cpd 0to Cpd I, and what are the energetic aspects of the process? Heretheory can play a more active role than it has done so far. But thiswould require a Herculean effort using DFT/MM studies of theCpd 0 → Cpd I conversion in WT,52 T252A and T252A/D251Nmutants.

7 Conclusions

The role of theory is not to merely generate “accurate” numbers(something which is anyway still impossible for systems like P450),but rather to paint global pictures and generate new ideas thatcan hopefully lead to new and more meaningful experiments. Thepresent review highlights this role of theory and shows that theaccessiblity of the spin states of Cpd I, its electron deficiencyand dense orbital manifold lead to TSR and MSR scenarios,which describe the simultaneous reactivity of many states. Thesescenarios lead to intriguing consequences, and generate, in somecases, reactivity patterns as though belonging to two or moredifferent oxidants. So far, many of the experimental data can beunderstood in terms of the TSR paradigm without a need to invokethe reactivity of Cpd 0 for the WT enzymes.7,16,18,60,100–103 The spinstates of Cpd I emerge from generally trustworthy computationalprocedures,36 and their degeneracy can be reasoned a priori also bytheoretical considerations of the nature of Cpd I.22,64 Model Cpd Isystems are abundant,5,103 and are known to have two closely lyingspin states. As such, if Cpd I is the active species of P450, as isindeed suggested by experiment, then these spin states also exist“in the flasks” of the experimentalist.

The problem is: how precisely can one probe these states? Anessential element, which is still missing in the TSR (MSR) scheme,is some quantitative information on the rate of spin crossoverbetween the two spin states vis-a-vis the radical rearrangmentand rebound rates. Understanding this factor may provide themechanistic chemists with additional means to articulate theconcept and design experiments for testing it. Based on previousexperience104 the spin–orbit coupling that is needed to induce spin-state transitions should be small in cases like the doublet andquartet states of Cpd I and of the radical complex intermediates,2,4Irad (Fig. 6). In such a case, the interconversion of the 4Cpd Iand 2Cpd I will still be competitive with the slower process ofH-abstraction. However, the interconversion of the 4Irad and 2Irad

species will be slow compared with rebound, and will be compet-itive with rearrangement for very slow clocks, but incompetitivewith the rearrangement of the ultrafast clocks (Fig. 2b). Thus, theTSR scenario is likely to have different features/manifestationsdepending on the competition of all these events. The advent ofspin–orbit calculations and a proper rate theory will be needed tomake further progress on this issue.

As we showed above for benzene hydroxylation, and desatura-tion reactions, the enzyme sometimes uses one of the spin statesselectively. Recently, it was shown that N-dealkylation proceedsonly via the low-spin state.105 It was further demonstrated thatthe spin-state degeneracy in Cpd I and in its species alongboth C–H hydroxylation and C=C epoxidation pathways, can beaffected dramatically by external electric fields.106 These resultsalong with the recent demonstration of magnetic-field effectsin oxidative reactions by horseradish peroxidase107 pose someexciting prospects of probing and affecting spin-selective chemistryby P450s.

Our attempts to gauge the reactivity of Cpd 0 led to a clearconclusion: Cpd 0 is less reactive than Cpd I for C=C epoxidation,C–H hydroxylation and sulfoxidation. Thus, when Cpd I is presentin the cycle, Cpd 0 will be silent, unless of course, the barrier forthe conversion of Cpd 0 to Cpd I is higher than the barriers tooxidation by Cpd 0. Since this does not seem to be the case,31,52

theory does not support a scenario whereby Cpd 0 and Cpd Icompete for oxidation of substrates. We are aware of the mostrecent study of Cryle and De Voss,108 with P450BM3, which stronglyimplies that sulfur oxidation is effected by Cpd 0 in both the WTand T268A mutant. However, all our studies so far exclude thisinterpretation. Electrophilic reactivity of Cpd 0 is deemed to bevery low since the species is an anion.69 The reactivity throughthe proximal oxygen atom of Cpd 0 may be promoted by generalacid catalysis (“pull” by water molecules) and/or by nucleophilicsubstrates, like reactive olefins.5,71 Alternatively, Cpd 0 may reactvia initial O–O bond homolysis and generation of a reactive OH•

radical, in which case one would expect to find the distal oxygenof Cpd 0 in the oxidized substrate. In the case of Cpd 0 reactingwith an olefin, via the homolytic mechanism, we find that a mostlikely product is the gem-diol; its presence can be a marker reactionof Cpd 0, if the latter were reactive. Despite these possibilities forthe reactivity of Cpd 0, there is still a lingering question: do theP450 mutants really lack Cpd I?

The status of Cpd I in the WT and mutant enzymes, the precisemechanism of conversion of Cpd 0 to Cpd I in P450s and thecomplete characterization of Cpd I are still the major unknownsin the P450 field. The field, by now distraught by controversies,


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


is begging for a focused effort to resolve these unknowns. Theorymight be a helpful partner to experiment in this endeavor, e.g., indesigning new mutants with promising properties, as the recentlypostulated selenocysteine mutant,109 or by careful scrutiny of theeffects of side chains, residues and additives on the structure andreactivity of P450 species.72,110,111 Recent experimental observationsof Cpd I in P450cam (CYP101) and P450BM3, using oxygen surrogatedonors,112,113 will hopefully also enable the study of the substrateoxidation processes by these enzymes.

8 Acknowledgements

The research was supported in part by a DIP grant (DIP-G.7.1) and an ISF grant (16/06). HH is a JSPS PostdoctralFellow for Research Abroad. SS thanks D. Ballou, I. Denisovand S. G. Sligar for critical reading of the section on Cpd I,and J. H. Dawson, J. T. Groves, and J. P. Jones, for many helpfuldiscussions. Special thanks go to all the coworkers in the P450 field:H. Schwarz, D. Schroder, N. Harris, M. Filatov, F. Ogliaro, S. P.de Visser, P. K. Sharma, S. Cohen, S. Kozuch, E. Derat, C. Hazan,K. Cho, C. Li, W. Wu, W. Thiel, A. Altun, J. Schoneboom,H. Lin, J. Zheng, D. Wang, and N. Reuter.

9 References

1 Cytochrome P450: Structure, Mechanism and Biochemistry, ed.P. R. Ortiz de Montellano, Plenum Press, New York, 2nd edn, 1995.

2 Cytochrome P450: Structure, Mechanism and Biochemistry, ed.P. R. Ortiz de Montellano, Plenum Press, New York, 3rd edn, 2005.

3 M. Sono, M. P. Roach, E. D. Coulter and J. H. Dawson, Chem. Rev.,1996, 96, 2841.

4 T. M. Makris, I. G. Denisov, I. Schlichting and S. G. Sligar, Activationof Molecular Oxygen by Cytochrome P450, in Cytochrome P450:Structure, Mechanism and Biochemistry, ed. P. R. Ortiz de Montellano,Plenum Press, New York, 3rd edn, 2005, ch. 5, pp. 149–181.

5 J. T. Groves, Models and Mechanisms of Cytochrome P450 Action,in Cytochrome P450: Structure, Mechanism and Biochemistry, ed.P. R. Ortiz de Montellano, Plenum Press, New York, 3rd edn, 2005,ch. 1, pp. 1–44.

6 M. Newcomb and P. H. Toy, Acc. Chem. Res., 2000, 33, 449.7 P. R. Ortiz de Montellano and J. J. De Voss, Nat. Prod. Rep., 2002, 19,

477.8 W.-D. Woggon, Top. Curr. Chem., 1996, 184, 39.9 I. G. Denisov, T. M. Makris, S. G. Sligar and I. Schlichting, Chem.

Rev., 2005, 105, 2253.10 Y. Watanabe, J. Biol. Inorg. Chem., 2001, , 6, 846.11 J. T. Groves, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 3555.12 F. P. Guengerich, Chem. Res. Toxicol., 2001, 14, 611.13 T. L. Poulos and E. F. Johnson, Structure of Cytochrome P450 En-

zymes, in Cytochrome P450: Structure, Mechanism and Biochemistry,ed. P. R. Ortiz de Montellano, Plenum Press, New York, 3rd edn,2005, ch. 3, pp. 87–114.

14 T. M. Makris, K. von Koenig, I. Schlichting and S. G. Sligar, J. Inorg.Biochem., 2006, 100, 507.

15 S. Shaik, S. P. de Visser and D. Kumar, J. Biol. Inorg. Chem., 2004, 9,661.

16 S. Shaik, D. Kumar, S. P. de Visser, A. Altun and W. Thiel, Chem. Rev.,2005, 105, 2279.

17 B. Meunier, S. P. de Visser and S. Shaik, Chem. Rev., 2004, 104, 3947.18 S. Shaik, S. Cohen, S. P. de Visser, P. K. Sharma, D. Kumar, S. Kozuch,

F. Ogliaro and D. Danovich, Eur. J. Inorg. Chem., 2004, 35, 207.19 S. Shaik and S. P. de Visser, Computational Approaches to Cy-

tochrome P450 Function, in Cytochrome P450: Structure, Mechanismand Biochemistry, ed. P. R. Ortiz de Montellano, Plenum Press, NewYork, 3rd edn, 2005, ch. 2, pp. 45–85.

20 G. H. Loew and D. L. Harris, Chem. Rev., 2000, 100, 407.21 D. L. Harris, Curr. Opin. Chem. Biol., 2001, 5, 724.

22 S. Shaik, S. P. de Visser, F. Ogliaro, H. Schwarz and D. Schroder, Curr.Opin. Chem. Biol., 2002, 6, 556.

23 R. Davydov, T. M. Makris, V. Kofman, D. E. Werst, S. G. Sligar andB. M. Hoffman, J. Am. Chem. Soc., 2001, 123, 1403.

24 I. Schlichting, J. Berendzen, K. Chu, A. M. Stock, S. A. Maves, D. E.Benson, R. M. Sweet, D. Ringe, G. A. Petsko and S. G. Sligar, Science,2000, 287, 1615.

25 R. Rutter, L. P. Hager, H. Dhonau, M. Hendrich, M. Valentine andP. Debrunner, Biochemistry, 1984, 23, 6809.

26 A. Zaks and D. R. Dodds, J. Am. Chem. Soc., 1995, 117, 10419.27 M. N. Bhakta, P. F. Hollenberg and K. Wimalasena, J. Am. Chem.

Soc., 2005, 127, 1376.28 V. Schunemann, C. Jung, J. Terner, A. X. Trautwein and R. Weiss,

J. Inorg. Biochem., 2002, 91, 586.29 T. Spolitak, J. H. Dawson and D. P. Ballou, J. Biol. Chem., 2005, 280,

20300.30 T. S. Dowers, D. A. Rock, D. A. Rock and J. P. Jones, J. Am. Chem.

Soc., 2004, 126, 8868.31 D. G. Kellner, S.-C. Hung, K. E. Weiss and S. G. Sligar, J. Biol. Chem.,

2002, 277, 9641.32 D. L. Harris, G. Loew and L. Waskell, J. Inorg. Biochem., 2001, 83,

309.33 K. L. Stone, R. K. Behan and M. T. Green, Proc. Natl. Acad. Sci.

U. S. A., 2005, 102, 16563.34 S. H. Kim, R. Perera, L. P. Hager, J. H. Dawson and B. M. Hoffman,

J. Am. Chem. Soc., 2006, 128, 5598.35 J. C. Schoneboom, H. Lin, N. Reuter, W. Thiel, S. Cohen, F. Ogliaro

and S. Shaik, J. Am. Chem. Soc., 2002, 124, 8142.36 J. C. Schoneboom, F. Neese and W. Thiel, J. Am. Chem. Soc., 2005,

127, 5840.37 A. D. N. Vaz, D. F. McGinnity and M. J. Coon, Proc. Natl. Acad. Sci.

U. S. A., 1998, 95, 3555.38 S. Jin, T. M. Makris, T. A. Bryson, S. G. Sligar and J. H. Dawson,

J. Am. Chem. Soc., 2003, 125, 3406.39 T. Hishiki, H. Shimada, S. Nagano, T. Egawa, Y. Kanamori, R.

Makino, S.-Y. Park, S. Adachi, Y. Shiro and Y. Ishimiura, J. Biochem.,2000, 128, 965.

40 T. Murakami, K. Yamaguchi, Y. Watanabe and I. Morishima, Bull.Chem. Soc. Jpn., 1998, 71, 1343.

41 M. Newcomb, R. Zhang, R. E. P. Chandrasena, J. A. Halgrimson,J. H. Horner, T. M. Makris and S. G. Sligar, J. Am. Chem. Soc., 2006,128, 4580.

42 R. Zhang, R. E. P. Chandrasena, E. Martinez, II, J. H. Horner andM. Newcomb, Org. Lett., 2005, 7, 1193.

43 Z. Pan, P. R. Zhang and M. Newcomb, J. Inorg. Biochem., 2006, 100,524.

44 R. Zhang and M. Newcomb, J. Am. Chem. Soc., 2003, 125, 12418.45 D. Ballou, private communication.46 J. T. Groves, Z. Gross and M. K. Stearns, Inorg. Chem., 1994, 33,

5065.47 W. Nam, S.-E. Park, I. K. Lim, M. H. Lim, J. Hong and J. Kim, J. Am.

Chem. Soc., 2003, 125, 14674.48 H. Hirao, D. Kumar and S. Shaik, J. Inorg. Biochem., 2006, 100,

2054.49 C. B. Brewer and J. A. Peterson, J. Biol. Chem., 1988, 263, 791.50 M. I. Page and W. P. Jencks, Proc. Natl. Acad. Sci. U. S. A., 1971, 68,

1678.51 F. Ogliaro, S. P. de Visser, J. T. Groves and S. Shaik, Angew. Chem.,

Int. Ed., 2001, 40, 2874.52 J. Zheng, D. Wang, W. Thiel and S. Shaik, J. Am. Chem. Soc., 2006,

128, 13204.53 A. Bassan, M. R. A. Blomberg, P. E. M. Siegbahn and L. Que, Jr.,

Angew. Chem., Int. Ed., 2005, 44, 2939.54 P. T. de Oliveira, A. Chanda, D. Banerjee, X. Shan, S. Mondal, L.

Que, Jr., E. L. Bominaar, E. Munck and T. J. Collins, Science, 2007,315, 835.

55 J. F. Berry, E. Bill, E. Bothe, S. D. George, B. Mienert, F. Neese andK. Wieghardt, Science, 2006, 312, 1937.

56 S. P. de Visser, D. Kumar, R. Newmann and S. Shaik, Angew. Chem.,Int. Ed., 2004, 43, 5661.

57 P. K. Sharma, S. P. de Visser, F. Ogliaro and S. Shaik, J. Am. Chem.Soc., 2003, 125, 2291.

58 J. T. Groves and G. A. McClusky, J. Am. Chem. Soc., 1976, 98, 859.59 P. R. Ortiz de Montellano and R. A. Stearns, J. Am. Chem. Soc., 1987,

109, 3415.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


60 P. R. de Montellano and J. J. De Voss, Substrate Oxidation by Cyto-chrome P450 Enzymes, in Cytochrome P450: Structure, Mechanismand Biochemistry, ed. P. R. Ortiz de Montellano, Plenum Press, NewYork, 3rd edn, 2005, ch. 6, pp. 183-245.

61 M. Newcomb, D. Aebisher, R. Shen, R. E. P. Chandrasena, P. F.Hollenberg and M. J. Coon, J. Am. Chem. Soc., 2003, 125, 6064.

62 P. R. Ortiz de Montellano, Acc. Chem. Res., 1998, 31, 543.63 L. Avila, H.-w. Huang, C. O. Damaso, S. Lu, P. Moenne-Loccoz and

M. Rivera, J. Am. Chem. Soc., 2003, 125, 4103.64 S. Shaik, M. Filatov, D. Schroder and H. Schwarz, Chem.–Eur. J.,

1998, 4, 193.65 S. P. de Visser, D. Kumar, S. Cohen, R. Shacham and S. Shaik, J. Am.

Chem. Soc., 2004, 126, 8362.66 J. C. Schoneboom, S. Cohen, H. Lin, S. Shaik and W. Thiel, J. Am.

Chem. Soc., 2004, 126, 4017.67 E. Derat, D. Kumar, H. Hirao and S. Shaik, J. Am. Chem. Soc., 2006,

128, 473.68 R. D. Bach and O. Dmitrenko, J. Am. Chem. Soc., 2006, 128, 1474.69 F. Ogliaro, S. P. de Visser, S. Cohen, P. K. Sharma and S. Shaik, J. Am.

Chem. Soc., 2002, 124, 2806.70 T. Kamachi, Y. Shiota, T. Ohta and K. Yoshizawa, Bull. Chem. Soc.

Jpn., 2003, 76, 721.71 P. K. Sharma, S. P. de Visser and S. Shaik, J. Am. Chem. Soc., 2003,

125, 8698.72 J. Harvey, C. M. Bathelt and A. J. Mulholland, J. Comput. Chem.,

2006, 27, 1352.73 C. M. Bathelt, J. Zurek, A. J. Mulholland and J. N. Harvey, J. Am.

Chem. Soc., 2005, 127, 12900.74 F. Ogliaro, S. Cohen, S. P. de Visser and S. Shaik, J. Am. Chem. Soc.,

2000, 122, 12892.75 F. Ogliaro, S. Cohen, M. Filatov, N. Harris and S. Shaik, Angew.

Chem., Int. Ed., 2000, 39, 3851.76 H. Hirao, D. Kumar, W. Thiel and S. Shaik, J. Am. Chem. Soc., 2005,

127, 13007.77 S. Cohen, PhD Thesis, The Hebrew University of Jerusalem, in

preparation.78 S. Cohen, S. Kozuch, C. Hazan and S. Shaik, J. Am. Chem. Soc., 2006,

128, 11028.79 S. P. de Visser, F. Ogliaro, P. K. Sharma and S. Shaik, J. Am. Chem.

Soc., 2002, 124, 11809.80 S. Shaik and A. Shurki, Angew. Chem., Int. Ed., 1999, 38, 586.81 D. Kumar, S. P. de Visser, P. K. Sharma, S. Cohen and S. Shaik, J. Am.

Chem. Soc., 2004, 126, 1907.82 D. Kumar, S. P. de Visser and S. Shaik, Chem.–Eur. J., 2005, 11, 2825.83 S. P. de Visser and S. Shaik, J. Am. Chem. Soc., 2003, 125, 7413.84 C. M. Bathelt, L. Ridder, J. Mulholland and J. N. Harvey, J. Am.

Chem. Soc., 2003, 125, 15004.85 C. Li, C. Zhang, W. Wu and S. Shaik, yet unpublished data.

86 D. Kumar, S. P. de Visser and S. Shaik, J. Am. Chem. Soc., 2004, 126,5072.

87 J. C. Hackett, R. W. Brueggemeier and C. M. Hadad, J. Am. Chem.Soc., 2005, 127, 5224.

88 A. E. Rettie, M. Boberg, W. A. Rettenmeier and T. A. Baillie, J. Biol.Chem., 1988, 263, 13733.

89 M. D. Wand and J. A. Thompson, J. Biol. Chem., 1986, 261, 14049.90 W. M. Atkins and S. G. Sligar, J. Am. Chem. Soc., 1987, 109, 3754.91 E. J. Mueller, P. J. Loida and S. G. Sligar, Twenty-five Years of

P450cam Research, in Cytochrome P450: Structure, Mechanism andBiochemistry, ed. P. R. Ortiz de Montellano, Plenum Press, New York,2nd edn, 1995, ch. 3, pp. 83–124.

92 Y. Goto, T. Matsui, S. Ozaki, Y. Watanabe and S. Fukuzumi, J. Am.Chem. Soc., 1999, 121, 9497.

93 M. J. Park, J. Lee, Y. Suh, J. Kim and W. Nam, J. Am. Chem. Soc.,2006, 128, 2630.

94 K. Lemma and A. Bakac, Inorg. Chem., 2004, 43, 6224.95 R. E. White, S. G. Sligar and M. J. Coon, J. Biol. Chem., 1980, 255,

11108.96 A. J. Allentoff, J. L. Bolton, A. Wilks, J. A. Thompson and P. R. Ortiz

de Montellano, J. Am. Chem. Soc., 1992, 114, 9744.97 J. T. Groves and Y. Watananbe, J. Am. Chem. Soc., 1988, 110, 8443.98 P. K. Sharma, R. Kevorkiants, S. P. de Visser, D. Kumar and S. Shaik,

Angew. Chem., Int. Ed., 2004, 116, 1129.99 C. Colas and P. R. Ortiz de Montellano, Chem. Rev., 2003, 103, 2305.

100 X. He and P. R. Ortiz de Montellano, J. Biol. Chem., 2004, 279, 39479.101 M. J. Cryle, J. M. U. Stuthe, P. R. Ortiz de Montellano and J. J. De

Voss, Chem. Commun., 2004, 512.102 K. Auclair, Z. Hu, D. M. Little, P. R. Ortiz de Montellano and J. T.

Groves, J. Am. Chem. Soc., 2002, 124, 6020.103 J. T. Groves, J. Inorg. Biochem., 2006, 100, 434.104 D. Danovich and S. Shaik, J. Am. Chem. Soc., 1997, 119, 1173.105 C. Li, W. Wu, D. Kumar and S. Shaik, J. Am. Chem. Soc., 2006, 128,

394.106 S. Shaik, S. P. de Visser and D. Kumar, J. Am. Chem. Soc., 2004, 125,

11746.107 M. S. Afanasyeva, M. B. Taraban, P. A. Purtov, T. V. Leshina and

C. B. Grissom, J. Am. Chem. Soc., 2006, 128, 8651.108 M. J. Cryle and J. J. De Voss, Angew. Chem., Int. Ed., 2006, 45, 8221.109 S. Cohen, D. Kumar and S. Shaik, J. Am. Chem. Soc., 2006, 128, 2649.110 A. Altun, S. Shaik and W. Thiel, J. Comput. Chem., 2006, 27, 1324.111 J. Zurek, N. Foloppe, J. N. Harvey and A. J. Mulholland, Org. Biomol.

Chem., 2006, 4, 3931.112 T. Spolitak, J. H. Dawson and D. P. Ballou, J. Inorg. Biochem., 2006,

100, 2034.113 G. M. Raner, J. I. Thompson, A. Haddy, V. Tangham, N. Bynum,

G. R. Reddy, D. P. Ballou and J. H. Dawson, J. Inorg. Biochem., 2006,100, 2045.


Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

29 S

epte

mbe

r 20

12Pu

blis

hed

on 2

3 A

pril

2007

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

6041

92M

View Online


Reactivity patterns of cytochrome P450 enzymes: multifunctionality of the active species, and the...

Documents

Transcript of Reactivity patterns of cytochrome P450 enzymes: multifunctionality of the active species, and the...