Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate

Article abstract of DOI:10.1016/j.abb.2010.11.001

The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 × 10¹⁰ s^-1, 7.2 × 10¹⁰ s ^-1 and 1.0 × 10¹² s^-1. For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 × 10¹⁰ s^-1 were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [Fe^IV-OH ^-R] can compete with cage collapse to form a C-O bond. The results could be significant with regard to our understanding of iron-catalyzed C-H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.

Full text of DOI:10.1016/j.abb.2010.11.001

Archives of Biochemistry and Biophysics 507 (2011) 111–118
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Molecular probes of the mechanism of cytochrome P450. Oxygen traps
a substrate radical intermediate
⇑
Harriet L.R. Cooper, John T. Groves
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 12 November 2010
The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three
drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1.
The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller
amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol,
was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol
also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a
ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios
of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respec-
tively, corresponding to rebound rates of 0.9 ꢀ 1010 sꢁ1, 7.2 ꢀ 1010 sꢁ1 and 1.0 ꢀ 1012 sꢁ1. For the model
iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 ꢀ 1010 sꢁ1 were determined.
These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and
radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron por-
phyrin suggest that for these systems a considerable amount of the intermediate carbon radical is
trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon
Keywords:
Radical
Rebound
Mechanism
Oxidation
Hydroxylation
Å
radical in [FeIV–OH R] can compete with cage collapse to form a C–O bond. The results could be signif-
icant with regard to our understanding of iron-catalyzed C–H hydroxylation, the observation of P450-
dependent peroxidation and the development of oxidative stress, especially for CYP2E1.
Ó 2010 Elsevier Inc. All rights reserved.
Introduction
notions of chemical reaction mechanisms, electronic structure and
reactivity [7–10].
Cytochrome P450 enzymes (CYP) comprise a large, important
and highly versatile class of heme-thiolate proteins [1]. Under-
standing the mechanisms of action of cytochrome P450 with its
substrates continues to be a central concern with regard to phase
I drug metabolism and xenobiotic toxicity [2,3]. Metabolites may
be toxic or they may have enhanced bioactivity [4]. Indeed, mod-
ern drug discovery requires the identification and characterization
of toxic metabolites early in the development pipeline [5,6]. CYP
enzymes are known to mediate a wide variety of oxidative trans-
formations, including N-, O- and S-dealkylations, heteroatom oxy-
genations, desaturations, deformylations and epoxidations.
However, the ability of CYPs to catalyze the oxygenation of even
strong C–H bonds, which is chemically the most extraordinary,
have received the greatest mechanistic [7] and theoretical [8]
attention. More generally, cytochrome P450 stands as a paradigm
of oxidative catalysis mediated by a first-row transition metal with
many lessons to offer regarding C–H bond activation and our basic
The consensus mechanism for C–H hydroxylation by cyto-
chrome P450 is shown in Scheme 1. There is abundant evidence
for the early stages of the reaction cycle, leading from the resting
ferric form of the heme site through the reduced, oxygenated, per-
oxo and hydroperoxo states. The accumulated spectroscopic evi-
dence has been extensively reviewed [7,9,11].
The oxygen rebound mechanism for P450-mediated hydroxyl-
ations, involving C–H abstraction by a ferryl heme intermediate
(compound I) first emerged from our studies with model iron sys-
tems [12,13]. In 1978 [14], we showed in collaboration with Jud
Coon that a reconstituted P450 system hydroxylated the simple
but informative hydrocarbon substrate norbornane at C2 with sig-
nificant loss of stereochemistry at the oxygenation site [15–17].
We interpreted this outcome to be indicative of a hydrogen
abstraction-radical recombination mechanism. In subsequent
years, aliphatic hydroxylations by P450 enzymes were found to
be accompanied by a range of molecular rearrangements support-
ing the idea of intermediate, but transient, carbon-centered radi-
cals. Modes of rearrangements that were observed include allylic
rearrangement [18], cyclopropyl-carbinyl-homoallyl rearrange-
ments [19], C–C fragmentation [20], and epimerizations [21]. In
⇑
Corresponding author.
E-mail address: jtgroves@princeton.edu (J.T. Groves).
0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2010.11.001

112
H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
O
the UV difference spectra used in these studies. Green has presented
R-H
R-H
O
O2
an analysis of these methods with the conclusion that the ferric-pro-
tein-like intermediate reported by Newcomb is not compound I of
P450 [33] Very recently, Rittle and Green have reported clear evi-
dence that the actual reactive intermediate of CYP119 does, indeed,
look like the oxoFe(IV) porphyrin radical cation (compound I) of
chloroperoxidase [34]. A high yield of the CYP119 compound I was
obtained by carefully tuning the peroxyacid oxidation of the resting
ferric enzyme. The authentic CYP119 compound I had a strongly
blue-shifted Soret band with diminished intensity and a broad
absorption near 700 nm, as is characteristic of oxoFe(IV) porphyrin
radical cations. The Mössbauer spectrum of freeze-quenched sam-
ples of the intermediate was similar to that of CPO compound I.
The EPR consistent with an S = 1 ferryl [Fe(IV)@O] spin coupled to
a porphyrin radical to afford a net S = 1/2 ground state. Significantly,
this CYP119compound I intermediate was able to hydroxylateunac-
tivated C–H bonds with very fast apparent rate constants in the
range of 104–107 Mꢁ1 sꢁ1. Clearly, the study of the nature of active
oxygen species involved in cytochrome P450 catalysis continues to
be a lively and informative field.
In the present study we have compared the reactions of several
P450 isozymes with the diagnostic substrate probe, tetramethylcy-
clopropane (TMCP)1. CYP3A4 is the most abundant P450 enzyme in
the human liver and is responsible for the phase I metabolism of
ꢂ50% of prescription drugs [35]. The P450 enzymes CYP2C9, 2C19,
2D6 and CYP3A4 together account for 95% of drug metabolism [36].
Human CYP2E1, found largely in hepatocytes and epithelial cells, is
known to oxidize a wide variety of low molecular weight xenobiotics
as well as signaling fatty acids such as arachidonic acid. CYP2E1
metabolism is largely responsible of the cytotoxicity of acetamino-
phen and is implicated in oxidative stress and liver damage resulting
from alcohol exposure [37,38]. Similarly, CYP2B1 is a drug metaboliz-
ing, phenobarbital-induced isozyme of rat liver microsomes.
Tetramethylcyclopropane was first used as a P450 substrate by
Ingold et al. in their pioneering development of radical clock sub-
strates [39,40]. The ring-opening rate constant for the cyclopro-
pylcarbinyl radical from TMCP was found to be moderately fast,
2 ꢀ 109 sꢁ1. Rat liver microsomes and a reconstituted CYP2B4 sys-
tem both gave about 1% ring-opened, homoallylic alcohol, in that
study, corresponding to an inferred radical lifetime of 4 ps in the
oxygen rebound process.
FeII
S
FeII
e-
S
e-
Cys
Cys
R-H
O
R-H
O
FeIII
S
FeIII
S
Cys
Cys
H
H2O
RH
H+
H
OH
peroxide shunt
HOOH
R-H
RH
O
O
FeIII
S
FeIII
uncoupling
S
Cys
Cys
ROH
*ROH
H2O
(R*)R
H
O
O
FeIV
FeIII
rebound
R
S
S
Cys
Cys
H
O
compound I
FeIV
S
cage escape
Cys
caged radical
H
H
O
O
FeIV
*R
R
FeIV
radical rearrangement
S
S
Cys
Cys
Scheme 1. Cytochrome P450 catalytic cycle, with emphasis on the oxygen rebound
step, showing the rearrangement of the radical intermediate, which could occur
either within the initial cage or after cage escape.
parallel model studies, we showed that synthetic oxoiron(IV) por-
phyrin radical cation complexes [22,23], as well as oxochromium,
oxomanganese and oxoruthenium analogs [10,24], could be pre-
paredandspectroscopicallycharacterizedbyavarietyoftechniques.
These studies showed that such species mimicked P450 reactivity in
a variety of ways. Significantly, recent studies with model, water-
soluble iron porphyrins have shown that a very reactive oxoiron(IV)
porphyrin radical cation [O@FeIVP+] could be prepared via peroxy-
acid oxidation of iron(III) precursors that are able to hydroxylate
C–H bonds at extraordinary rates (>106 Mꢁ1, sꢁ1) [25].
The reactive P450 intermediates after the ferric-hydroperoxo
state have proven to be much more elusive. Studies of P450 reac-
tions with peroxyacids by Egawa [26], Dawson and Ballou [27],
and Sligar [28] have supported the view that the oxidizing species
of P450 is a particularly reactive, thiolate-ligated, oxoiron(IV)
porphyrin radical cation [O@FeIVP+] analogous to the well-charac-
terized compound I of chloroperoxidase [29], although the prepa-
ration of the P450 compound I intermediate in those studies was
difficult to characterize. Newcomb et al. have offered evidence that
a reactive CYP119 species with a visible spectrum very much like
the resting ferric protein could be generated by laser flash
photolysis [30]. However, there is debate over the nature of the
precursor that was photolyzed, since the method of production
via peroxynitrite oxidation could lead either to a compound II
species, PFeIV–OH [31], or a ferric nitrosyl, FeIII–NO [32]. There
has also been debate regarding the method of data analysis for
The rationale for our reexamination of TMCP as a diagnostic P450
substrate was to see if this small substrate could afford evidence of
the radical cage phenomena we had seen in the non-heme diiron
hydroxylase AlkB [41]. In that case a slower radical clock produced
more rearranged product than the faster clock. Such a result can be
accommodated if cage-escape of an incipient radical is competitive
with rebound to afford hydroxylated product. The results show that
the apparent radical lifetime varies over two orders of magnitude
depending upon the particular P450 isozyme used. Significantly, a
new product, diacetone alcohol, was also detected. We compare
these results for the three CYP enzymes with a benchmark model
iron porphyrin/iodosylbenzene system, which afforded the same
products.
Results
Hydroxylation of tetramethylcyclopropane with CYP enzymes
1,1,2,2-Tetramethylcyclopropane 1 (TMCP) was reacted with
human CYP3A4 and CYP2E1, and rat CYP2B1 in a reconstituted sys-
tem that also contained P450 reductase and cytochrome b5 [42].
1
Abbreviations used: TMCP, tetramethylcyclopropane; NADPH, Nicotinamide ade-
nine dinucleotide phosphate; CYP, Cytochrome P450 enzymes.

H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
113
Nicotinamide adenine dinucleotide phosphate (NADPH) was used
as the electron donor. TMCP turned out to be a very good substrate
for the CYP enzymes employed here, with activities ranging from
90 to 2000 nmol/min/nmol P450, ꢂ5-fold more active than is typ-
ical for standard substrates. The structures of TMCP and the ob-
served products are depicted in Fig. 1. Products 2 and 3 were
identified by comparison to synthesized authentic standards. Prod-
ucts 4 and 5 were identified by comparison to commercially avail-
able materials. Yields were determined by comparing response
factors of reaction mixtures to these standards vs. dodecane as
an internal standard.
chloride solvent as we have previously described [43]. With the
FeIII-4-TMPyP catalyst the oxidation of TMCP gave 75% of the unrear-
ranged primary alcohol 2, 2% of the rearranged tertiary alcohol 4 and
12% of the autoxidation product 5 in a 1% overall conversion with ex-
cess substrate. A rearranged epoxide 6 (11%) was also detected
(Fig. 4). By contrast no diacetone alcohol (5) was detected with Ru(-
CO)TPFPP as the catalyst. These results afford an apparent radical
lifetime of 81 ps and a radical rebound rate of 1.2 ꢀ 1010 sꢁ1
(160 ps if 6 is counted as a rearrangement product).
Table 1 shows the results of oxidation of tetramethylcyclopro-
pane by the three CYP proteins. The GC-MS TIC traces of the indi-
vidual reactions are shown in Figs. 2 and 3. In the oxidation of
TMCP by CYP2E1, the unrearranged alcohol 2 was the major prod-
uct (80%). The only other product detected was a very significant
amount of diacetone alcohol 5 (20%). This product was completely
absent in control runs lacking NADPH (blue line). For the oxidation
of TMCP by rat CYP2B1, again the unrearranged alcohol 2 was the
major product (99.7%), along with 0.1% unrearranged aldehyde 3.
The rearranged alcohol 4 (0.2% relative yield) and diacetone alco-
hol 5 (trace) were also detected. As such, this run served as a neg-
ative control for the production of 5 since it had all of the
ingredients as the CYP2E1 run except a different P450 protein.
For the oxidation of TMCP by CYP3A4 (Fig. 3), the major product
was the unrearranged alcohol 2 (91%), accompanied by 1.5% unre-
arranged aldehyde 3. The rearranged alcohol 4 was present in 1.0%
relative yield, and 1.5% diacetone alcohol 5 was also detected.
In control experiments (full reaction mixture without NADPH),
products 4 and 5 were stable under the reaction conditions and
were not converted to any products. TMCP was filtered through ba-
sic alumina before use to ensure removal of oxygenated impurities.
Control reactions (either without enzyme, without NADPH or with-
out both) were free of any products including 5, which could be an
autoxidation product of TMCP. Each reaction was run in the pres-
ence of both catalase (1000 units) and MnSOD (1000 units). Under
these conditions the amount of diacetone alcohol product (5) was
reduced to 7.5% for the CYP2E1 reaction. There was no effect on the
CYP3A4 or CYP 2B1 reactions.
Discussion
Tetramethylcyclopropane 1 (TMCP) was a good substrate for
the three P450 isozymes investigated and the model iron porphy-
rin FeTMPyP in this study, affording reaction mixtures with two
unrearranged products, the alcohol 2 and the corresponding alde-
hyde 3; and products derived from cyclopropylcarbinyl-homo-allyl
rearrangement, the homoallylic alcohol 4 and diacetone alcohol
(5). From the ratios of (4 + 5)/(2 + 3) apparent radical lifetimes of
1-120 ps were calculated (Table 1), corresponding to radical re-
bound rates varying from 1.0 ꢀ 1012 to 0.9 ꢀ 1010 sꢁ1. These values
closely match the reported range for other aliphatic cyclopropanes
for P450-catalyzed hydroxylation reactions: bicyclopentane
(ꢂ50 ps) [19], methylcyclopropanes (4–100 ps) [39,40], norcarane
(16–50 ps) [44], thujone (8ꢂ142 ps) [21] and bicyclohexane
(74ꢂ252 ps) [45]. Our observed radical lifetime (1 ps) for rat
CYP2B1 was somewhat shorter than that reported by Ingold et al.
[39,40] (4.0 ps) for rat liver microsomes, which are known to con-
tain mostly CYP2B1 and CYP2B2. By contrast, the apparent radical
lifetimes reported for phenylmethylcyclopropanes are notably
shorter [46,47].
Diacetone alcohol (5) as an oxygen-trapped product
The appearance of diacetone alcohol (5) among the products
can be rationalized by trapping of the rearranged homo-allyl radi-
cal by molecular oxygen (Scheme 2). In the metalloporphyrin-
catalyzed oxidation of TMCP, product 5 was not formed in the
absence of oxygen or in the presence of oxygen-radical scavengers.
Control reactions without enzyme, without NADPH or without
both showed no or almost no production of 5, ruling out significant
contribution of autoxidation processes to its formation. Although,
diminished yields of 5 with CYP2E1 in the presence of catalase
and MnSOD suggest that some of this product may result from
uncoupling of the oxygen reduction process from substrate oxy-
genation (Scheme 1). However, TMCP is a rather stable substrate
and not prone to extensive spontaneous autoxidation due to the
strong methyl C–H bonds (97 kcal/mol) [48]. Indeed, the reaction
of CYP2B1 with TMCP in the presence of NADPH showed almost
no 5. Accordingly, the production of 5 is most probably due to
the action of the enzyme and not by non-enzymatic reactions or
side reactions.
Iron porphyrin-catalyzed oxidation of 1,1,2,2-
tetramethylcyclopropane (TMCP)
The oxidation of TMCP (1) by a model iron metalloporphyrin was
also investigated. The reaction was carried out using iron(III)
tetrakis-5,10,15,20-(N-methyl-4-pyridyl)-porphyrin (FeIII-4-TMPyP)
catalystand iodosylbenzene as the oxidant in acetonitrile [25]. As a
control of the analytical method, carbonylruthenium(II) tetrakis-
5,10,15,20-pentafluorophenyl-porphyrin (CO)RuII(TPFPP) was used
with 2,6-dichloropyridine N-oxide as the oxidant in methylene
OH
O
The role of CYP2E1 in the production of radicals
CYP2E1 is involved in the metabolism of low molecular weight
compounds such as ethanol, acetone, benzene and chlorobenzene
[49]. CYP2E1 is induced by ethanol, and is thought to be a major
contributor to liver disease among alcoholics via ethanol-induced
lipid peroxidation [38,50]. The observation of 5, the oxygen-
trapped product from the TMCP radical, among the CYP2E1 metab-
olites suggests that radicals are formed in the catalytic cycle of this
P450 enzyme. There is evidence that CYP2E1 is ‘leaky’, allowing
radicals to escape from the active site before the completion of
the oxygenation reaction. Radicals from a variety of substrates,
1
2
3
5
O
HO
HO
4
Fig. 1. Oxidation products from tetramethylcyclopropane (TMCP, 1).

114
H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
Table 1
Results of P450 catalyzed oxidation of TMCP.
Enzyme
Human CYP2E1
Human CYP3A4
Rat CYP2B1
Product
2
3
4
Yield (%)a
Yield (%)a
96
1.6
Yield (%)b
99.7
0.1
0.2
80
ndc
ndc
1.1
5
20
1.6
Trace
Conversion
Activityd
U/Re
Rebound rate
Radical lifetime
4%
90
4.5 1.5
17%
800
36
86%
2000
2
750 250
0.9 ꢀ 1010 0.3 ꢀ 1010 sꢁ1
120 40 ps
7.2 ꢀ 1010 0.4 ꢀ 1010 sꢁ1
1 ꢀ 1012 1 ꢀ 1011 sꢁ1
13 1 ps
1
0.3 ps
a
b
c
Average of 3 runs.
Average of 2 runs.
nd, not detected.
nmol/min/nmol P450.
Ratio of unrearranged (2 + 3) to rearranged (4 + 5) products.
d
E
Fig. 2. GC-MS total ion traces for the TMCP oxidation product region for (A) CYP2E1,
(B) CYP2B1. In each trace the product run is in black and the control run, without
NADPH, is in blue.
Fig. 3. GC-MS total ion trace for the TMCP oxidation product region for CYP3A4
(insets are 5ꢀ).
depressed. That observation, the fact that TMCP has strong C–H
bonds and the detection of 5 among the products with FeTMPyP/
iodosylbenzene argue that some of the substrate radicals derive
from turnover at the active site and may involve the reactive ferryl
species.
including ethanol [51–53], propanol and butanol [54], carbon tet-
rachloride [55], and ciprofloxacin [56], have been trapped with
spin traps such as 4-POBN [a-(4-pyridyl-1-oxide)-N-tert-butylnit-
rone], as shown by EPR. It has been inferred that the ethanol-de-
rived radicals may derive from the iron dioxygen complex of
CYP2E1 (FeIII–O–OÅ) [38]. However, it has also been suggested that
reactive oxygen species such as superoxide and hydrogen peroxide,
rather than substrate radicals, are escaping from the active site, as
evidenced by the fact that the addition of the antioxidant trolox
can suppress radical formation [56]. The present study does not al-
low a definitive assignment of the origin of the diacetone alcohol
product 5. However, carbon–carbon bond scission at the b-position
of alkoxy and peroxy radicals is a common process in olefin autox-
idation [57,58], which in this case would lead to 5 and formalde-
hyde. The presence of MnSOD and catalase did not prevent the
formation of 5 under conditions for which concentrations of both
superoxide and hydrogen peroxide should have been greatly
The crystal structure of CYP2E1 has been determined recently,
both with a small, coordinating molecule at the active site and a
longer fatty acid [59,60]. The substrate binding site is delineated
by the heme and five nearby phenylalanines (106, 116, 207, 298,
and 478) defining a volume of 190 Å3. A second, smaller cavity is
immediately adjacent, separated only by Phe298 and Phe106 and a
simple rotation of the phenyl appendage of Phe298 with no change
in the I-helix, which was observed upon binding of fatty acids, more
than double the size of the active site [60]. TMCP, with a molecular
volume of 160 Å3 fits within the active site cavity but not with much
room to spare (Fig. 5). However, rotation of Phe298 could explain the
apparent facility with which reduced oxygen species and substrate
radicals escape from the active site of CYP2E1.

H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
115
Fig. 5. Active site of CYP2E1 with
a
modeled substrate, tetramethylethylene
(medium blue). The figure illustrates constrictions between the active site, the
small distal void, and the substrate access channel (top left). The phenyl substituent
of F298 in the I helix (dark blue) forms a cap above the active site. Rotation of this
group by about 90°, which has been observed for longer substrates, opens the active
site to the substrate channel. The heme (light blue), iron (orange), axial thiolate
ligand (yellow) and threonine hydroxyl (red) are also depicted. Created with
MacPyMol from 3E61.pdb, cf. Refs. [55,56].
Fig. 4. Oxidation of TMCP oxidized by PhIO catalyzed by Fe-4-TMPyP in
acetonitrile.
R
R-H
H2O
R-H
H2O
H2O
H
O
FeIV
O
FeIV
O
FeIV
substrate C-H encounter
O2
S
S
S
Cys
Cys
Cys
compound I
O
O
HAT transition state
O2
*R-OO
oxygen trapping
R
R
*R
H2O
H2O
H2O
H
H
H
O
FeIV
O
FeIV
O
FeIV
cage escape
radical rearrangement
O
O
O
O
S
S
S
Cys
Cys
Cys
compound II with
H-bonded radical
O2
O2
H
*R
H
R
O
H2O
H2O
O
O
O
O
FeIII
FeIII
O
O
O
O
O
S
S
Cys
Cys
Scheme 3. Depiction of a cage recombination-cage escape scenario for oxygen
rebound by cytochrome P450. The intermediates, in sequence, are compound I
(oxoiron(IV) porphyrin radical cation) with the substrate and peroxide-derived
*
p –approach to the ferryl; the hydrogen
O
OH
water at the active site; C–H encounter via
abstraction (HAT) transition state; compound II with a hydrogen bond to the
incipient substrate radical RÅ; cage escape of the radical and rearrangement or
oxygen trapping; oxygen rebound to form either rearranged or unrearranged
product alcohol coordinated to the heme iron(III).
5
Scheme 2. Possible oxygen-trapping pathways to diacetone alcohol (5).
CYP2B1, bacterial P450CAM or P450BM3 [44], consistent with the re-
sults reported here.
Suppression of intramolecular deuterium isotope effects for
4,40-dimethylbiphenyl as compared to xylenes with CYP2E1 have
shown that there is not enough space in the active site for the long-
er molecule to turn, and therefore for the two ends were not full
competition with each other [61–63].
Thus, upon ring-opening of the substrate probe, the resulting
homo-allyl radical may encounter restrictions in turning to com-
plete the oxygen rebound process. Auclair et al. have reported that
for norcarane, CYP2E1 showed more rearrangement than did rat
Oxygen rebound as a cage recombination process
In light of the results presented here indicating that some
TMCP-derived radicals live long enough to be captured by diffusion
of molecular oxygen, especially for CYP2E1, it is pertinent to dis-
cuss the P450 C–H hydroxylation process in terms of a hydrogen
abstraction-radical cage recombination process, as depicted in
Scheme 1. To frame this discussion, the time scale of molecular
vibrations is in the range of 10–100 fs and the frequency of passage

116
H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
O
CH3
~50 ps
74~252 ps
16~50 ps
8~142 ps
1~ 120 ps
80~200 fs
phenylmethyl-
cyclopropane [46]
1 [39] and
this work
bicyclopentane [19] bicyclohexane [41] norcarane [44] thujone [21]
Fig. 6. Apparent lifetimes of intermediate cyclopropylcarbinyl radicals as determined from rearranged products in reactions mediated by cytochrome P450 enzymes.
of a molecular reaction such as hydrogen abstraction through a
transition state is less than 200 fs [64]. A well-studied example
of cage recombination and cage escape is the rebinding of NO to
the heme iron of myoglobin after photodissociation [65,66]. The bi-
phasic kinetics have been interpreted to result from very rapid NO
recombination from the distal pocket within 10 ps and a slower
(200 ps) binding process of NO molecules that have diffused into
neighboring protein cavities. Thus, cage escape of NO competes
with NO rebinding even though there is a negligible barrier to
the rebinding process. Statistical mechanics simulations have
shown that 5% of the NO molecules were still found in the distal
pocket and in these cavities even after 1 ns. The kinetic barrier to
this slower recombination phase has been associated with entropic
considerations, solvent effects and the dynamics of conformational
gating for diffusive return to the distal pocket.
depicted water molecule has been suggested to play a role in
lowering the barrier to the hydrogen atom transfer process
through hydrogen bonding [8]. Completion of the hydrogen
atom transfer from carbon to the ferryl oxygen will produce a
hydroxoiron(IV) intermediate (compound II) [72]. The strength
of the newly formed FeIVO–H bond of P450 compound II can
be estimated to be ꢂ100 kcal/mol based on the rates of hydro-
gen atom abstraction of a model oxoiron(IV) porphyrin cation
radical [25]. Such a mechanism would suggest that the incipient
substrate carbon radical would be hydrogen bonded to the oxy-
gen of compound II. Indeed, experimental [73] and computa-
tional evidence for hydrogen bonds to alkyl radicals, especially
acidic H-bond donors [74], has been reported. The major interac-
tion has been attributed to charge transfer from the carbon rad-
*
ical into the H-bond donor O–H
r orbital. Thus, one expects the
A similar scenario can be envisioned for C–H hydroxylation by
iron-containing oxygenases. The diiron alkane hydroxylase (AlkB)
is a non-heme iron enzyme that is thought to have a sterically re-
stricted active site, since it is designed to hydroxylate the terminal
methyl group of n-alkanes [67]. AlkB shows a large amount of rear-
ranged products with typical mechanistic probes such as norcarane
and bicyclohexane, indicating a long-lived (ꢂ10 ns) radical inter-
mediate [41]. A revealing aspect of that study was the observation
that bicyclohexane, which is a relatively slow radical clock, affor-
ded more radical rearranged product than norcarane. While this
result is inconsistent with the typical manner that radical clock
data is interpreted, the outcome is fully consistent with a compe-
tition between oxygen rebound and cage escape, or recoil
[68,69], of the incipient radical, since the recoil/rebound rate ratio
will determine the extent of radical rearrangement. Such a process
has the effect of masking the radical ring-opening rate. A similar
substrate carbon radical to be stabilized by the hydrogen bond
to the hydroxyl of compound II as in [R—HO–FeIVP]. Completion
of the oxygen rebound process would appear to require a dis-
ruption of this hydrogen bond, such as by cage escape of the
radical. As is typical of such stochastic processes, some radicals
would escape the cage and rearrange while others would not.
Further, the small confines of the active site, as is seen in
CYP2E1, may make reencounter of the escaped radical with com-
pound II unfavorable with respect to trapping by molecular oxy-
gen. A hydrogen bond to the intermediate substrate radical
could also explain the very short apparent radical lifetimes ob-
served for phenylmethylcyclopropane and similar substrates,
since
a strong hydrogen bond could drastically change the
ring-opening rate.
Å
phenomenon is observed in the cage escape of NO2 from [FeIV@O
Summary and conclusions
ÅNO2] at the active site of myoglobin during its reaction with per-
oxynitrite [70,71].
We have examined the behavior of tetramethylcyclopropane as
a diagnostic substrate with three important drug-metabolizing
cytochrome P450 isozymes – human CYP2E1, human CYP3A4
Applied to the cytochrome P450 enzymes in this study, CYP2B1
has shown a very short radical lifetime (1–4 ps) but with rear-
ranged products that were still detectable. Longer apparent radical
lifetimes of 13 ps for CYP3A4 and 120 ps for Cyp2E1, as well as
81 ps for the model iron porphyrin, FeTMyP, are all in the same
time zone for recombination as those observed for NO recombina-
tion to myoglobin. But it is important to recognize that these rad-
ical lifetimes are all averages, with some radicals that have escaped
the cage living very much longer, which could allow for trapping
by freely diffusing molecular oxygen.
A more detailed description of the oxygen rebound/cage es-
cape scenario is shown in Scheme 3. The sequence begins with
the formation of the oxoiron(IV) porphyrin radical cation (com-
pound I) intermediate (Scheme 1) with the substrate in place.
The nearby water molecule is the one that is mechanistically de-
rived from O–O bond heterolysis of the hydroperoxoiron(III) pre-
cursor (compound 0). The hydrogen abstraction is initiated by
and rat CYP2B1. The ring-opening rate of TMCP (2 ꢀ 109 Mꢁ1 sꢁ1
)
has revealed radical lifetimes over the range of 1–120 ps. Signifi-
cantly, the model iron porphyrin FeTMPyP/iodosylbenzene system
showed amounts of substrate rearrangement commensurate with
an 81 ps radical lifetime with this same substrate. The similarity
of the observed behavior of the model system and the active en-
zymes indicate that similar mechanisms and similar intermediates
are involved in both cases. These values compare well with radical
lifetimes on the order of 1–250 ps observed with a variety of en-
zymes and radical clock substrates (Fig. 6). The large variation in
apparent radical lifetime observed for TMCP with these CYP en-
zymes shows that subtle differences in the active sites can signifi-
cantly change the outcome. Both CYP2E1 and the model iron
porphyrin catalyst produced a significant amount of an oxygen-
trapped product, diacetone alcohol (5). Cage escape of the radical
intermediate competing with radical recombination is suggested
to explain these results.
approach of the scissile C–H bond to the singly-occupied Fe–O
*
p
orbital leading to the HAT transition state. Interestingly, the

H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
117
[9] P.R. Ortiz de Montellano, J.J. De Voss, in: P.R. Ortiz de Montellano (Ed.),
Cytochrome P450 Structure, Mechanism and Biochemistry, Klewer Academic/
Plenum, New York, 2005, pp. 183–245.
[10] J.T. Groves, in: P.R. Ortiz de Montellano (Ed.), Cytochrome P450: Structure,
Mechanism, and Biochemistry, Klewer Academic/Plenum, New York, 2005, pp.
1–44.
[11] I.G. Denisov, T.M. Makris, S.G. Sligar, I. Schlichting, Chem. Rev. 105 (2005)
2253–2277.
[12] J.T. Groves, G.A. McClusky, J. Am. Chem. Soc. 98 (1976) 859–861.
[13] J.T. Groves, M. Van Der Puy, J. Am. Chem. Soc. 98 (1976) 5290.
[14] J.T. Groves, G.A. McClusky, R.E. White, M.J. Coon, Biochem. Biophys. Res.
Commun. 81 (1978) 154–160.
Experimental
General
Tetramethylcyclopropane and authentic standards for their oxi-
dation products 2–6 were purchased or synthesized by literature
methods.
Cytochrome P450 assays
[15] J.T. Groves, S. Krishnan, G.E. Avaria, T.E. Nemo, Illus (1980) P277–290.
[16] J.T. Groves, Proc. Natl. Acad. Sci. USA 100 (2003) 3569–3574.
[17] J.T. Groves, J. Inorg. Biochem. 100 (2006) 434–447.
[18] J.T. Groves, D.V. Adhyam, J. Am. Chem. Soc. 106 (1984) 2177–2181.
[19] P.R. Ortiz de Montellano, J. Am. Chem. Soc. 109 (1987) 3415–3420.
[20] V. Stanjek, M. Miksch, P. Lueer, U. Matern, W. Boland, Angew. Chem. Int. Ed. 38
(1999) 400–402.
[21] Y.Y. Jiang, X. He, P.R. Ortiz de Montellano, Biochemistry 45 (2006) 533–542.
[22] J.T. Groves, T.E. Nemo, R.S. Myers, J. Am. Chem. Soc. 101 (1979) 1032–1033.
[23] J.T. Groves, R.C. Haushalter, M. Nakamura, T.E. Nemo, B.J. Evans, J. Am. Chem.
Soc. 102 (1981) 2884–2886.
[24] J.T. Groves, W.J. Kruper, R.C. Haushalter, J. Am. Chem. Soc. 102 (1980) 6377–
6380.
[25] S.R. Bell, J.T. Groves, J. Am. Chem. Soc. 131 (2009) 9640–9641.
[26] T. Egawa, H. Shimada, Y. Ishimura, Biochem. Biophys. Res. Commun. 201
(1994) 1464–1469.
Cytochrome P450 enzymes were purchased in insect cell micro-
somes from BD Biosciences (Woburn, MA). The microsomes also
contained cytochrome P450 reductase (activity 2200 nmol/
(min mg protein)) and cytochrome b5 (610 pmol/mg protein). For
each reaction, 100 lL of Supersomes containing 100 pmol P450,
540 pmol cytochrome b5, cytochrome P450 reductase of
200 nmol/min activity as received was added to a mixture of buffer
(0.5 mL, 100 mM potassium phosphate, pH 7.4), NADPH (8 mg,
10
bated in a water bath at 37 °C for 30 min. The reaction was stopped
by the addition of dichloromethane (250 L) and centrifuged for
lmol), and substrate (1 lL, 7 lmol). This mixture was incu-
l
[27] T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309.
[28] D.G. Kellner, S.C. Hung, K.E. Weiss, S.G. Sligar, J. Biol. Chem. 277 (2002) 9641–
9644.
three minutes at 13,750 rpm. The organic layer was separated
and dried with MgSO4, then subjected to GC-MS analysis.
[29] K.L. Stone, R.K. Behan, M.T. Green, Proc. Natl. Acad. Sci. USA 102 (2005) 16563–
16565.
[30] Q. Wang, X. Sheng, J.H. Horner, M. Newcomb, J. Am. Chem. Soc. 131 (2009)
10629–10636.
Oxidation of TMCP by synthetic metalloporphyrins
The substrate 1 (10
stock solution of the porphyrin (65
l
L) in acetonitrile (200
lL) was added to a
[31] M. Newcomb, J.A. Halgrimson, J.H. Horner, E.C. Wasinger, L.X. Chen, S.G. Sligar,
Proc. Natl. Acad. Sci. USA 105 (2008) 8179–8184.
[32] R.K. Behan, L.M. Hoffart, K.L. Stone, C. Krebs, M.T. Green, J. Am. Chem. Soc. 129
(2007) 5855–5859.
[33] J. Rittle, J.M. Younker, M.T. Green, Inorg. Chem. 49 (2010) 3610–3617.
[34] J. Rittle, M.T. Green, Science 330 (2010) 933–937.
[35] T. Shimada, H. Yamazaki, M. Mimura, Y. Inui, F. Guengerich, J. Pharmacol. Exp.
Ther. 270 (1994) 414–423.
[36] J.A. Williams, R. Hyland, B.C. Jones, D.A. Smith, S. Hurst, T.C. Goosen, V.
Peterkin, J.R. Koup, S.E. Ball, Drug Metab. Dispos. 32 (2004) 1201–1208.
[37] D.T. Trafalis, E.S. Panteli, A. Grivas, C. Tsigris, P.N. Karamanakos, Exp. Opin.
Drug Metabol. Toxicol. 6 (2010) 307–319.
lL of 5.6 mM in acetonitrile
[3.93 mg/mL Fe-4-TMPyP]), which was purchased from Mid-Cen-
tury Chemical. Solid iodosylbenzene (5 mg) was added in five por-
tions over 15 min with efficient stirring, then stirred for an
additional 15 min. The reaction mixture was filtered through a
short plug of silica and activated charcoal, which was rinsed with
dichloromethane (200 lL). The solution was diluted for GC-MS
(around 1:100). The ruthenium porphyrin catalyst, Ru(TPFPP)(CO),
was prepared and the oxidation carried out as previously described
[43].
[38] M. Comporti, C. Signorini, S. Leoncini, C. Gardi, L. Ciccoli, A. Giardini, D.
Vecchio, B. Arezzini, Genes Nutr. 5 (2010) 101–109.
[39] J.K. Atkinson, K.U. Ingold, Biochemistry 32 (1993) 9209–9214.
[40] V.W. Bowry, K.U. Ingold, J. Am. Chem. Soc. 113 (1991) 5699–5707.
[41] R.N. Austin, K. Luddy, K. Erickson, M. Pender-Cudlip, E. Bertrand, D. Deng, R.S.
Buzdygon, J.B. van Beilen, J.T. Groves, Angew. Chem. Int. Ed. 47 (2008) 5232–
5234.
[42] D. Pompon, Biochemistry 26 (2002) 6429–6435.
[43] C.Q. Wang, K.V. Shalyaev, M. Bonchio, T. Carofiglio, J.T. Groves, Inorg. Chem. 45
(2006) 4769–4782.
[44] K. Auclair, Z.B. Hu, D.M. Little, P.R. Ortiz de Montellano, J.T. Groves, J. Am.
Chem. Soc. 124 (2002) 6020–6027.
[45] R.N. Austin, D.Y. Deng, Y.Y. Jiang, K. Luddy, J.B. van Beilen, P.R. Ortiz de
Montellano, J.T. Groves, Angew. Chem. Int. Ed. 45 (2006) 8192–8194.
[46] M. Newcomb, P.F. Hollenberg, M.J. Coon, Arch. Biochem. Biophys. 409 (2003)
72–79.
GC-MS analysis
GC-MS analyses were run using an Agilent 7890A GC coupled to
a 5975C Inert MSD with a Rtx-5Sil MS (30 m length, 0.25 mm ID,
0.25 lm film) column. Two GC-MS thermal gradient methods were
used: Method 1 started at 30 °C, holding for 6 min, ramping to
230 °C at 10 °C/min and Method 2 started at 50 °C, holding for
2 min, ramping to 230 °C at 10 °C/min. Dodecane (13 ng/mL) was
added as an internal standard.
[47] M. Newcomb, P.H. Toy, Acc. Chem. Res. 33 (2000) 449–455.
[48] R.D. Bach, O. Dmitrenko, J. Am. Chem. Soc. 126 (2004) 4444–4452.
[49] F.P. Guengerich, D.H. Kim, M. Iwasaki, Chem. Res. Toxicol. 4 (1991) 168–179.
[50] Z.Y. Cheng, Y.Z. Li, Chem. Rev. (Washington, D.C., United States) 107 (2007)
748–766.
Acknowledgment
We are grateful for support of this research by the National
Institutes of Health (2R37 GM036298).
[51] L.A. Reinke, E.K. Lai, C.M. Dubose, P.B. McCay, Proc. Natl. Acad. Sci. USA 84
(1987) 9223–9227.
[52] E. Albano, A. Tomasi, L. Goria-Gatti, M.U. Dianzani, Chem. Biol. Interact. 65
(1988) 223–234.
[53] K. Minakata, E. Okuno, M. Nakamura, H. Iwahashi, J. Biochem. 142 (2007) 73–
78.
[54] E. Albano, A. Tomasi, J.O. Persson, Y. Terelius, L. Goriagatti, M.
Ingelmansundberg, M.U. Dianzani, Biochem. Pharmacol. 41 (1991) 1895–1902.
[55] D.A. Stoyanovsky, A.I. Cederbaum, Chem. Res. Toxicol. 12 (1999) 730–736.
[56] A. Gurbay, B. Gonthier, D. Daveloose, A. Favier, F. Hincal, Free Radic. Biol. Med.
30 (2001) 1118–1121.
[57] A. Boto, C. Betancor, E. Suarez, Tetrahedron Lett. 35 (1994) 5509–5512.
[58] E.R. Birnbaum, J.A. Labinger, J.E. Bercaw, H.B. Gray, Inorg. Chim. Acta 270
(1998) 433–439.
References
[1] P.R. Ortiz de Montellano, Cytochrome P450: Structure, Mechanism and
Biochemistry, Academic/Plenum Publishers, New York, 2004.
[2] H. Veith, N. Southall, R.L. Huang, T. James, D. Fayne, N. Artemenko, M. Shen, J.
Inglese, C.P. Austin, D.G. Lloyd, D.S. Auld, Nat. Biotechnol. 27 (2009) 1050–
U1123.
[3] F.P. Guengerich, Chem. Biol. 16 (2009) 1215–1216.
[4] D.C. Lamb, M.R. Waterman, S.L. Kelly, F.P. Guengerich, Curr. Opin. Biotechnol.
18 (2007) 504–512.
[5] R.S. Obach, Chem. Res. Toxicol. 23 (2010) 42.
[59] P.R. Porubsky, K.M. Meneely, E.E. Scott, J. Biol. Chem. 283 (2008) 33698–33707.
[60] P.R. Porubsky, K.P. Battaile, E.E. Scott, J. Biol. Chem. 285 (2010) 22282–22290.
[61] K.R. Iyer, J.P. Jones, J.F. Darbyshire, W.F. Trager, Biochemistry 36 (1997) 7136–
7143.
[6] C.B. Frederick, R.S. Obach, Clin. Pharmacol. Ther. 87 (2010) 345–350.
[7] P.R. Ortiz de Montellano, Chem. Rev. 110 (2010) 932–948.
[8] S. Shaik, S. Cohen, Y. Wang, H. Chen, D. Kumar, W. Thiel, Chem. Rev. 110 (2010)
949–1017.

118
H.L.R. Cooper, J.T. Groves / Archives of Biochemistry and Biophysics 507 (2011) 111–118
[62] C. Audergon, K.R. Iyer, J.P. Jones, J.F. Darbyshire, W.F. Trager, J. Am. Chem. Soc.
121 (1999) 41–47.
[68] V. Guallar, M.H. Baik, S.J. Lippard, R.A. Friesner, Proc. Natl. Acad. Sci. USA 100
(2003) 6998–7002.
[63] J.P. Harrelson, K.R. Henne, D.O.V. Alonso, S.D. Nelson, Biochem. Biophys. Res.
Commun. 352 (2007) 843–849.
[69] V. Guallar, B.F. Gherman, W.H. Miller, S.J. Lippard, R.A. Friesner, J. Am. Chem.
Soc. 124 (2002) 3377–3384.
[64] A.H. Zewail, J. Phys. Chem. A 104 (2000) 5660–5694.
[65] D. Ionascu, F. Gruia, X. Ye, A. Yu, F. Rosca, C. Beck, A. Demidov, J.S. Olson, P.M.
Champion, J. Am. Chem. Soc. 127 (2005) 16921–16934.
[66] R.A. Goldbeck, S. Bhaskaran, C. Ortega, J.L. Mendoza, J.S. Olson, J. Soman, D.S.
Kliger, R.M. Esquerra, Proc. Natl. Acad. Sci. USA 103 (2006) 1254–1259.
[67] J.B. van Beilen, T.H.H. Smits, F.F. Roos, T. Brunner, S.B. Balada, M. Röthlisberger,
B. Witholt, J. Bacteriol. 187 (2005) 85–91.
[70] J. Su, J.T. Groves, Inorg. Chem. 49 (2010) 6317–6329.
[71] J. Su, J.T. Groves, J. Am. Chem. Soc. 131 (2009) 12979–12988.
[72] R.K. Behan, L.M. Hoffart, K.L. Stone, C. Krebs, M.T. Green, J. Am. Chem. Soc. 128
(2006) 11471–11474.
´
[73] J.M. Merritt, S. Rudic, R.E. Miller, Chem. Phys. 124 (2006) 084301.
[74] S. Hammerum, J. Am. Chem. Soc. 131 (2009) 8627–8635.