Cytochrome P450-catalyzed hydroxylation of mechanistic probes that distinguish between radicals and cations. Evidence for cationic but not for radical intermediates

Article abstract of DOI:10.1021/ja994106+

Oxidation of the mechanistic probes trans,trans-2-methoxy-3- phenylmethylcyclopropane and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that different structural rearrangeme

Full text of DOI:10.1021/ja994106+

VOLUME 122, NUMBER 12
MARCH 29, 2000
©
Copyright 2000 by the
American Chemical Society
Cytochrome P450-Catalyzed Hydroxylation of Mechanistic Probes
that Distinguish between Radicals and Cations. Evidence for Cationic
but Not for Radical Intermediates
Martin Newcomb,*, Runnan Shen,1a Seung-Yong Choi, Patrick H. Toy,
1a
1a
1a
Paul F. Hollenberg,*, Alfin D. N. Vaz, and Minor J. Coon*
1b
1c
,1c
Contribution from the Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202,
Department of Pharmacology, The UniVersity of Michigan, Ann Arbor, Michigan 48109, and Department
of Biological Chemistry, The UniVersity of Michigan, Ann Arbor, Michigan 48109
ReceiVed NoVember 23, 1999
Abstract: Oxidation of the mechanistic probes trans,trans-2-methoxy-3-phenylmethylcyclopropane and
methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical
and cationic species in that different structural rearrangements occur for the two types of intermediates. The
P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from
rabbit), the expressed truncated isozymes P450 ∆2B4 and P450 ∆2E1 (ethanol-inducible, from rabbit), and
mutants of the latter two in which an active site threonine was replaced with alanine, ∆2B4 T302A, and ∆2E1
T303A. Cationic rearrangement products were found from both probes. Oxidations of trans,trans-2-methoxy-
3
-phenylmethylcyclopropane gave small amounts of radical-derived rearrangement products indicating that
hydroxylation occurs via insertion reactions with transition state lifetimes in the 80-200 fs range. A mechanistic
description of cytochrome P450-catalyzed hydroxylations that is in accord with the present and previous radical
probe results is presented. This description incorporates the recent demonstrations that two electrophilic oxidants
are produced in the natural course of P450 oxidation reactions and that both electrophilic oxidant forms can
effect hydroxylation reactions. Following production of a peroxo-iron species, protonation gives a hydroperoxo-
iron species. Protonation of the hydroperoxo-iron species gives an iron-oxo species and water. Hydroxylations
by both the hydroperoxo-iron and iron-oxo species occur by insertion reactions. The hydroperoxo-iron species
+
inserts the elements of OH producing protonated alcohol products that can react in solvolysis-type reactions
to give cationic rearrangement products. The iron-oxo species reacts by insertion of an oxygen atom.
The cytochrome P450 enzymes (P450s) are ubiquitous in
nature and effect numerous oxidations of physiologically
important natural and foreign substrates including the remark-
ably difficult hydroxylation of unactivated C-H bonds in
from protein cysteine serving as a fifth ligand to iron. Many of
the steps in the sequence of the oxidation reactions are well
characterized, but the identity of the final oxidant(s) and the
reaction mechanisms are still poorly understood. The well-
characterized portion of the sequence involves substrate binding,
reduction of the resting ferric form of the enzyme to a ferrous
state, binding of oxygen to give a superoxide complex, and a
second reduction step. Although subsequent oxidation of
substrate is fast, attempts at spectral identification of the ultimate
2
hydrocarbons and other compounds. The engine for oxidation
is an iron-protoporphyrin IX complex (heme) with a thiolate
(1) (a) Wayne State University. (b) Department of Pharmacology,
University of Michigan. (c) Department of Biological Chemistry, University
of Michigan.
(2) Cytochrome P450 Structure, Mechanism, and Biochemistry, 2nd ed.;
Ortiz de Montellano, P. R., Ed.; Plenum: New York, 1995.
(3) Blake, R. C., II; Coon, M. J. J. Biol. Chem. 1989, 264, 3694-3701.
1
0.1021/ja994106+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/11/2000

2678 J. Am. Chem. Soc., Vol. 122, No. 12, 2000
Newcomb et al.
Figure 1. The hydrogen abstraction, oxygen-rebound mechanism for
P450-catalyzed hydroxylation. Abstraction of hydrogen atom by the
iron-oxo species is followed by a homolytic displacement of OH that
returns the resting enzyme.
oxidant in P4503,4 and the related nitric oxide synthase have
met with partial success, and recent developments in transient
state crystallographic techniques appear to have identified such
5
Figure 2. Substrate 1 and its putative products from P450-catalyzed
oxidations.
6
a species in the reaction cycle of P450cam. The formal addition
of two electrons and two protons to oxygen produces the
equivalent of hydrogen peroxide, and these monooxygenases
produce water in addition to the oxidized substrate.
As the mechanistic picture became more complicated, so also
did the view of the active oxidant(s) in P450. Studies with
expressed P450 isozymes and their mutants lacking a conserved
threonine in the active site indicated that multiple oxidizing
species are produced in the natural course of P450 oxida-
The active electrophilic oxidant in P450 usually has been
assumed to be a high-valent iron-oxo species, structurally similar
to the intermediate Compound I that is known to form in
reactions of the heme-containing peroxidase enzymes with
13,14
tions.
That work implicated two electrophilic oxidant species,
one a preferential epoxidizing agent and the other a preferential
hydroxylating agent, and a subsequent study indicated that both
of these could effect hydroxylation reactions when epoxidation
7
hydrogen peroxide. A consensus view of the mechanism of
P450-catalyzed hydroxylations by an iron-oxo species evolved
over the past two decades, primarily based on the results of
1
5
was not possible.
2
,7
mechanistic probe studies and kinetic isotope effects. In that
mechanism, the iron-oxo species abstracts a hydrogen atom from
substrate to give an iron-hydroxy species and an alkyl radical
intermediate; the alkyl radical then displaces hydroxy from iron
In this work, we report oxidations of two hypersensitive
radical probe/clock substrates with wild-type and mutant P450
isozymes. In their capacity as mechanistic probes, both substrates
have the potential to differentiate between radical and cationic
intermediates, and cationic rearrangement products were found
from both. One substrate also serves as a radical clock, and in
this mode we find “radical” lifetimes that are too short for a
true radical intermediate. We present a mechanistic description
of P450-catalyzed hydroxylation reactions that is consistent with
the present and previous results. The new features of this
description are (1) two electrophilic oxidants, a hydroperoxo-
iron complex and an iron-oxo complex, effect hydroxylations
via insertion processes and (2) cationic rearrangements occur
from solvolytic-type reactions of protonated alcohols, the first-
formed products from hydroxylation by the hydroperoxo-iron
species.
8
in a process termed “oxygen-rebound” (Figure 1).
Results of the past few years indicate that the hydroxylation
reaction is more complex than previously thought. The mecha-
9
nistic picture began to cloud when ultrafast “radical clocks”
were used in attempts to “time” the oxygen rebound step; the
amounts of rearranged products did not correlate with the radical
rearrangement rate constants. Moreover, hydroxylation of a
probe that could distinguish between radical and cationic species
by one P450 isozyme indicated that cationic rearrangements
were complicating studies with probes/clocks, and the results
suggested that hydroxylation occurred by an insertion reaction
instead of abstraction and recombination.10 The latter study
presented a new paradox in that it provided evidence that cations
could be formed in hydroxylation reactions whereas it is well
established that carbocations cannot be requisite intermediates.
As discussed later, possible explanations for these results were
presented recently.1
Results
Substrates and Oxidation Products. One of the probe
substrates used in this work was trans,trans-2-methoxy-3-
phenylmethylcyclopropane (1) which is shown in Figure 2 with
its oxidation products. The mechanisms of the rearrangement
reactions are discussed later. Oxidation of the cyclopropyl
methyl group in 1 can give the unrearranged alcohol 2, two
diastereomers of benzylic alcohol 3 derived from rearrangement
of a cyclopropylcarbinyl radical, and aldehyde 4 derived from
rearrangement of a cyclopropylcarbinyl cation. Oxidation of the
phenyl group in probe 1 would give phenol 5 or other
regioisomeric phenols. Oxidation of the methoxy methyl group
in probe 1 would produce a formaldehyde hemiacetal that
hydrolyzes to give the demethylated cyclopropanol product, but
we did not attempt to identify this product.
0-12
(
4) Egawa, T.; Shimada, H.; Ishimura, Y. Biochem. Biophys. Res.
Commun. 1994, 201, 1464-1469.
5) Bec, N.; Gorren, A. C. F.; Voelker, C.; Mayer, B.; Lange, R. J. Biol.
Chem. 1998, 273, 13502-13508.
6) Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Sweet, R. M.;
(
(
Ringe, D.; Petsko, G. A.; Davies, M.; Mueller, E. J.; Benson, D.; Sligar, S.
FASEB J. 1997, 11, A769. Schlichting, I.; Berendzen, J.; Chu, K.; Stock,
A. M.; Maves, S. A.; Benson, D. E.; Sweet, R. M.; Ringe, D.; Petsko, G.
A.; Sligar, S. G. Science. In press.
(
7) White, R. E.; Coon, M. J. Annu. ReV. Biochem. 1980, 49, 315-356.
Groves, J. T.; Han, Y.-Z. In Cytochrome P450 Structure, Mechanism and
Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum: New York,
995; pp 3-48. Guengerich, F. P.; Macdonald, T. L. FASEB J. 1990, 4,
(12) Shaik, S.; Filatov, M.; Schroder, D.; Schwarz, H. Chem. Eur. J.
1998, 4, 193-199.
(8) Groves, J. T.; McClusky, G. A. J. Am. Chem. Soc. 1976, 98, 859-
(13) Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 3555-3560.
(
9) Griller, D.; Ingold, K. U. Acc. Chem. Res. 1980, 13, 317-323.
(10) Newcomb, M.; Le Tadic-Biadatti, M. H.; Chestney, D. L.; Roberts,
(14) Vaz, A. D. N.; Pernecky, S. J.; Raner, G. M.; Coon, M. J. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 4644-4648.
E. S.; Hollenberg, P. F. J. Am. Chem. Soc. 1995, 117, 12085-12091.
11) Collman, J. P.; Chien, A. S.; Eberspacher, T. A.; Brauman, J. I. J.
Am. Chem. Soc. 1998, 120, 425-426.
(
(15) Toy, P. H.; Newcomb, M.; Coon, M. J.; Vaz, A. D. N. J. Am. Chem.
Soc. 1998, 120, 9718-9719.

Cytochrome P450-Catalyzed Hydroxylations
J. Am. Chem. Soc., Vol. 122, No. 12, 2000 2679
Scheme 1a
in the preparation of 6,19 and compound 9 is produced by acid-
2
0
catalyzed rearrangement of 8. The regioisomeric methylcu-
banols 7 were detected as products from oxidation of 6 by a
2
1
methane monooxygenase (MMO) system, but they were not
isolated. In the P450-catalyzed oxidations of 6 conducted in
this work, we observed by GC three products that had mass
spectra the same as those reported for compounds 7 in the MMO
2
1
study in addition to products 8 and 9.
The identities of the products 7, as well as those of products
8
and 9, were verified by treatment of the P450 product mixtures
with acetic anhydride and pyridine to give the corresponding
acetates followed by GC and mass spectral analyses. Oxidation
of probe 6 by one P450 isozyme, specifically P450 2B1,
followed by derivatization to the acetates was previously shown
to give the acetates 7a-OAc, 7c-OAc and 8-OAc, as determined
by comparison to authentic samples, and another acetate
a
3 3 2 3 2
Conditions: (a) CH OCH(Li)P(Ph) ; (b) Et Zn, CH CHI ; (c) NaSEt.
Products 2, 3, and 4 were identified in product mixtures from
P450 oxidations of probe 1 by comparison of the GC retention
times and mass spectral fragmentation patterns to those of
authentic samples. Compounds 2 and 4 were previously
known. A mixture of diastereomers of 3 was prepared by
reaction of a methoxyallyl-tin reagent with benzaldehyde by
16
10
1
9
product. The fourth acetate product found in that work was
identified as 7b-OAc on the basis of the similarity of its GC
retention time and mass spectral fragmentation pattern to those
1
7
the general procedure reported by Koreeda and Tanaka.
1
9
of acetates 7a-OAc and 7c-OAc. The same acetates were
obtained from derivatization of the product mixtures obtained
with all P450 isozymes used in this work. In addition, acetate
An authentic sample of phenol 5 was not isolated as a pure
compound, but it was possible to demonstrate that phenol 5
was not a significant product in the P450-catalyzed oxidations
of substrate 1. A mixture of 5 and its three diastereomeric
isomers was prepared by the method shown in Scheme 1. Thus,
reaction of p-anisaldehyde with the methoxymethyl Wittig
reagent gave a ca. 1:1 mixture of cis- and trans-1-methoxy-2-
9-OAc was present after derivatization of product mixtures from
reactions with the ∆2B4 and ∆2E1 isozymes and their mutants;
an authentic sample of 9-OAc was prepared from 9.
From analyses of the alcohol products from 6 and their acetate
derivatives, we conclude that cubanols 7 were partially decom-
posed during the GC analysis. In several cases, the GC peak
shapes for 7 were not symmetrical, but more importantly, the
ratios of products 7 to product 8 obtained from the analyses of
the alcohols and the acetates were not the same. Furthermore,
product 9 appeared to be partially decomposed in the acetate
derivatization procedure; again, this was deduced from the ratios
of products observed before and after derivatization. Because
the ratios of the products from oxidation of the methyl group
in substrate 6 were most important for this work, we quantitated
the alcohol mixtures with the result that the yields of cubanols
(
p-methoxyphenyl)ethene that was treated with an ethyl car-
benoid reagent to give a mixture of four diastereomeric products.
Demethylation of these compounds with sodium ethylthiolate
gave 5 and its diastereomers which were isolated as a mixture.
GC analysis of the mixture showed four components with
baseline resolution, all with similar mass spectral fragmentation
patterns and strong molecular ions. GC analyses of the P450
oxidation products of 1 demonstrated that none of the compo-
nents of the mixture containing 5 was present in significant
amounts, and separation of 5 from its diastereomers was deemed
pointless.
7
discussed below are variable. We emphasize that the ratios
The second probe substrate used in this work was methyl-
cubane (6) which is shown below with its possible oxidation
products. Again, the mechanisms of the rearrangement reactions
of products from methyl group oxidation of substrate 6 (i.e.
products 8 and 9) are not affected by partial loss of products 7.
P450-Catalyzed Oxidation Reactions. Probes 1 and 6 were
oxidized by various hepatic P450 isozymes and mutants. P450
2
B1 and P450 2B4 are the phenobarbital-inducible isozymes
from rat and rabbit, respectively. The truncated isozyme P450
2B4 is the expressed rabbit liver enzyme with N-terminal
amino acids 2-27 deleted, and P450 ∆2B4 T302A is the Thr-
∆
14
302 to Ala mutant of this isozyme. P450 ∆2E1 is the expressed
rabbit liver ethanol-inducible isozyme with N-terminal amino
acids 3-29 deleted, and P450 ∆2E1 T303A is the Thr-303 to
are discussed later. Oxidation of the cubyl positions in 6 gives
three regioisomeric methylcubanol products (7). Oxidation of
the methyl group in 6 could give unrearranged cubylmethanol
13
Ala mutant of this isozyme.
The enzyme-catalyzed oxidations were performed under
15
conditions similar to those employed in previous studies.
(8) or the cube-expanded product 1-homocubanol (9) from a
Following the oxidation reactions, the products were extracted
into CH2Cl2. Product quantitation was accomplished by GC
analysis on a Carbowax column with FID detection. Product
identifications were achieved as noted above. For some oxidation
reactions, the turnovers were so small that the amounts of
products were not sufficient for high-quality GC-mass spectral
analyses, and the identities of the products in those cases rely
cationic rearrangement process. Several radical-derived rear-
rangement products from 6 are possible in principle, all in which
the cube skeleton has been destroyed;1 these products are
expected to be highly unstable (see below).
8
Compounds 8 and 9 were identified in the product mixtures
from P450-catalyzed oxidation of probe 6 by comparison of
the GC retention times and mass spectral fragmentation patterns
to those of authentic samples. Compound 8 is an intermediate
(19) Choi, S. Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard, S.
J.; Newcomb, M.; Putt, D. A.; Upadhyaya, S. P.; Xiong, Y. J. Am. Chem.
Soc. 1996, 118, 6547-6555.
(20) Della, E. W.; Janowski, W. K. J. Org. Chem. 1995, 60, 7756-
7759.
(16) Le Tadic-Biadatti, M. H.; Newcomb, M. J. Chem. Soc., Perkin
Trans. 2 1996, 1467-1473.
(
(
17) Koreeda, M.; Tanaka, Y. Tetrahedron Lett. 1987, 28, 143-146.
18) Eaton, P. E.; Yip, Y. C. J. Am. Chem. Soc. 1991, 113, 7692-7697.
(21) Jin, Y.; Lipscomb, J. D. Biochemistry 1999, 38, 6178-6186.

2680 J. Am. Chem. Soc., Vol. 122, No. 12, 2000
Newcomb et al.
Table 1. Products from Cytochrome P450-Catalyzed Oxidations of
a
Probe 1
isozyme
B1
B4
2
3
4
turnoverb
2
2
85
90
93
90
89
79
11
7
5
5
9
4
3
2
5
2
24
166
336
20
44
22
∆
∆
∆
∆
2B4
2B4 T302A
2E1
2E1 T303A
9
12
a
Average relative percentage yields of 2, 3, and 4 from duplicate
b
or triplicate runs. Average turnover values.
Figure 3. Time course study for oxidation of 1 with P450 2B1. The
on the GC retention times and the expectation that similar
product mixtures were formed with all enzymes. Small peaks
with GC retention times consistent with phenol 5 and its
diastereomers were observed in most reactions of 1, but we
could not confirm the identity of 5 in the enzyme product
mixtures by GC-mass spectral analysis due to the small amounts
of materials.
Table 1 summarizes the results from P450-catalyzed oxida-
tions of probe 1; complete results are given in the Supporting
Information. Unrearranged alcohol 2, radical-derived rearrange-
ment products 3, and cation-derived rearrangement product 4
were produced in all cases. As noted, phenol product 5 was not
produced in significant amounts in these oxidations, but small
peaks with appropriate retention times for product 5 were present
in the GC traces. If we assume that the largest peaks observed
in this region of the GC trace were due to 5, then the amount
of 5 formed in various P450 oxidations was in the range of
tenths of nanomoles.
yields of each product (in nmol) are given; the thick line is the ratio of
(2 + 4) to 3.
Table 2. Products from Cytochrome P450-Catalyzed Oxidations of
Probe 6a
isozyme
(7a + 7b + 7c)
8
9
turnoverb
2
2
2
∆
∆
B1 (batch 1)c
B1 (batch 2)c
B4
2B4
2B4 T302A
31
36
11
19
31
42
24
29
68
66
89
80
48
48
70
56
0d
nd
24
98
61
12
21
105
14
d
0
0
d
1
21
10
6
15
c
∆2E1 (batch 1)
∆
∆
2E1 (batch 2)c
2E1 T303A
a
Average percentage yields of 7-9 from duplicate or triplicate runs.
b
c
Average turnover value; nd ) not determined. Results from two
samples of enzymes. d Trace of product 9 (<1%) detected in GC-MS
analysis.
the conclusion that substrate 1 also protects product 4 by
Products 2-4 were stable to the GC conditions used for
analysis. Control reactions were performed to determine whether
these products were stable to the enzyme reaction conditions
and could be recovered in good yield. The detailed results of
the control reactions are given in the Supporting Information.
Samples of the authentic oxidation products in amounts similar
to those found in the oxidations of substrate 1 were employed
with (A) reaction mixtures containing fully competent enzyme
oxidation systems, (B) mixtures lacking the enzymes, and (C)
mixtures lacking NADPH, and the amounts of material remain-
ing after incubation, reaction, and workup were determined. In
the case of cyclic alcohol 2 and the acyclic alcohols 3, the test
substrates were returned in high yields. Acyclic alcohols 3 and
aldehyde 4 were not produced in the control reactions for
product 2 which indicates that 3 and 4 were not formed in
secondary reactions.
Aldehyde 4 was unstable when tested in the absence of a
cosubstrate. A reaction with the fully competent P450 2B1
enzyme system containing 29 nmol of 4 resulted in complete
loss of the aldehyde. However, when aldehyde 4 (25 nmol) was
admixed with probe 1 in a reaction with the 2B1 isozyme, no
loss of product was observed. Given that 4 was obtained in a
yield of only 0.5 nmol from the oxidation of 1 by the 2B1
isozyme, this result suggests that substrate 1 served as a
competitive inhibitor for enzyme-catalyzed consumption of 4,
although it is possible in principle that the amounts of 4
produced and destroyed in the control reaction were the same.
To test the latter possibility, a reaction was conducted with the
competitive inhibition.
As a further test of stabilities of the products from substrate
1
, a series of reactions with the 2B1 isozyme was conducted
over varying times. Figure 3 shows the yields in nanomoles of
products 2, 3, and 4 for a series of reactions conducted between
5
and 40 min. Apparently, an enzyme was destroyed or NADPH
was consumed within 10 min of initiating the reactions. It is
possible that small amounts of products were lost on extended
standing, but most importantly, the ratio of ([2] + [4])/[3],
indicated by the heavy line in Figure 3, was invariant in the
time course study. This ratio provides the lifetime of the putative
radical formed by hydrogen atom abstraction as discussed later.
Table 2 contains a summary of results from P450-catalyzed
hydroxylations of methylcubane (6); complete results are given
in the Supporting Information. Because methylcubane is quite
volatile, we were unable to determine percent recoveries of
unreacted substrate. The three methylcubanol products (7) and
cubylmethanol (8) were observed in all oxidations. 1-Homocu-
banol (9) was observed by GC using FID detection in reactions
with four of the six isozymes. For the two cases where product
9
was not observed with FID detection, the 2B1 and 2B4
2
B1 isozyme in which trans-2-(p-methoxyphenyl)methylcyclo-
isozymes, GC-MS analyses of the reaction mixtures showed
that traces of 9 (<1% relative yield) were present.
The GC and GC-mass spectral analyses of the product
mixtures before and after acetate derivatization provided firm
identifications of the products. The results from oxidations of
probe 6 are qualitatively meaningful. They are, however, less
reliable in a quantitative sense than those from oxidations of
2
2
propane (10) was used as a surrogate for substrate 1, and
aldehyde 4 was present in a small amount (1.6 nmol). Aldehyde
23
4
was stable in this test reaction indicating that substrate 10
was a competitive inhibitor for consumption of 4 and reinforcing
(
22) Newcomb, M.; Choi, S.-Y.; Toy, P. H. Can. J. Chem. 1999, 77,
1
123-1135.

Cytochrome P450-Catalyzed Hydroxylations
J. Am. Chem. Soc., Vol. 122, No. 12, 2000 2681
Scheme 2
Scheme 3
probe 1. Synthetic routes for production of authentic samples
of the methylcubanols (7) are not available, so GC response
factors could not be determined. We made the reasonable
assumption that the flame ionization response factors for
compounds 7 were equal to that for the isomeric compound 8.
A further complication in quantitation resulted from the apparent
partial decomposition of compounds 7 on the GC column as
noted above.
Control reactions with products 8 and 9 were conducted in a
manner similar to those conducted with the products from
substrate 1. Results are given in the Supporting Information.
Good consistency in product recoveries was observed, but the
yields were somewhat reduced from those found with products
2-4. The slight reductions in recoveries were independent of
the presence of enzymes, and we conclude that these compounds
were not appreciably oxidized. The loss apparently is due to
either the distribution of products 8 and 9 between the buffer
and CH2Cl2 used for extraction or, more likely, some evapora-
tion of the products during the solvent removal step preceding
analysis. The recovery-stability results present yet another
caution regarding the precision of the quantitative data for
studies with substrate 6. Most importantly, however, the control
experiments demonstrate that product 9 was not formed from 8
in reactions of fully competent enzyme systems.
that shortcoming was that all probes from which rearranged
products had been obtained would suffer the same skeletal
reorganization from reactions of radicals or cations.28 Both of
the substrate probes used in the present work display distinctly
different rearrangement patterns for radicals and cations.29
The reaction manifolds for the cyclopropane-based probe 1
are shown in Scheme 2. The cyclopropylcarbinyl radical 11
rearranges with high regioselectivity, 160:1 at ambient temper-
1
6,28
ature,
to the benzylic radical 12; in the context of enzyme-
catalyzed hydroxylations, benzyl alcohols 3 are produced from
this pathway. An incipient cyclopropylcarbinyl cation (13) opens
Discussion
28
with even higher regioselectivity, >1000:1, to oxonium ion 14.
The hydroxylation product from the cationic route is an unstable
How the Probes Work. The hydrogen abstraction-oxygen
rebound mechanism for P450-catalyzed hydroxylations shown
in Figure 1 was generally accepted at the beginning of the 1990s.
It is qualitatively consistent with the observations of rearranged
products from oxidations of substrates that are precursors to
highly reactive radicals, but it is not quantitatively consistent.
For example, oxidations of kinetically calibrated hypersensitive
radical probes gave apparent rebound barriers ranging from 4
hemiacetal (15) that hydrolyzes to the â,γ-unsaturated aldehyde
1
6 that, in turn, was found to isomerize to aldehyde 4 in the
1
0
enzyme buffer medium. In addition to the high fidelity of the
two rearrangement pathways, relative rate constants for the
radical ring opening of 11 in competition with PhSeH trapping
were determined. Using the product ratios from that work and
the recently recalibrated rate constants for reactions of PhSeH
with alkyl radicals, one computes a rate constant for ring
opening of 11 at 37 °C of 6 × 10 s .
16
3
0
kcal/mol found with bicyclo[2.2.0]pentane2
found with a constrained aryl-substituted cyclopropane probe.
4-26
to 0 kcal/mol
1
1
-1
27
The reaction manifolds for oxidation of the methyl group
We proposed that the inconsistencies might be the result of
multiple oxidation pathways, with both radical and cationic
rearrangements possible, and a shortcoming of the probe designs;
methylcubane are shown in Scheme 3. The cubylcarbinyl radical
8,31
(
16) experiences a cascade of bond-cleavage reactions.1 The
first rearranged radical (17) has not been trapped, and it is
(
23) Substrate 6 was previously studied with microsomes from livers of
phenobarbital-treated rats, which contain mainly P450 2B1, and the purified
B1 isozyme (ref 19). In that study, the alcohol products were converted
to acetates that were analyzed, and the amounts of products 7-OAc and
-OAc found were comparable. Given the apparent decomposition of
(28) Newcomb, M.; Chestney, D. L. J. Am. Chem. Soc. 1994, 116, 9753-
9754.
2
(29) It is noteworthy that the concept of studying P450 hydroxylations
with a probe that can differentiate between radicals and cations was put
forth by Groves. Specifically, bicyclo[4.1.0]heptane was used as a probe
because radicals and cations at the cyclopropylcarbinyl position of this
species give different rearrangement products. P450-catalyzed hydroxylation
of this probe, however, resulted in no rearrangement products. See: White,
R. E.; Groves, J. T.; McClusky, G. A. Acta Biol. Med. Ger. 1979, 38, 475-
489.
(30) Newcomb, M.; Choi, S.-Y.; Horner, J. H. J. Org. Chem. 1999, 64,
1225-1231.
(31) Choi, S.-Y.; Eaton, P. E.; Newcomb, M.; Yip, Y.-C. J. Am. Chem.
Soc. 1992, 114, 6326-6329.
8
alcohols 7 in GC analyses, the yields of 7 relative to 8 found here are in
reasonable agreement with those previously reported.
(
24) Ortiz de Montellano, P. R.; Stearns, R. A. J. Am. Chem. Soc. 1987,
09, 3415-3420.
25) Newcomb, M.; Manek, M. B.; Glenn, A. G. J. Am. Chem. Soc.
991, 113, 949-958.
26) Bowry, V. W.; Lusztyk, J.; Ingold, K. U. J. Am. Chem. Soc. 1991,
13, 5687-5698.
27) Newcomb, M.; Le Tadic, M. H.; Putt, D. A.; Hollenberg, P. F. J.
Am. Chem. Soc. 1995, 117, 3312-3313.
1
1
1
(
(
(

2682 J. Am. Chem. Soc., Vol. 122, No. 12, 2000
Newcomb et al.
possible that simultaneous bond cleavages occur taking 16
directly to 18. Radical 18 can be trapped, but in the absence of
1
8
reactive trapping agents, it further reacts to give 19. An
incipient cubylcarbinyl cation (20) suffers a bond migration to
give the 1-homocubyl cation (21).2
0,32,33
The regioselectivities
of the two pathways apparently are high, none of the “wrong”
rearrangement product has been reported for either pathway,
but the relatively high reactivities of the observed and putative
products have precluded precise determinations of regioselec-
tivity. Rate constants for rearrangement of radical 16 to radical
10
-1
31
Figure 4. Percentages of cationic rearrangement products relative to
total methyl group oxidation found in P450-catalyzed hydroxylations
of substrates 1 (unfilled) and 6 (filled).
1
8 are known (e.g., 3 × 10
s
at ambient temperature), but
it is unlikely that radical-derived rearrangement products from
hydroxylation of 6 would be stable. The strained tricycloocta-
diene products from hydrogen atom trapping of 18 are not stable
upon standing at ambient temperature,18 and the corresponding
alcohols should also be subject to solvolytic reactions in the
enzyme medium. A recent report21 that a radical-derived
rearrangement product was obtained by methane monooxyge-
nase (MMO) oxidation of 6 appears to be in error as the
published2 mass spectrum of the putative radical rearrangement
mocubanol (9) was formed in appreciable amounts with several
of the P450 isozymes. Given the differences in the structural
features of probes 1 and 6 and their different modes of
rearrangements, it is difficult to formulate any rationalization
wherein the rearrangement products 4 and 9 are produced other
than from cationic species. The demonstrations that authentic
samples of unrearranged alcohols 2 and 8 gave no detectable 4
and 9, respectively, when tested with the enzymes indicates that
the rearrangement products were produced in the hydroxylation
reactions. One appears to be left only with the explanation that
cationic species were produced in part in the enzyme-catalyzed
reactions. Multiple reaction pathways are strongly suggested.
Moreover, the patterns in the product distributions for the
two probes are roughly correlated in a manner consistent with
multiple reaction pathways. Figure 4 shows the percentages of
cationic rearrangement products from 1 and from oxidation of
the methyl group in 6 for the various enzymes studied. Increased
amounts of cationic rearrangement products were found with
the mutants, which were designed to disrupt the normal sequence
of events in P450 oxidations. Later in discussion, we will
attribute the cationic rearrangement products to reaction of a
specific P450 oxidant species.
Radical Lifetimes in P450-Catalyzed Hydroxylations. That
both radical- and cationic-type rearrangements can occur in
P450-catalyzed hydroxylations is important for understanding
the results of previous mechanistic probe studies. Both pathways
occurred for the probes studied here, and there is little reason
to believe that they could not also occur when other, “non-
distinguishing” probes were employed. Because both radical and
cationic rearrangements of those other probes ultimately would
give the same products, one has no method for determining how
much of any observed rearranged product should be ascribed
to a radical pathway. Therefore, all “radical lifetimes” calculated
from the product distributions found in P450-catalyzed hy-
droxylations of those probes can only be upper limits.
1
3
4
product is the same as that of authentic 1-homocubanol (9),
and 9 was shown to be a product from MMO oxidations of 6
in a more recent work.34
Probe 6 was also oxidized at the cube positions giving
products 7. Formation of cubanols 7 requires an electrophilic
35
oxidant, and alkoxyl radical abstraction from 6 occurs on the
cube.1
9,36
Whereas the formation of products 7 speaks to the
character of the oxidizing species, it does not provide readily
interpreted mechanistic information.
Multiple Reaction Pathways in P450-Catalyzed Hydroxy-
lations. That radical and cationic rearrangement pathways occur
28
in P450 hydroxylation reactions is clearly demonstrated by
the results with probe 1 listed in Table 1. The discrimination
factor for this probe, the product of the regioselectivities for
the two types of intermediates, exceeds 100 000, and there is
little doubt about the radical origin of product 3 and the cationic
origin of product 4. A similar demonstration of two distinct
pathways for rearrangement was previously reported for the 2B1
isozyme with a related probe.10 Specifically, oxidations of probe
2
2 by microsomes from livers of phenobarbital-treated rats,
which contain mainly P450 2B1, and by the purified P450 2B1
isozyme gave mixtures of unrearranged product, radical-derived
rearrangement product, and cation-derived rearrangement prod-
uct in a product distribution similar to those found here with
probe 1.
From the design of probe 1, one is confident that benzylic
alcohol products 3 are not from cations; they must be produced
in a radical process. Table 3 lists the ratios of non-radical-
derived hydroxylation products (2 and 4) to radical-derived
products 3 for each isozyme studied. These ratios can be used
with the rate constant for rearrangement of the cyclopropyl-
carbinyl radical 11 to compute the apparent “rebound” rate
constants and the lifetimes (1/k), both of which are also listed
in Table 3. The consistency of the values for both of the wild-
type and mutant pairs is satisfying. When compared to results
from other probes, these results clearly demonstrate the impor-
tance of separating radical-derived and cation-derived rear-
The observations of cationic rearrangement products in the
P450 hydroxylations are the unexpected results because there
is no pathway for formation of these species in what is, or was,
clearly the consensus view of P450-catalyzed hydroxylation (i.e.,
Figure 1). Thus, the results from oxidations of methylcubane
3
7
(6) are important. The cationic rearrangement product 1-ho-
(32) Eaton, P. E. Angew. Chem., Int. Ed. Engl. 1992, 31, 1421-1436.
(33) Smith, B. J.; Tsanaktsidis, J. J. Org. Chem. 1997, 62, 5709-5712.
(34) Choi, S.-Y.; Eaton, P. E.; Kopp, D. A.; Lippard, S. J.; Newcomb,
M.; Shen, R. J. Am. Chem. Soc. 1999, 121, 12198-12199.
35) Hrovat, D. A.; Borden, W. T. J. Am. Chem. Soc. 1994, 116, 6459-
460.
36) Della, E. W.; Head, N. J.; Mallon, P.; Walton, J. C. J. Am. Chem.
Soc. 1992, 114, 10730-10738.
(37) The radical lifetimes were calculated by a conventional competition
kinetics. The apparent rebound rate constant is equal to the ratio of
nonradical-derived to radical-derived products, ([2] + [4])/[3], times the
(
6
1
1 -1
(
rate constant for ring opening of radical 11, 6 × 10
s . See: Newcomb,
M. Tetrahedron 1993, 49, 1151-1176.

Cytochrome P450-Catalyzed Hydroxylations
J. Am. Chem. Soc., Vol. 122, No. 12, 2000 2683
Table 3. Radical Lifetimes in Cytochrome P450-Catalyzed
Oxidations of Probe 1a
c
lifetime (fs)d
isozyme
B1e
B4
non-rad/radb
k
ox
12
2
2
8.1 ( 0.7
13.0 ( 0.6
20.0 ( 0.1
19.2 ( 0.4
9.6 ( 1.7
9.8 ( 3.3
5 × 10
8 × 10
200
130
80
80
170
170
1
2
1
1
3
3
∆
∆
∆
∆
2B4
2B4 T302A
2E1
1.2 × 10
1.2 × 10
Figure 5. The consensus pathway for production of the iron-oxo
species from the peroxo-iron species where Fe represents heme-bound
iron.
1
1
2
2
6 × 10
2E1 T303A
6 × 10
a
b
formulate a mechanism wherein cationic rearrangements are
possible, but carbocations are not required. The latter condition
is absolute because requisite carbocations would result in ring-
opened products from all methylcyclopropane probes.
Average values from duplicate runs unless noted. Ratio of (2 +
c
4
) to 3; stated error is one standard deviation. Apparent oxygen
d
rebound rate constant. Lifetime of radical in femtoseconds. e Average
value from eight runs.
Some recent mechanistic descriptions of P450 hydroxylations
have attempted to address the origin of cationic products. Those
explanations include rearrangement of the probe while bound
rangement products. For example, hydroxylations of a hyper-
sensitive probe in which no epoxidation pathway was available
with the wild-type and mutant pairs used here resulted in ratios
of rearranged to unrearranged products that varied dramati-
11
to iron in an agostic complex, competing production of a spin-
paired complex (insertion) and non-spin-paired complex (radical
1
5
cally.
12
intermediate) with electron transfer from the radical, and
The computed radical lifetimes of 80-200 fs are in good
+
insertion of the elements of OH followed by solvolysis of the
agreement with the value of 100 fs found in oxidation of probe
resulting protonated alcohol in competition with deprotona-
2
2 with the P450 2B1 isozyme.38 Previous results with other
1
0,39
tion,
the latter implying that the ultimate oxidant was iron-
probes are also seen to be consistent with these lifetimes when
one understands that the values computed from those studies
can only be upper limits. Some of those upper limits are quite
short, for example, lifetimes of <200 fs were found in P450-
catalyzed hydroxylations probes of 23-25.2 Lifetimes of 80-
complexed hydrogen peroxide instead of iron-oxo. Elements of
these mechanistic proposals might explain cationic rearrange-
ments, but they contained shortcomings that have been dis-
3
9
cussed. We believe that a problem with these descriptions is
that they have been cast in terms of a single oxidizing species,
whereas a unified mechanistic view must incorporate recent
evidence that two distinct electrophilic oxidants are active in
P450-catalyzed reactions.
7,39
200 fs are too short for a true intermediate, and the correspond-
ing rate constants listed in Table 3 are in the range expected
for vibrational rate constants, not for chemical rate constants.
That is, the lifetimes are those of transition states.40 True radical
intermediates are not formed.
The sequence of events in P450-catalyzed oxidation reactions
involves substrate binding, reduction of the ferric form of the
enzyme, oxygen binding to give a ferrous-dioxygen species, and
a second reduction step to give a peroxo-iron species that
ultimately gives oxidizing species. Neither the active oxidant-
(s) in P450 nor any intermediate following the ferrous-dioxygen
species has been characterized unambiguously, but the common
belief has been that the oxidant is an iron-oxo species related
to the spectroscopically observed Compound I of peroxidase
2
chemistry. A commonly accepted route to the iron-oxo species,
If no radical intermediates are formed, then the hydroxylation
reactions must be insertion processes. The small amounts of
radical rearrangement products found reflect a bifurcation in
the transition state between collapse without ring opening and
ring opening followed by collapse. Although dynamicists might
have no problem with the concept of a bifurcation from a
transition structure, many chemists and biochemists find this
notion unusual. Another way to perceive the process is that
collapse of the “radical” formed by abstraction occurs with no
barrier but still requires an exquisitely short period of time (a
vibrational period) during which some ring opening occurs.
Multiple Cytochrome P450 Oxidants and the Origins of
the Cation Species. The implication of a cationic species
resulting in rearranged products is clear from the results with
the substrates studied in this work and those found previously
with probe 22.10 That a cationic species can be formed in the
hydroxylation reaction might explain why other probes gave
conflicting results in regard to the apparent lifetimes of radicals,
but it also presents a new mechanistic puzzle. One must
supported by recent computational work by Harris and Loew,41
is shown in Figure 5. Initial protonation of the distal oxygen in
the peroxo-iron complex gives a hydroperoxo-iron species, and
a subsequent protonation of the distal oxygen gives a species
that loses water to give an iron-oxo complex similar to
Compound I of the peroxidases. The computational results
further indicated that the loss of water from the penultimate
complex shown in Figure 5 to give the iron-oxo species has no
barrier, and that initial protonation of the proximal oxygen in
the peroxo-iron species would give a considerably higher energy
species than that formed by distal oxygen protonation.41
Despite the widespread belief that an iron-oxo species is the
active oxidant in P450 reactions, the diverse oxidations effected
by these enzymes suggest that different types of oxidants are
involved in some specific functions. For example, the iron-oxo
species is an electrophilic oxidant that would seem to be the
appropriate character for epoxidation and hydroxylation reac-
tions. Alternatively, the deformylation reaction effected by P450
aromatase and similar deformylations of various xenobiotic
42,43
(38) On the basis of the recalibration of PhSeH trapping kinetics (ref
aldehydes by microsomal P450s
are consistent with nucleo-
3
0), the rate constant for rearrangement of the radical derived from probe
2
2 (ref 16) must be adjusted. This, in turn, results in an adjustment of the
(41) Harris, D. L.; Loew, G. H. J. Am. Chem. Soc. 1998, 120, 8941-
8948.
(42) Vaz, A. D. N.; Roberts, E. S.; Coon, M. J. J. Am. Chem. Soc. 1991,
radical lifetime in the 2B1 oxidation of 22 (ref 10) from 70 to 100 fs.
39) Toy, P. H.; Newcomb, M.; Hollenberg, P. F. J. Am. Chem. Soc.
998, 120, 7719-7729.
40) The lifetime of a transition state at ambient temperature from
classical transition state theory is 170 fs.
(
1
113, 5886-5887.
(
(43) Coon, M. J.; Vaz, A. D. N.; Bestervelt, L. L. FASEB J. 1996, 10,
428-434.

2684 J. Am. Chem. Soc., Vol. 122, No. 12, 2000
Newcomb et al.
philic oxidants that react in Baeyer-Villiger-type processes. In
fact, a nucleophilic peroxo-iron porphyrin was recently shown
4
4
by Valentine and co-workers to mimic aromatase behavior.
The nucleophilic oxidizing species produced in reactions of P450
is not responsible for oxidations studied here, however, because
the oxidant must have electrophilic character.
The mutants used in this work were designed to study the
potential disruption of the sequence of electrophilic oxidant
formation in Figure 5. Threonine is highly conserved in the
active sites of P450 enzymes45 indicating a specific role for the
hydroxy group, and in crystal structures of bacterial P450
enzymes, threonine is located within hydrogen-bonding distance
of a putative hydroperoxo-iron species.46 One proposed role for
the conserved threonine is protonation of the hydroperoxo-iron
species, possibly via a relay.47
Recent studies with both ∆2B4 and ∆2E1 and their respective
mutants indicated that two electrophilic oxidants are produced
in the natural course of P450 oxidations.13 In that work, the
ratios of epoxidation to allylic oxidation products for a series
of alkenes was found to differ for the isozymes and their
respective mutants. The conclusions were that both the hydro-
peroxo-iron species and the ultimate oxidant were electrophilic
oxidizing entities and that the epoxidation was the preferred
reaction of the former whereas hydroxylation was the preferred
reaction of the latter. The proposed effect of substitution of
alanine for threonine in the mutants was either to slow the
overall rate of formation of the ultimate oxidant or to affect an
equilibrium reaction involving protonation;13 on the basis of the
computational finding of a barrierless loss of water following
the second protonation,41 one would now favor the former
possibility.
Figure 6. Likely mechanism for hydroxylation by the P450 hydrop-
eroxo-iron species where Fe represents heme-bound iron and first-
formed protonated alcohols from various probes.
but hydroxylation by the hydroperoxo-iron species would
+
involve insertion of the elements of OH (Figure 6). The latter
reaction gives protonated alcohol products such as 28 and 29
for the two probes studied in this work or 30 and 31 for probes
used in previous studies (see Figure 6). Intermediates 28-30
would be expected to be highly reactive with respect to
solvolytic reactions with anchimeric assistance, and the bicyclo-
[2.1.0]pentyl system is also known to be quite reactive in
cationic processes that cleave the central C-C bond.48 It would
appear that all probes that have been found to give rearranged
hydroxylation products in P450 oxidations are reasonable
candidates for cationic rearrangements from first-formed pro-
tonated alcohols.
We suggest, therefore, that cationic rearrangement products
derive from hydroxylation reactions effected by the hydroper-
oxo-iron species. If the formulation of the effects of the Thr to
Ala replacement in the mutant enzymes proposed here is correct,
then larger percentages of hydroxylation by the hydroperoxo-
iron species should have occurred with the mutants, resulting
in larger amounts of cationic rearrangement products. In general,
this is the case as shown in Figure 4. Supporting evidence also
is available in the results from oxidations of probe 27 which
cannot react by arene epoxidation; specifically, substantially
more rearranged products were produced with the mutants than
with the wild-type isozymes suggesting a larger percentage of
More recent studies with the same wild-type and mutant
enzymes supported the conclusion that two electrophilic oxidant
forms exist and, moreover, indicated that both species were
15
capable of effecting hydroxylation. Those studies involved the
hypersensitive probes 26 and 27. Consistent ratios for the
rearranged and unrearranged products from methyl group
oxidation in probe 26 (where an arene epoxidation pathway
exists) were found for the wild-type and mutant pairs, but the
ratios of rearranged to unrearranged products varied dramatically
for probe 27 (where arene epoxidation is not possible). The
deduction from those observations was that each electrophilic
oxidant was displaying a characteristic product ratio for methyl
group oxidation, that most of the methyl group oxidation in
probe 26 was attributable to one oxidizing species, and that both
oxidizing species effected methyl group hydroxylation in probe
1
5
cationic rearrangement products.
A Unified Mechanistic View of P450-Catalyzed Hydroxy-
lations. A unified description of P450-catalyzed hydroxylation
reactions thus involves the following features. (1) Both the
hydroperoxo-iron complex and the iron-oxo species can effect
hydroxylation reactions. (2) Hydroxylations by both species
involve insertion processes, that is, no radical intermediates are
produced in the reactions. (3) Hydroxylation by the iron-oxo
species involves insertion of an oxygen atom giving a neutral
alcohol product directly. (4) Hydroxylation by the hydroperoxo-
15
2
7.
+
iron species involves insertion of the elements of OH , and the
first formed product is a protonated alcohol. (5) Protonated
alcohols formed in the latter process can rearrange by cationic
routes. Figure 7 summarizes the properties of the oxidants in
P450 implicated by the present work and previous studies.1
3-15
The demonstration of two active electrophilic oxidant forms
(46) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195,
87-700. Ravichandran, K. G.; Boddupalli, S. S.; Hasemann, C. A.;
6
for P450, the hydroperoxo-iron species and the iron-oxo,
provides a straightforward explanation for the production of
cation-derived rearrangement products. Hydroxylation by the
iron-oxo species would involve insertion of an oxygen atom,
Peterson, J. A.; Deisenhofer, J. Science 1993, 261, 731-736. Hasemann,
C. A.; Ravichandran, K. G.; Peterson, J. A.; Deisenhofer, J. J. Mol. Biol.
1994, 236, 1169-1185. Cupp-Vickery, J. R.; Poulos, T. L. Nat. Struct.
Biol. 1995, 2, 144-153.
(47) Imai, M.; Shimada, H.; Watanabe, Y.; Matsushima-Hibiya, Y.;
(
44) Wertz, D. L.; Sisemore, M. F.; Selke, M.; Driscoll, J.; Valentine, J.
Makino, R.; Koga, H.; Horiuchi, T.; Ishimura, Y. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 7823-7827. Martinis, S. A.; Atkins, W. M.; Stayton, P.
S.; Sligar, S. G. J. Am. Chem. Soc. 1989, 111, 9252-9253.
(48) Wiberg, K. B.; Williams, V. Z., Jr.; Friedrich, L. E. J. Am. Chem.
Soc. 1970, 92, 564-567.
S. J. Am. Chem. Soc. 1998, 120, 5331-5332.
(
45) Nelson, D. R.; Strobel, H. W. Biochemistry 1989, 28, 656-660.
Gotoh, O.; Fujii-Kuriyama, Y. In Frontiers in Biotransformation; Ruckpaul,
K., Rein, H., Eds.; Akademie: Berlin, 1989; Vol. 1, pp 195-243.

Cytochrome P450-Catalyzed Hydroxylations
J. Am. Chem. Soc., Vol. 122, No. 12, 2000 2685
barrier-free process.55 A ramification of this result is that
hydroxylation of an alkane by an iron-oxo species in a low-
spin ensemble is predicted to occur effectively via a “concerted
insertion” process. High-level computations of hydroxylations
by methane monooxygenase enzymes also appear to be con-
verging on the conclusion that collapse of the “radical” with
5
6
an Fe-OH complex is barrier-free or that the hydroxylation
5
7
reaction occurs by concerted insertion.
The unified view also is consistent with the results that
originally led to the conclusion that hydroxylation occurred by
hydrogen abstraction and oxygen rebound (i.e., Figure 1). The
most important early evidence came from mechanistic probe
Figure 7. Multiple oxidants generated by cytochrome P450 enzymes;
the character and typical reactions of each species are listed.
7
studies. Examples include deuterium epimerization in oxidation
5
8
59
A seemingly unusual feature of this mechanistic picture is
the insertion of OH+ in the reaction of the hydroperoxo-iron
species, but there is good evidence for such reactions. Acid-
catalyzed hydroxylation of hydrocarbons by hydrogen peroxide
of 32, allylic rearrangement in oxidation of 33, and some
ring opening in the hydroxylation of bicyclo[2.1.0]pentane
2
4
(34). The epimerizations and allylic shifts do not require
discrete intermediates as these rearrangements should be fast
enough to compete with collapse of transition structures. The
rearrangement of 34 does require an intermediate, but as noted
above, the species that rearranges could be the protonated
alcohol 31.
+
49
has long been thought to involve OH insertion, and such an
insertion is supported by computational results.50 Perhaps a more
meaningful analogy is found in the mechanism of reaction of
heme oxygenase, the enzyme that oxidizes heme to biliverdin.
On the basis of successful H2O2 shunting of the reaction and
the failure of other oxidants such as m-CPBA to effect reactions,
Wilks and Ortiz de Montellano concluded that the heme
oxygenase “ferric peroxide” species (structurally the same as
the hydroperoxo-iron in Figures 5 or 7) reacted by insertion of
+
51
OH into a heme C-H bond. The formulation of this aspect
of the heme oxygenase reaction sequence5 is the same as that
shown in Figure 6 for reaction of the hydroperoxo-iron species
in P450.
2
The other evidence often cited for the abstraction/rebound
mechanism is the observation of primary kinetic isotope effects
(KIEs) in P450-catalyzed hydroxylations that are similar to those
observed in hydrogen atom abstractions by alkoxyl radicals. For
example, a recent such study by Dinnocenzo, Jones, and co-
workers demonstrated a high correlation between KIEs in P450-
catalyzed hydroxylations with those found in reactions of the
+
It is possible that OH insertion involves reaction of an iron-
hydrogen peroxide complex instead of a hydroperoxo-iron
complex, and we cannot exclude this possibility. Such a reaction
is more akin to acid-catalyzed insertion reactions of hydrogen
peroxide noted above. Reaction of iron-complexed hydrogen
peroxide was a speculation offered previously to account for
6
0
same substrates with the tert-butoxyl radical. Whereas the
observed correlations are undoubtedly accurate, the inference
that such similarities in KIEs result from the same mechanisms
can be questioned. One important lesson from modern compu-
tational studies of KIEs is that the assumption that a single
vibrational mode is isolated in the transition state of a hydrogen
atom transfer reaction is a gross oversimplification.61 Moreover,
Shaik and co-workers point out that the KIE for oxygen atom
insertion from an iron-oxo species reacting with an alkane on
a low spin surface is expected to mirror that of a hydrogen atom
1
0
production of cationic species in P450 hydroxylations, and
Pratt et al. reported evidence suggesting that such a species was
5
3
involved in at least some P450-catalyzed oxidations. If our
formulation of the effects of the Thr to Ala replacement in the
mutants is correct, however, the hydroperoxo-iron species is
the “other” oxidant, and some evidence supports this view.
Specifically, the mutant enzymes reacted with probe 26 to give
predominantly phenol products,15 but phenols were not formed
when the same probe was oxidized with the P450-like enzyme
chloroperoxidase activated with H2O2.54
5
5
abstraction reaction. That is, the insertion reaction does
resemble a hydrogen abstraction to a point and should have a
similar KIE, even though no intermediate radical is produced.
In summary, the results from oxidations of probes 1 and 6
by the various P450 isozymes used in this work suggest that
production of cationic intermediates apparently is a common
feature of P450-catalyzed hydroxylations. We believe these
species are most likely protonated alcohols produced by insertion
The unified view appears to be consistent with most of the
recent high-level computational studies of P450-catalyzed and
related hydroxylations, the only real difference being the
incorporation of two active oxidants. We noted earlier the
computational study of the oxidants in P450 (Figure 5) that
provided barriers for the various processes.41 In a quite recent
work, the Shaik group found that reaction of an alkyl radical
with a porphyrin radical cation/hydroxy-iron(III) complex on a
low spin surface gives an alcohol and prophyrin/iron(III) in a
+
of OH in reactions of the hydroperoxo-iron species formed in
the natural course of P450 oxidation reactions. The results from
(55) Harris, N.; Cohen, S.; Filatov, M.; Ogliaro, F.; Shaik, S. Angew.
Chem., Int. Ed. In press.
(
56) Siegbahn, P. E. M. Inorg. Chem. 1999, 38, 2880-2889.
(
49) Olah, G. A.; Parker, D. G.; Yoneda, N. Angew. Chem., Int. Ed.
(57) Yoshizawa, K.; Ohta, T.; Yamabe, T. Bull. Chem. Soc. Jpn. 1998,
71, 1899-1909.
Engl. 1978, 17, 909-931.
(
(
50) Bach, R. D.; Su, M. D. J. Am. Chem. Soc. 1994, 116, 10103-10109.
51) Wilks, A.; Ortiz de Montellano, P. R. J. Biol. Chem. 1993, 268,
(58) Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J. Biochem.
Biophys. Res. Commun. 1978, 81, 154-160.
2
2357-22362.
(59) Groves, J. T.; Subramanian, D. V. J. Am. Chem. Soc. 1984, 106,
2177-2181.
(
(
52) Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543-549.
53) Pratt, J. M.; Ridd, T. I.; King, L. J. J. Chem. Soc., Chem. Commun.
(60) Manchester, J. I.; Dinnocenzo, J. P.; Higgins, L. A.; Jones, J. P. J.
Am. Chem. Soc. 1997, 119, 5069-5070.
1
995, 2297-2298.
54) Toy, P. H.; Newcomb, M.; Hager, L. P. Chem. Res. Toxicol. 1998,
1, 816-823.
(
(61) Liu, Y. P.; Lynch, G. C.; Truong, T. N.; Lu, D. H.; Truhlar, D. G.;
Garrett, B. C. J. Am. Chem. Soc. 1993, 115, 2408-2415.
1

2686 J. Am. Chem. Soc., Vol. 122, No. 12, 2000
Newcomb et al.
oxidations of probe 1 also indicate that hydroxylations by both
the iron-oxo and hydroperoxo-iron species occur by insertions,
not by initial hydrogen abstractions that give radical intermedi-
ates. The unified mechanistic picture of P450-catalyzed hy-
droxylation, two electrophilic oxidants reacting by insertions,
appears to be consistent with virtually all previous results.
extracted with CH
dried over MgSO
internal standard. The mixtures were concentrated under a nitrogen
stream to a volume of ca. 0.2 mL and analyzed by GC on Carbowax
2
Cl
2
(3 × 2 mL). The combined organic phases were
4
, filtered, and mixed with a solution containing an
2
0M bonded-phase columns. Quantitation was achieved on an FID-
equipped GC using a 15 m × 0.5 mm column. Product identification
was achieved by the use of a GC equipped with a mass selective detector
using a 25 m × 0.25 mm column. The results are summarized in Tables
Experimental Section
1
and 2, and detailed listings of product yields are provided in the
Materials. trans,trans-2-Methoxy-3-phenylmethylcyclopropane (1),
alcohol 2, and aldehyde 4 were prepared as previously reported.16
Supporting Information.
Control reactions were designed to evaluate the stability of the
products and the amounts of products that could be recovered. Typically,
the product under evaluation was added to reaction mixtures in
approximately the amount produced by enzyme-catalyzed oxidation of
the parent probe, and the reactions were worked-up and analyzed in
the same method as described above. Detailed results from these studies
are given in the Supporting Information.
2
-Methoxy-1-phenyl-3-buten-1-ol (3) was prepared following the
1
7
procedure of Koreeda and Tanaka, and the preparation of a 17:1
mixture of erythro and threo diastereomers of 3 is reported in the
Supporting Information. trans,trans-2-(p-Hydroxyphenyl)-3-methoxy-
1
-methylcyclopropane (5) was obtained as a mixture with its three
diastereomers from the reaction sequence shown in Scheme 1; details
of the preparation are given in the Supporting Information. Methylcu-
bane (6) and cubylmethanol (8) were prepared by reported methods.1
8,19
Acknowledgment. We thank the National Institutes of
Health for support of this work through grants GM-48722
(M.N.), CA-16954 (P.F.H.), and DK-10339 (M.J.C.). We thank
Prof. P. E. Eaton for a gift of cubanecarboxylic acid. We are
grateful to Prof. S. Shaik for providing us with a manuscript
in advance of publication. We are grateful to both Prof. Shaik
and Prof. P. E. M. Siegbahn for informative discussions.
1
-Homocubanol (9) was prepared by acid-catalyzed reaction of 8 in a
2
0
slight modification of a reported method.
Enzyme-Catalyzed Oxidations. The preparations of the truncated
1
3,14
and mutant P450 isozymes have been reported.
2
by the method previously described with the exceptions that the
amounts of P450 isozymes and reductase were 0.2 and 0.4 nmol,
respectively, and the incubation period prior to addition of substrate
and NADPH was reduced from 5 to 2 min. For the oxidations by P450
Oxidations with
B4, ∆2B4, ∆2B4 T302A, ∆2E1, and ∆2E1 T303A were conducted
55
1
3
2
B1, 0.6 nmol of P450 and 0.6 nmol of reductase were used in
Supporting Information Available: Synthetic details for
the preparation of 3 and 5, NMR and mass spectra for 5, and
tables of results from enzyme-catalyzed oxidation studies and
control studies (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
39
procedures that were otherwise the same as previously reported.
Approximately 1.3 µmol of substrates 1 and 6 was used in each reaction.
Enzyme-catalyzed oxidation reactions were conducted at 37 °C. With
the exception of the time course study of 1 with P450 2B1, all reactions
were allowed to proceed for 30 min. The reactions were quenched by
placing the reaction mixtures in an ice bath. The reaction mixtures were
JA994106+