Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB

Article abstract of DOI:10.1021/ja3059149

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d ₄, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ～20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

Full text of DOI:10.1021/ja3059149

Article
pubs.acs.org/JACS
Parallel and Competitive Pathways for Substrate Desaturation,
Hydroxylation, and Radical Rearrangement by the Non-heme Diiron
Hydroxylase AlkB
Harriet L. R. Cooper,§ Girish Mishra,† Xiongyi Huang,§ Marilla Pender-Cudlip,‡ Rachel N. Austin,‡
John Shanklin,*,† and John T. Groves*,§
§Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
†Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, United States
‡Department of Chemistry, Bates College, Lewiston, Maine 04240, United States
S
* Supporting Information
ABSTRACT: A purified and highly active form of the non-
heme diiron hydroxylase AlkB was investigated using the
diagnostic probe substrate norcarane. The reaction afforded
C2 (26%) and C3 (43%) hydroxylation and desaturation
products (31%). Initial C−H cleavage at C2 led to 7% C2
hydroxylation and 19% 3-hydroxymethylcyclohexene, a re-
arrangement product characteristic of a radical rearrangement
pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-
d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol
was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both
the C−H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at
C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C−H hydrogen abstraction from C2 or C3 and that
the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and
competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the
desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these
norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate
leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation
products. The results indicate that C−H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon
radicals that diverge at the product-forming step.
carboxylate-rich diiron binding sites of sMMO,8 TMO,5 and
INTRODUCTION
■
AurF9 and likely has even more coordinating histidines than the
The alkane monooxygenase AlkB from Pseudomonas putida
(formerly P. oleoverans) is an integral membrane, non-heme
iron hydroxylase that initiates the terminal hydroxylation of
recently characterized CmlA10 and deoxyhypusine hydroxy-
lase.11 Iron-binding sites similar to that of AlkB have also been
inferred in epoxidases, acetylenases, conjugases, ketolases,
linear alkanes.1 AlkB is a member of a large and environ-
ribonucleotide reductases, decarbonylases, and methyl oxidases,
mentally important family of particulate alkane hydroxylases
although no determined structural analogies have appeared.12
(pAHs) that allows microbial growth on petroleum,2 making
Obtaining a crystal structure for AlkB is a coveted goal, to
this enzyme group of interest for bioremediation and
biocatalysis.3 AlkB has been classified as a diiron protein on
the basis of its signature Mossbauer spectra, which show
interpret the available biochemical data in a structural context
and to serve as a reference structure for this functionally diverse
̈
class of integral membrane class of enzymes.6a,13 However,
despite the long history of AlkB and its relative importance in
the global carbon cycle, the structure of this hydroxylase and
the molecular determinants of its activity are unknown.
Homology modeling has suggested that the protein scaffold
contains six transmembrane helices with the diiron site
coordinated to conserved histidines located in loop regions
on the cytoplasmic side of the helix termini (Figure 1).14 The
evidence of an exchange-coupled dinuclear iron center.4 The
spectrum of the diferric form of the enzyme is very similar to
that of the well-characterized toluene monooxygenase.5
However, the reduced, diferrous form of AlkB, which is stable
to air in the absence of substrate, was found to display an
unusually low isomer shift (δ = 1.05−1.15), suggesting a
distinct, histidine-rich diiron binding motif similar to that of
particulate fatty acid desaturases.4,6 Alanine scanning muta-
genesis has revealed eight essential histidines7 that are thought
to comprise a hemerythrin-like active site. As such, the catalytic
site of AlkB appears to be distinctly different from the
Received: June 18, 2012
Published: November 16, 2012
© 2012 American Chemical Society
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Article
RESULTS
■
Synthesis of 3,3,4,4-Norcarane-d4. 3,3,4,4-Norcarane-d4
was synthesized from 4,4,5,5-cyclohexene-d4 by a method
previously reported for the non-deuterated analogue.20 4,4,5,5-
Tetradeuteriocyclohexene was purchased from C/D/N Iso-
topes (99.8% D content), and its extent of deuteration was
confirmed by conversion to the corresponding trans-1,2-
dibromocyclohexane by treatment with bromine in dichloro-
methane. The deuterium content of this 1,2-dibromocyclohex-
ane was determined by observing the prominent m-Br ion (m/z
165) in the mass spectrum (Figures S1−S5 and Table S1). The
relative abundance of the m-Br-d3 ion (m/z 164) was 2.0%,
while in the non-deuterated control spectrum the intensity of
m/z 160 was 1.7% that of m/z 161. Accordingly, the very small
excess indicated that the extent of tetradeuteration was 99.8%.
The high degree of deuteration and the position of deuteration
in 3,3,4,4-norcarane-d4 were further confirmed by NMR (no
detectable C3 proton resonances).
Purification of AlkB and Determination of Activity.
Alkane hydroxylase (AlkB) was over-expressed in E. coli as
previously described4 and was purified by differential ultra-
centrifugation in the presence of 10% (v/v) ethylene glycol.
Hydroxylase activity was determined using an octane-depend-
ent NADPH consumption assay and monitoring the
production of 1-octanol by GC-MS. Typical activity obtained
for purified AlkB was in the range of 4.3 units/mg, where one
unit of activity is defined as the consumption of 1 μmol of
NADPH per min at 22 °C. The activities of AlkB toward
norcarane (1) and 3,3,4,4-norcarane-d4 (1-d4) compared to
octane were measured spectrophotometrically by monitoring
the rate of NADPH oxidation in the presence of excess
recombinant P. putida rubredoxin-2 and maize ferredoxin
NADPH reductase. Norcarane showed 30% of the activity of
octane, and deuterated norcarane showed 70% of the activity of
norcarane (Figure S6).
Figure 1. Depiction of the structure of AlkB showing six trans-
membrane helices, the histidine-rich diiron active site and a long, inter-
helix substrate binding channel from the homology model of van
Beilen et al.14
crystal structure of a member of an independently evolved
enzyme, the acyl−acyl carrier protein desaturase from Hedera
helix (English ivy), was recently reported at 1.95 Å resolution.15
Comparison of the active-site structure in the homologous Δ9
stearoyl−acyl carrier protein desaturase substrate complex from
Ricinus communis (castor oil plant)6b was recently used to
identify the structural basis for the differences in the
regioselectivity observed for these enzymes.6c
The mechanistic relationships between desaturase and
hydroxylase activity in these enzymes are not understood.
Desaturases are capable of acting as hydroxylases,6,12e,15 and
hydroxylases are capable of acting as desaturases.16 Apparently,
even small structural changes can affect the activity of these
enzymes; for instance, mutating a single amino acid residue,
threonine 199, to glutamic acid switched the reactivity of castor
Δ9-18:0-desaturase from a desaturase to that of an oxygenase.17
We have previously shown that AlkB hydroxylates saturated
mechanistic probes such as norcarane and bicyclo[3.1.0]hexane,
both in whole cell cultures and in cell-free extracts, via a
relatively long-lived (1−170 ns) substrate radical intermedi-
ate.18 The timing has indicated a hydrogen abstraction−radical
rebound−cage escape mechanistic scenario in which the
incipient carbon radical can either be captured within the
substrate−active site encounter complex or can migrate away
from the iron center via diffusive processes.18a Interestingly,
small amounts of desaturation products were also detected in
that work.19 Here we report results for the reaction of
norcarane as a substrate with highly purified and highly active
AlkB. Norcarane and its deuterated analogue 3,3,4,4-norcarane-
d4 were found to be excellent substrates for purified AlkB,
showing rapid turnover and high conversions. The enzyme
both hydroxylates and desaturates these substrates. By
comparing the product distribution of the deuterated norcarane
to that of the non-deuterated norcarane, we were able to
determine that the desaturation products from norcarane derive
from hydrogen abstraction at C3, while reaction at C2 results in
hydroxylation and radical rearrangement of the substrate.
Table 1. Results of NADPH Consumption Assay
rate (dA/
min)
result
activity
specific activity
a
compound
acetone
(units/min)
(units)
(units/mg AlkB)
−0.024
−0.061
3.97
9.89
nd
nd
norcarane-
d4
5.9
0.59
norcarane
−0.086
−0.29
14.0
47.0
10
43
1
b
octane
4.3
a
Corrected for acetone activity; one unit of activity is defined as the
b
consumption of 1 μmol of NADPH per min at 22 °C. Octane was
oxidized in 72% conversion.
Table 2. Parameters of Purified AlkB Activity toward
Norcarane (1) and Deuterated Norcarane (1-d4)
conversion
(%)
product yield
(nmol)
turnovers/
a
b
turnovers
s
1-d0
1-d4
57
66
70
476
529
458
1480
1642
1421
4.9
5.5
4.7
1.4 d0 and 1.0
d4
a
For 0.322 nmol of AlkB protein. Counting the secondary oxidations
b
would add about 10% to the TON for 1-d0 and 1% to 1-d4. Substrate
oxidations were run for 5 min.
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AlkB Product Analyses. AlkB oxidations and product
distribution analyses were performed with norcarane (1),
3,3,4,4-norcarane-d4 (1-d4), a 1.4:1 mixture of 1 and 1-d4, and
bicyclo[3.1.0]hexane as substrates. Each run was carried out
using 75 μg of AlkB enzyme in the presence of excess
recombinant P. putida rubredoxin-2 and maize ferredoxin
NADPH reductase. Substrates were introduced in acetone
solution. NADPH was added to initiate the reaction. Octane in
acetone was used as positive control and acetone alone as a
negative control. The reactions were carried out for 5 min at 25
°C, and then extracted with dichloromethane, which also served
to stop the reactions. The products found for the oxidations of
1 and 1-d4 by AlkB are shown in Scheme 1.
Scheme 1. Products from Norcarane Hydroxylation and
Desaturation Mediated by AlkB
Figure 2. Total ion chromatograms of norcarane-derived products
detected upon reaction with purified AlkB. (A) Oxidation of norcarane
(1); hydrocarbon products determined by GC method 1. (B)
Oxidation of 3,3,4,4-norcarane-d4 (1-d4); hydrocarbon products
determined method 1. (C) Oxidation of 1; alcohol products
determined by GC method 2. The small peak after endo-9 was also
present in the control. (D) Oxidation of 1-d4; alcohol products
determined by method 2. The small peak (∼0.5%) near 7.1 min was a
mixture and was disregarded (see Experimental Section).
to be a mixture and was not considered in the analysis.
Desaturation products made up only 4.8% of the products.
Norcarenes (2-d2 and 3-d3) and toluene (4-dn) were observed,
along with traces of 5-d2 and 6-d2. Fully deuterated 2-norcarene
(2-d4) was not observed. No ketones (2-norcaranone or 3-
norcaranone) were observed. Product identifications for the
deuterated products were facilitated by the consistent and
typical isotope effect on the retention time, with the deuterated
products appearing about 0.1 min before the protio cases.23
The observed mass spectral fragmentation of these products
was also typical of alicyclic alcohols, being dominated by
expected α-cleavage reactions, McLafferty rearrangements, γ-
hydrogen abstractions, and hydride shifts.24 Response factors
for the starting materials and products were within 3% and were
not corrected. The full product distributions are shown in
Table 3. Mass spectra of key products are shown in Figure 3
(also see Supporting Information). A graphical depiction of the
changes in product distribution upon deuteration is presented
in Figure 4. For bicyclo[3.1.0]hexane as the substrate, 3-
hydroxymethylcyclopentene, 4-hydroxycyclohexene, and 2-
hydroxy-bicyclo[3.1.0]hexanes (exo- and endo-) were detected
in a relative ratio of 11.1:0.27:1.0, respectively (Table S2 and
Figure S7).
The products in the organic extract (2−9) were identified
and quantified by comparison to authentic standards in the case
of norcarane products as has been previously described,19 and
by comparison to the undeuterated counterparts in the case of
deuterated products. Mass spectral total ion current traces for
product analyses for the AlkB-mediated oxidation of norcarane
and 3,3,4,4-norcarane-d4 are shown in Figure 2 and Figures S11
and S12. The major product of norcarane hydroxylation was
endo-3-norcaranol (endo-9), with a small amount of exo-3-
norcaranol (exo-9). The radical rearranged alcohol (7) and the
unrearranged alcohol (endo-8) were also observed in a 2.7:1
ratio. There was negligible exo-8, as determined by inspection
of the mass spectrum of endo-9, with which it co-eluted. The
ring expansion product 3-cycloheptenol, which could be readily
detected by this assay,21 was not detected in any of the AlkB
runs. This product was also absent in our earlier analysis of
toluene monoxygenase, T4MO.22 The desaturation products 2-
and 3-norcarene (3 and 2), toluene (4) and norcarenols (5 and
6) accounted for 31% of total products. In the case of 3,3,4,4-
norcarane-d4, the radical rearranged alcohol 7-d4 was the major
product, observed in a 3:1 ratio with the unrearranged alcohol
endo-8-d4. endo-9-d3 was a minor product. A very small GC-MS
peak (∼ 0.5%) at the time expected for exo-9-d3 was discerned
Isotope Effect Determination. The primary isotope effect
kH/kD (KIE) for C−H bond cleavage at C3 was calculated from
the change in regioselectivity observed for the undeuterated
and C3-deuterated norcarane. The general scheme for this
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Table 3. Distribution of Products from the AlkB Oxidation of Norcarane and 3,3,4,4-Norcarane-d4
a
b
a
b
ac
,
b
norcarane (1) products
% of products
5.6
norcarane-d4 (1-d4) products
2-d2
% of products
nd
mixture of 1 and 1-d4
% of products
2
2
1.4
nd
2-d4
3
3
13.6
5.1
3-d3
3.3
0.7
4.5
1.0
4.8
0.2
nd
3-d3
4
d
4
4-dn
c
4-dn
5
1.0
5-d2
0.6
5
5-d2
nd
6
5.4
6-d2
0.2
6
0.5
nd
6-d2
7
19.4
7.2
7-d4
65.2
21.7
7
12.9
29.8
7-d4
g
endo-8
endo-8-d4
endo-8
endo-8-d4
exo-9
endo-9
endo-9-d3
4.8
g
9.8
exo-9
4.2
exo-9-d3
nd
2.3
26.7
1.3
endo-9
38.6
endo-9-d3
8.3
eh
,
R/U
radical lifetime ,
2.7(0.2)
3.0(0.2)
fh
13.5(1) ns
15(1) ns
a
b
Compound numbers refer to Scheme 1; nd = not detected. Negligible amounts of exo-8 eluted with endo-9. Averages of at least three runs.
c
d
e
Products from 1.4:1 mixture of norcarane and norcarane-d4. In 4-dn, n = 1−3. R/U is the ratio of rearranged to unrearranged products, specifically
f
g
the ratio of 7 or 7-d4 over 8 or 8-d4. Radical lifetime is the inverse of R/U times the radical ring-opening rate of 2 × 108 s−1. Estimated by height of
h
m/z 81 vs 85. Numbers in parentheses represent the error.
Figure 4. Grouped product distribution of norcarane (top) and
norcarane-d4 (bottom) oxidation by AlkB.
Figure 3. Mass spectra of key products from the oxidation of
norcarane (1) and 3,3,4,4-norcarane-d4 (1-d4) by purified AlkB: (A) 7,
hydrogen isotope effect should be fully expressed, or nearly
(B) 7-d4, (C) 3 (the mass spectrum of 2 is almost identical), and (D)
so.27 Since there is only one substrate present, the measure-
ment of the intramolecular isotope effect by this method can be
3-d3.
determined from the product ratios and is independent of
kinetic parameters. Products can be grouped into C2
hydroxylated products (7 and 8), C3 hydroxylated products
(9), and desaturation products (2−6). Thus, the data afford
two independent measures of the same isotope effect (Scheme
situation is shown in Scheme 2, where k2/kH is the product
branching ratio for C2- and C3-derived products as described
by Miwa and Northrop et al.25 Oxidation of a 1.4:1 mixture of
norcarane and norcarane-d4 confirmed that these two substrates
have similar reactivity (1.2:1 ratio of protio and deuterio
2). The ratio of products from C2 compared to C3
hydroxylation is 1:1.6 for the protio substrate, and 10.5:1 for
the deuterated substrate, affording an apparent KIE for C3
hydroxylation by AlkB of 17. The ratio of products from C2
compared to desaturation is 1:1.15 for the undeuterated
substrate, and 18.1:1 for the deuterated case. Thus, the
apparent KIE for desaturation is 21.
products), confirming a high commitment to catalysis (Figure
S12). The secondary isotope effect for the effect of C3
deuteration on hydrogen abstraction at C2 (k2/k′2) is presumed
to be close to 1.0.26 Since norcarane is a small, nearly spherical
and featureless molecule, we have also assumed that
conformations that present the adjacent hydrogens at C2 and
C3 of the substrate are in rapid equilibrium. In such a case the
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with the purified protein provides strong support for a
hydrogen abstraction mechanism for AlkB that was first
deduced from results from whole cell preparations.18
Scheme 2. Determination of the Kinetic Isotope Effect in an
Isotopically Sensitive Branching Pathway from the Product
a
Ratios
Kinetic Isotope Effect and Desaturation. The analysis
presented in Scheme 2 shows that the primary isotope effect,
kH/kD, for C−H bond cleavage at C3 can be determined from
the product ratios independent of kinetic parameters. The
similarity of the two determinations, kH/kD = 17 and 21 for
hydroxylation and desaturation, respectively, indicates that
these are two measurements of the same event. Accordingly, we
take the isotope effect for C−H cleavage at C3 to be about 20.
It is clear from the very large suppression of desaturation
products upon C3 deuteration that essentially all of the
desaturation products must derive from initial hydrogen
abstraction at the C3 position, and not from C2. Desaturation
deriving from initial C2 hydrogen abstraction could not have
displayed such a large isotope effect. Confirmation of this
conclusion derives from the data from oxidation of the mixture
of norcarane and norcarane-d4. As can be seen in Figure S12, all
of the desaturation products appear at retention times
characteristic of undeuterated species. Further, from the data
in Table 3 there was very little deuterium content in the
observed desaturation products. Another indication that the
desaturation products derive from C−H bond cleavage at C3 is
the similarity of the ratios of the sum of all desaturation
products (2−6) to those of C3 hydroxylation (9) for the
deuterated and undeuterated situations (0.6 and 0.7 respec-
tively). Thus, for norcarane, the C3 radical partitions in a ratio
of 1:1.6 between desaturation and hydroxylation in both the
non-deuterated and the deuterated substrates, as would be
expected to the analysis depicted in Scheme 2.
A KIE of ∼20 is much larger than those commonly reported
for fatty acid hydroxylases (6−8).26,31 Exceptions are the very
large KIEs that have been reported for particulate Δ9
desaturases from the moth Spodopter littoralis32 and from the
cyanobacterium Spirulina platensis.33 Very large hydrogen
isotope effects have also been observed for sMMO34 and the
mono-iron hydroxylase, TauD.35 Such large isotope effects are
substantially above the classical limit, indicating that hydrogen
tunneling may play a significant role.36 The tunneling
component would be expected to be important for a sterically
inaccessible active site.
Desaturation activity for hydroxylases is known but not
common.37 Gibson et al. have shown that the non-heme iron
dioxygenase naphthalene dioxygenase catalyzes both hydrox-
ylation and desaturation of indan. The desaturation product
was subsequently transformed in part to a cis-glycol, as is usual
for this enzyme.38 Similarly, parallel hydroxylation/desaturation
has been observed for clavaminate synthase.39 The iconic
CYP101A1 (P450cam) from P. putida40 and human liver
P450s41 afford up to 14% desaturation of α-thujone to give
7,8-dehydrothujone. Valproic acid desaturation is known to be
mediated by a cytochrome P45042 and a CYP102A1 variant has
been found to give up to 35% desaturation of alkylben-
zenes.43,44
a
For the case of AlkB oxidation of norcarane, C2 represents all
products derived from initial oxidation at C2, while C3 represents
products from the C3 oxidation. For the AlkB oxidation of norcarane-
d4, C2-dn represents products from C2 oxidation, C3-dn represents
products from the C3 radical. The ratio k2/k′2 was taken to be 1.
DISCUSSION
■
Oxidation of Norcarane by Purified AlkB. The highly
purified AlkB preparations examined here showed both
hydroxylase and desaturase activity in their oxidations of
norcarane. Significant amounts of rearranged products provided
evidence for long-lived substrate radical intermediates. These
observations are of particular interest since AlkB and the
particulate desaturases such as stearoyl Δ9 desaturase appear to
be related, having histidine-rich active sites.4,6 The dramatic
changes in the product distribution observed upon full
deuteration of C3 in norcarane are very revealing. Deuteration
of this site induced a very large 20-fold shift in regioselectivity
from C3 to C2 as would be expected to result from a large
hydrogen isotope effect.26 This phenomenon is well known for
cytochrome P450 enzymes.25,28 A more recent and very
pertinent example of product switching that results from
hydrogen abstraction by an iron oxo intermediate from adjacent
carbons is the case of the nonheme iron halogenase SyrB2.
Here, substitution of deuterium for hydrogen changed the
regiochemical preference for C−H(D)C−H(D) bond cleavage
and the preference for chlorination or hydroxylation.29
The high activity of the purified AlkB allowed us to use mild
conditions and short reaction times (5 min), which minimized
the amount of secondary oxidation products. The fact that no
ketones (2-norcaranone or 3-norcaranone) were observed in
these purified enzyme reactions simplified the analyses and
indicated that the ketones observed in whole cell oxidations
were the result of further processing by other enzymes in the
cells, such as alcohol dehydrogenases, and not oxidation by
AlkB. We have previously shown that the wild-type P. putida
cells harboring AlkB showed more substrate-derived ketones
than transformed E. coli cells expressing AlkB that are known to
have relatively low dehydrogenase activity.30
As can be seen from the product distribution presented in
Table 3, the hydrogens at C3 are more reactive toward AlkB
than those at C2, despite having a stronger C−H bond.
Accordingly, substrate binding and steric considerations appear
to outweigh selectivity based on the thermodynamics of C−H
bond energies. This regioselectivity is consistent with the
preference for AlkB to hydroxylate the terminal position of n-
alkanes. The major product of norcarane hydroxylation was
endo-3-norcaranol (endo-9). The appearance of 20% of the
radical rearranged product, 3-hydroxymethyl cyclohexene (7)
Kinetic simulation and pathway flux analysis (Scheme S1 and
Figure S9) with a KIE = 20 for the C−H bond at C3, and a
large commitment to catalysis, correctly predicted the shift in
the yields of all products. Here it is assumed that substrate
binding allows sufficient motion to present the adjacent
hydrogens on C2 and C3 to the reactive iron-oxo intermediate.
The simulation also showed that the norcarenes afforded by the
desaturation pathway have about the same reactivity as that of
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norcarane. Oxidation of a 1.4:1 mixture of norcarane and
norcarane-d4 confirmed that these two substrates have similar
reactivity (1.2:1 ratio of protio and deuterio products), as
would be expected for essentially irreversible substrate binding
to the active site and, thus, a high commitment to catalysis. This
conclusion is also supported by the NADPH consumption
assay, which showed similar turnover rates for the protio and
deuterio substrates.
Timing the Substrate Radical Intermediates. The AlkB-
mediated hydroxylation at C2 led to unrearranged 2-
hydroxynorcarane (U) and a relatively large amount of 3-
hydroxymethyl cyclohexene, 7, the radical rearrangement
product (R) (R/U = 2.7 0.4). The product expected from
a cationic ring expansion, 3-cycloheptenol, was not observed.
Consideration of the known 2-norcaranyl ring-opening rate
constant (kr = 2 × 108 s−1) and application of a radical clock
analysis45 of the data, indicates a long-lived (13.5 ns) substrate
radical intermediate for the C2 hydroxylation of norcarane by
purified AlkB. Thus, oxygenation of the intermediate radical
occurs with a rebound rate of 8 × 107 s−1. For deuterated
norcarane, the ratio of rearranged to unrearranged products was
does encounter a barrier to the rebound process affording a
longer-lived radical intermediate. In this scenario, the apparent
radical lifetime would be a weighted average of the population
of radicals that followed one trajectory or the other, and this
averaging might well differ from substrate to substrate.
However, the very large amounts of rearranged products
observed with AlkB, and the very long apparent lifetimes of the
intermediate carbon radicals require additional considerations.
Stochastic behavior of the incipient substrate radical in the
enzyme active site would lead to a non-equilibrium situation
wherein some radicals collapse rapidly within bonding distance
of the active-site iron−oxo species whereas others escape
diffusively into the enzyme active site. Radical lifetimes in the
10−300 ns range are long enough to accommodate significant
diffusive migrations of the substrate radical within the enzyme
active site. Further, such lifetimes are much longer than the low
ps correlation times typical for spin state interconversions in
transition metal complexes.53 It could well be that the escaping
radicals are those that traverse the high-spin energy landscape
and encounter the larger recombination barrier. In the case of
AlkB, homology modeling has indicated that there is likely an
elongated substrate binding cavity that is able to accommodate
an alkane of medium chain length, such as octane (Figure 1).2a
The very long radical lifetime we have seen for bicyclo[3.1.0]-
hexane as an AlkB substrate and the lack of correlation with the
ring-opening rate constant would be the predicted result of
excursions of some radicals away from the active-site iron.18a
Accordingly, the interpretation of data for radical clock
rearrangements that occur within the confines of an enzyme
active site need to account for the kinetics of these excursions.
Mechanism of Competitive Desaturation, Hydroxyla-
tion, and Rearrangement of Norcarane by AlkB. A
substrate oxidation mechanism for AlkB that accommodates the
3-norcaranyl radical partitioning into desaturation and hydrox-
ylation, while the 2-norcaranyl radical leads to hydroxylation
and substrate rearrangement, is shown in Figure 5. The bis-μ-
oxo structure for the active diiron species has been depicted in
analogy to compound Q of MMO, although the reactive
intermediate for AlkB has not been observed and could be a
more extended structure.54 The large intramolecular deuterium
isotope effect, as reflected in the changes in product ratios upon
C3 deuteration, indicates that the transition states for C3 and
C2 hydrogen abstraction, TS-C3 and TS-C2, respectively, are
reversibly accessible and in effective competition. After the
initial hydrogen abstraction at C3 by the active iron oxidant,
either a normal rebound to afford endo-3-norcaranol, 9, or a
second hydrogen abstraction to give 3- or 2-norcarene, 3 and 2,
can occur. By contrast, hydrogen abstraction at C2 of norcarane
is shown to proceed to C2-hydroxylation and rearrangement of
the 2-norcaranyl radical. The lack of any detectable 3-
cycloheptenol among the products supports this view since
that product would require the formation of an intermediate 2-
norcaranyl cation. These divergent pathways seem to reflect
intrinsic differences in the behavior of the 3- and 2-norcaranyl
radicals.
3.0
0.1 (average of three runs), which affords a nearly
identical apparent rebound rate of 7 × 107 s−1 and a radical
lifetime of 15 ns. The apparent radical lifetime (τ) is
determined from the radical rearrangement rate constant (kr)
and the ratio of rearranged (R) and unrearranged (U) products
according to the relationship τ = R/(U kr). The net rebound
rate is simply the reciprocal of the apparent radical lifetime (τ).
Likewise, for bicyclohexane (kr = 2.9 × 107 s−1), the ratio of 3-
hydroxymethylcyclopentene to 2-hydroxybicyclo[3.1.0]hexane
was 11.4 2.5:1 (average of three runs), corresponding to an
apparent radical lifetime of 390 ns, which is unusually long for a
hydroxylase-generated carbon radical intermediate.46 Thus,
paradoxically, the substrate with a 7-fold slower radical
rearrangement rate has afforded 4-fold more of the rearranged
product. These results with the purified protein are consistent
with those we have found for whole cell and cell free extract
assays with AlkB.18,30,47 Accordingly, the variation in substrate
radical lifetimes is an intrinsic part of the enzyme mechanism,
rather than the result of effects deriving from complications
inherent to whole cell metabolism. While substrate radical
lifetimes of 10−15 ns are probably not long enough to allow
significant escape of the substrate radicals from the active site,
the very long apparent radical lifetimes for bicyclohexane-
derived radicals makes that a possibility. We have recently
reported that substrate radicals do escape the active site of
CYP2E1, leading to capture by oxygen and unique carbonyl
products.46 The absence of carbonyl products observed with
AlkB make this scenario unlikely. The results support the view
derived from substrate rearrangements that a hydrogen
abstraction leading to a substrate radical intermediate is the
predominant pathway for C−H hydroxylation among other
iron hydroxylases (toluene monooxygenase,22 soluble methane
monooxygenase,48 and XylM,30 naphthalene dioxygenase49)
and cytochrome P450.40,45,46,50
Shaik et al. have proposed a two-state hypothesis to explain
the curious radical clock timing50c,51 and unusual isotope effect
behavior52 of high-valent iron species for cytochrome P450
hydroxylations. In this interpretation, nearly degenerate,
multiple spin states of the reactive iron oxo species may lead
to a situation wherein one reaction pathway, usually a low-spin
trajectory, can collapse to form a C−O bond of the product
alcohol without a significant barrier, whereas a high-spin state
The conformations and potential energies of the 2-
norcaranyl radical have been calculated using DFT methods
by Jager et al.55 We have reproduced those calculations with
̈
similar results and extended the analysis to include the 3-
norcaranyl radical (Figure 6). Both radicals have two stable
conformations that are either chair-like or boat-like with regard
to one methylene group (C4) in the six-membered ring. From
the data we know that the endo alcohols are the preferred
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products for both C3 and C2 hydroxylation. Therefore, the
substrate radical presumably approaches the iron-oxo inter-
mediate on the endo face in spite of potential steric interference
from the adjacent cyclopropyl ring. In the preferred
conformation of the 2-norcaranyl radical (conformation 2b in
Figure 6A), the C3 endo hydrogen is quasi-equatorial, and will
have poor overlap with the C2 radical center. Further, the C2
radical is expected to be partially delocalized into the
cyclopropyl ring (Figure 6B) as is suggested by the 3 kcal/
mol stabilization of the 2-norcaranyl radical with respect to the
C3 isomer. The boat to chair rearrangement of the 2-norcaranyl
radical was found to have a 5 kcal/mol barrier (Figure S8). By
contrast, we find the 3-norcaranyl radical to be very flexible,
with only a 2 kcal/mol barrier to chair-boat rearrangement. As
can be seen in Figure 6A, the more stable conformation of the
3-norcaranyl radical (3b) has pseudoaxial hydrogens adjacent to
the C3 radical on both C2 and C4. We suggest that the
conformational flexibility of the 3-norcaranyl radical and these
pseudoaxial hydrogens with good overlap with the radical
SOMO facilitate the desaturation pathway that affords both 2-
and 3-norcarene (Figure 6B).
The desaturation-rebound scenario described here is
analogous to the parallel reaction pathways observed for
radical−radical recombination and disproportionation that is
typical of carbon-centered radicals. Interestingly, resonance
stabilized radicals, such as the cumyl radical, exhibit a decreased
tendency for disproportionation as compared to tert-butyl
radicals.56 The effect has been attributed to a loss of resonance
energy upon hydrogen atom transfer and the resulting lower
reaction enthalpy change. Radical−radical recombinations
appeared to be insensitive to this effect. Applied to the
situation with the 3- and 2-norcaranyl radicals, one would
predict that the desaturation pathway for the 2-norcaranyl
radical would be less favorable than that for desaturation of the
3-norcaranyl radical due to the radical delocalization into the
cyclopropyl ring. Interestingly, Lipscomb has shown that the
oxidation of ethyl benzene by the diiron methane mono-
oxygenase (MMO) affords 9% of the desaturation product
styrene when the effector protein MMOB is present but almost
none in the absence of MMOB.16 The role of MMOB has been
suggested to control and limit substrate access to the reactive
diiron center.
Figure 5. Mechanism of parallel, competitive desturation, hydrox-
ylation and rearrangement of norcarane (1) mediated by AlkB. The
structure of the active species is modeled in analogy to compound Q
of sMMO.
Reactions of deuterated fatty acids with enzymes like the
yeast particulate Δ9 desaturase have shown that there is a
typical kH/kD of 7.1 for reaction in the C9 position, while a
small primary KIE of 1.03 was observed for reaction with
deuterium at the C10 position. This outcome is indicative of a
stepwise reaction, wherein a hydrogen is first abstracted from
the C9 site and then a second hydrogen atom is lost from the
adjacent C10 site (Figure 7).26 The data presented here for the
oxidation of norcarane by AlkB support a mechanism in which
the substrate reaction with the reactive iron oxo intermediate of
AlkB is initiated by hydrogen atom abstraction from either C2
or C3. The resulting C2 cyclopropylcarbinyl radical is long-
lived and rearranges to a large extent to the cyclohexenylmethyl
radical. Hydrogen abstraction from C3 results in the 3-
norcaranyl radical that either rebounds to form endo-3-
norcaranol (endo-9) or desaturates via a second hydrogen
atom abstraction from C2 or from C4 to afford 2- and 3-
norcarene (2 and 3). It has been shown that the preferred
stereochemistry for fatty acid desaturation by the castor Δ9 and
palmitoyl-ACP Δ4 desaturases are syn (pro-R at both carbons),
with the alkyl chain folded into an eclipsed conformation at the
Figure 6. (A) Lowest energy conformations of 2- and 3-norcaranyl
radicals as determined by DFT (kcal/mol). (B) Comparative Mulliken
spin densities.
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Figure 7. Comparison of fatty acid desaturation with 3,3,4,4-norcarane-d4 hydroxylation/desaturation.
positions of desaturation.57 Thus, it appears that the desaturases
favor the dehydrogenation pathway over hydroxylation in part
through favorable orientation of the incipient radical center
with the second scissile C−H bond, as shown in Figure 7.
The cells were harvested by centrifugation at 16000g for 6 min, and
AlkB purification was carried out following the protocol from Shanklin
et al.4 with several modifications. Cells (15 g) were resuspended in 50
mL of 50 mM Hepes (pH 8.0) containing 4 mM MgCl2, 2 mM
phenylmethylsulfonyl fluoride, 10 mg of DNase I, and 50 mg of
catalase. The cell suspension was disrupted by single passage through a
French pressure cell with a 70 MPa pressure drop. The resulting
extract was clarified by centrifugation at 43000g for 45 min. The
supernatant fraction was divided among four 14 × 89 mm polyallomer
SW41 centrifuge tubes (Beckman). The membrane pellets were
harvested after centrifugation at 285000g for 90 min and diluted to 15
mL of 25 mM Hepes (pH 7.5) containing 10% (v/v) ethylene glycol
(buffer A) by sonication. The resuspended material was applied in
three separate, equal volume portions at 1 mL/min to a 1 cm × 20 cm
column of POROS 20CM (Perseptive Biosystems, Cambridge, MA)
equilibrated with buffer A and eluted with a linear gradient of buffer A
containing 0−600 mM NaCl at 4 mL/min over 38 min. Fractions
enriched in AlkB were pooled and collected by centrifugation at
240000g for 1 h. The resulting pellet was resuspended by sonication in
an approximately equal volume of buffer. The final enzyme preparation
was frozen in liquid nitrogen and stored at −70 °C.
AlkB Activity Assays: Pseudomonas putida Alkane Hydrox-
ylase (AlkB) and NADPH Consumption. As previously described,58
the activity of AlkB toward various substrates was determined by
monitoring the drop in the absorbance at 339 nm in the presence of
excess recombinant P. putida rubredoxin-2 and maize ferredoxin
NADPH reductase (Figure S6). One unit of activity is defined as the
consumption of 1 μmol of NADPH per minute at 22 °C. For the
octane assay is based on that of McKenna and Coon:58 0.5 mL,
containing 50 mM Tris-HCl at pH 7.5, 20 μg of rubredoxin, 10 μg of
FNR (35 mU), 200 μM octane and 250 μM NADPH. AlkB was ∼10
μg (3 μL of 3 mg/mL).
CONCLUSIONS
■
A purified form of the non-heme diiron hydroxylase AlkB from
P. putida was investigated using the diagnostic substrate
norcarane and a selectively deuterated derivative. The AlkB-
catalyzed oxidation of norcarane revealed significant desaturase
activity in addition to the expected hydroxylase activity. The
same large deuterium isotope effect was observed both for
hydroxylation at C3 and desaturation, indicating that all of the
desaturation products resulted from initial hydrogen removal
from C3. This conclusion is confirmed by the observed low
deuterium inventory of the desaturation products derived from
a mixture of norcarane and norcarane-d4 and the insensitivity of
the ratio of desaturation and hydroxylation to deuteration at
C3. By contrast, hydrogen removal from C2 to form a long-
lived 2-norcaranyl radical led to C2 hydroxylation and
significant amounts of radical rearrangement product. These
results add new support to the view that the desaturation and
hydroxylation reaction pathways are very closely related, with
control of the reaction channels dictated both by the substrate
radical conformation and steric accessibility to the iron−oxo
active site. While the X-ray crystal structure of AlkB remains
elusive, it is apparent that a diiron center heavily coordinated
with histidines, rather than the carboxyl-rich coordination
sphere of sMMO and its relatives, can perform both of these
biologically important reactions.
Substrate Activity Assays. Product distribution assays were
carried out using 75 μg of AlkB for each substrate with octane in
acetone as positive control and acetone as negative control. Substrates
were introduced in acetone solution (5 μL in 1 mL of acetone). The
reactions were carried out for 5 min and then stopped with excess
dichloromethane. The products were identified and quantified by GC-
MS, using an Agilent 7890A GC coupled to a 5975C inert MSD with a
Rtx-5Sil MS (30 m length, 0.25 mm i.d., 0.25 μm 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/
EXPERIMENTAL SECTION
■
AlkB Expression and Purification. The alkane-hydroxylase gene
was amplified using a forward primer containing nde1 and reverse
having a BglII site. The amplified product was restricted with nde1 and
BglII and cloned into nde1 and BamH1 sites of pET9a vector. For
expression, pAlkB-9a was introduced into E. coli BL21(DE3)pLysS and
grown as described earlier.4 AlkB production was induced by addition
of isopropyl β-D-1-thiogalactopyranoside to 0.4 mM at 37 °C for 4 h.
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min. Method 1 gave excellent separation of the desaturation products
(Figures 2A and S11) but did not resolve hydroxymethylcyclohexene
(7) from endo-2-norcaranol (8). Method 2 gave excellent separation of
the hydroxylation products (Figure 2B). The small amounts of
secondary oxidation products from norcarenes (hydroxynorcarenes, 5
and 6) were adequately separated from the radical rearranged alcohol
and did not interfere with the determination of the radical lifetime,
consistent with our previous results50a,d and contrary to other
reports.59
The deuterium content of the 1,2-dibromocyclohexane was
determined by observing the parent Br ion of the mass spectrum.
The deuterium content was found to be 99.8%.
MS: m/z (relative abundance) 248 (2), 246 (4), 244 (2), 167 (30),
165 (31), 85 (100), 84 (44).
The exact breakdown in the parent Br peak (m/z 167/165) is
shown in Figure S1.
COMPUTATIONAL METHODS
■
The data in Table 3 were determined by digital integration of the
TIC in Figure 2 and Figures S11 and S12 using the Agilent
ChemStation software package. Controls showed that norcarane,
norcarenes and the product alcohols all had very similar response
factors ( 4%), so no corrections were used. There was negligible exo-
8, as determined by inspection of the mass spectrum of endo-9, with
which it co-eluted. These two isomers could be readily distinguished
by the parent ion regions since exo-8 showed a prominent m-1 peak at
m/z 111, while endo-9 had a significant parent ion at m/z 112.
Synthesis of 3,3,4,4-Norcarane-d4. Zinc−copper couple20 (0.12
g, 2 mmol) was stirred with diethyl ether (1 mL) and a crystal of I2 was
added to help initiate the reaction. CH2I2 (0.16 mL, 1.9 mmol) and
4,4,5,5-cyclohexene-d4 (0.08 g, 0.9 mmol) were added, and the mixture
was heated for 17 h at 60 °C in an oil bath. The reaction was
quenched, and the zinc salts dissolved, by the careful addition of
saturated aqueous ammonium chloride. The layers were separated, and
the aqueous layer was extracted with ether (3 × 0.5 mL). The
combined organic layers were washed successively with water (0.5
mL) and brine (0.5 mL), and then dried with solid magnesium sulfate.
After removal of the drying agent, the solvent was carefully blown off
under a stream of nitrogen, and the residue was subjected to
preparative gas chromatography (packed column, 10% RTX-1
Silcoport W 100/120 mesh, 2 m length, 2 mm i.d., 0.25 in o.d.,
oven temperature 130 °C, TCD detector temperature 200 °C, injector
temperature 200 °C). The final yield was 11 μL of 3,3,4,4-norcarane-
d4.
The DFT calculations were performed with the Gaussian03 suite of
programs (see Figure S8). The scan of the potential energy surfaces for
chair−boat interconversion at C4 started from conformers 2a and 3a
(Figure 6), by changing the dihedral angles (C6−C5−C4−H) in
conformers 2b and 3b by 6° at each point. All other degrees of
freedom were optimized without symmetry constraints. The critical
points from the potential surface scan were chosen for further
transition state optimization. The geometries for each conformer and
transition state were fully optimized using B3LYP/6-31G(d) methods.
Minima and transition states were confirmed by frequency analysis
with the same methods. The zero-point energies and the thermal
correction to Gibbs free energies derived from this analysis were used
for further correction of the single-point energies. Single-point
energies were calculated with a larger basis set, 6-311++G(2df, 2p).
The final free energy change was then obtained after correction for
ZPE (scaling factor 0.9806), thermal corrections (0→298 K), and the
entropy terms. The reference state for all of the calculated gas-phase
free energies was 1 atm and 298 K.
ASSOCIATED CONTENT
* Supporting Information
■
S
Mass spectra of starting materials; total ion current plots and
mass spectra of products; plot of NADPH consumption;
potential energy plots for 2- and 3-norcaranyl radicals;
comparisons of experimental and simulated product ratios.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1H NMR: δ (CDCl3) −0.05 (dd, 1H, J = 5.15 Hz, 4.27 Hz), 0.48
(td, 1H, J = 4.40, 8.80 Hz), 0.81 (m, 2H), 1.60 (d, 2H, J = 8.80 Hz),
1.80 (dd, 2H, J = 4.40 Hz, 13.24 Hz). 13C NMR: (CDCl3): 9.54
(cyclopropane CH2), 10.46 (cyclopropane CH), 23.75 (cyclohexane
methylene) ppm.
Neither evidence of isomerization nor protons in the deuterium
positions were observed by NMR.
MS: m/z (relative abundance) 100 (38), 85 (100), 84 (52) 71 (94),
70 (71), 57 (48), 56 (47).
AUTHOR INFORMATION
Corresponding Author
jtgroves@princeton.edu
■
Notes
The authors declare no competing financial interest.
Norcarane Authentic Standards and Determination of
Radical Lifetime. Norcarane and authentic standards of the
oxygenation and desaturation products were prepared, and the
determination of the radical lifetime was carried out as described
previously (Figures S10 and S13).47 The radical lifetime was
determined from the ration of 7 and endo-8 (Figure S15). Very
similar values were obtained from the ratio of 7-d4 and endo-8-d4. GC-
MS analysis indicated that the starting norcarane was completely free
of toluene, norcarenes, and oxygenated products. Starting materials
and products have nearly identical response factors and were not
corrected (Figure S14). Bicyclohexane and product standards were
prepared as previously described.50a
Bromination of 4,4,5,5-Cyclohexene-d4. 4,4,5,5-Cyclohexene-d4
was purchased from C/D/N Isotopes (99.8% D content), and its
extent of deuteration was checked by conversion to the corresponding
1,2-dibromocyclohexane in the following manner: Bromine was added
dropwise to 4,4,5,5-cyclohexene-d4 (10 μL) in dichloromethane (0.5
mL) until the brown color persisted. A separate bromination of
cyclohexene was used as a control. The resulting solutions were diluted
100-fold and subjected to GC-MS analysis, using an Agilent 7890A GC
coupled to a 5975C inert MSD with a Rtx-5Sil MS (30 m length, 0.25
mm i.d., 0.25 μm film) column. A GC-MS thermal gradient method
was used: starting at 30 °C, holding for 6 min, ramping to 230 °C at 10
°C/min.
ACKNOWLEDGMENTS
■
Support of this research by the National Science Foundation
(CHE-0616633 and CHE-1148597 to J.T.G.) and the National
Institutes of Health (2R15GM072506 to R.N.A.) is gratefully
acknowledged. J.S. and G.M. acknowledge support from the
office of Basic Energy Sciences of the United States
Department of Energy. M.P.C. received a Beckman Scholars
fellowship.
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