Use of perfluorocarboxylic acids to trick cytochrome P450BM3 into initiating the hydroxylation of gaseous alkanes

Article abstract of DOI:10.1002/anie.201007975

It has long been believed that the fatty acid hydroxylase wild-type P450BM3 is unable to oxidize gaseous alkanes. However, the simple addition of a perfluorocarboxylic acid as a dummy substrate to initiate the P450BM3 catalytic cycle enabled the efficient hydroxylation of butane and propane (see picture).

Full text of DOI:10.1002/anie.201007975

DOI: 10.1002/anie.201007975
Alkane Activation
Use of Perfluorocarboxylic Acids To Trick Cytochrome P450BM3 into
Initiating the Hydroxylation of Gaseous Alkanes**
Norifumi Kawakami, Osami Shoji, and Yoshihito Watanabe*
The catalytic hydroxylation of inert CÀH bonds under mild
initiated by fatty-acid binding to the heme cavity of
P450BM3. This binding induces a structural change in
P450BM3 and the removal of a water molecule coordinated
[1]
conditions is a major challenge in synthetic chemistry.
[2–4]
Despite important advances in this area,
the direct
3
+
activation of alkanes is still a key research topic. Such
transformations are of great value to the chemical industry. In
particular, the hydroxylation of gaseous alkanes has become
an increasingly important approach for producing liquid fuel
to the heme iron atom (Fe ). It results in a positive shift in
[
8,13,14]
the reduction potential of the heme iron atom,
followed
by electron transfer from NADPH (the reduced form of
3
+
nicotinamide adenine dinucleotide phosphate) to reduce Fe
[5]
2+
or chemical precursors from natural gas. Biocatalysts
provide an alternative to conventional chemical processes
to Fe . After the reductive activation of molecular oxygen to
generate a highly active oxidant species, the oxoferryl(IV)
[6]
[15]
for alkane hydroxylation. Iron-containing monooxygenases,
such as soluble methane monooxygenase (sMMO), alkane
w-hydroxylase (AlkB), and cytochrome P450s (P450s), show
remarkable catalytic activity towards the oxidation of a
porphyrin p cation radical (so-called compound I),
the
bound substrate is oxidized by compound I (Figure 1a).
According to this reaction mechanism of P450BM3, substrate
binding is crucial for initiation of the catalytic cycle. Thus,
substrates whose structures are different from that of the fatty
acid cannot enable the formation of compound I from
P450BM3. Therefore, P450BM3 does not catalyze the hy-
droxylation of gaseous alkanes, such as methane, ethane, or
propane, because gaseous alkanes cannot bind to the heme
active site of P450BM3. Since the substrate specificity in an
enzymatic reaction is governed by the local chemical environ-
ment of the enzyme active site, mutants of P450BM3 have
been prepared as biocatalysts for the hydroxylation of
[6,7]
variety of alkanes with dioxygen to give alcohols.
P450BM3 (CYP102A1) isolated from Bacillus megaterium is
a promising enzyme, as it has the highest catalytic rate
À1
reported so far for a P450 (> 15000 min with arachidonic
acid). The high catalytic ability of P450BM3 is attributed to
the location of its heme and reductase domains on a single
[
8]
polypeptide chain. The hydroxylation rate is even faster
À1
than those of sMMO (222 min with methane) and AlkB
À1
[9,10]
(
210 min with octane).
hydroxylation at the terminus of alkyl chains (w-1, w-2, and w-
; see Figure 1) of long-alkyl-chain fatty acids, such as myristic
acid and palmitic acid, as well as some unsaturated fatty
P450BM3 exclusively catalyzes
[
16,17]
gaseous alkanes.
3
Although the mutagenesis of P450BM3 to construct a
binding pocket suitable for gaseous-alkane hydroxylation is
regarded as a promising method, we propose a simple and
unique strategy for the hydroxylation of gaseous alkanes with
wild-type P450BM3 without the replacement of any amino
acid residues. We assumed that compound I could be formed
from P450BM3 by the addition of a “dummy substrate” with a
chemical structure similar to that of a fatty acid. Studies on
the binding of its inhibitors have shown that P450BM3 has a
large heme cavity that can accommodate two different
[
11,12]
acids.
The crystal structure of the bound form of P450BM3 with
palmitoleic acid shows that palmitoleic acid is fixed by two
major interactions: 1) the ionic interaction of the substrate
carboxylate group with Arg47 and Tyr51, and 2) the hydro-
phobic interaction of the alkyl chain with amino acids at the
substrate-binding site. The catalytic cycle of P450BM3 is
[
18,19]
molecules at the same time
and thus indicate that the
[
*] Dr. N. Kawakami, Prof. Dr. Y. Watanabe
Research Center for Materials Science, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81)52-789-2953
simultaneous binding of a gaseous alkane and a dummy
substrate would be possible. If the dummy substrate had
stable chemical bonds, such as CÀF bonds, that were not
[
20]
oxidizable, and if gaseous alkane molecules could penetrate
into the active site of P450BM3 in the presence of such a
dummy substrate, then gaseous alkane molecules could be
hydroxylated. Herein, we report that the addition of per-
fluorocarboxylic acids (PFs) as dummy substrates to wild-type
P450BM3 results in the hydroxylation of gaseous alkanes to
the corresponding alcohols.
E-mail: p47297a@nucc.cc.nagoya-u.ac.jp
Dr. O. Shoji
Department of Chemistry, Graduate School of Science
Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
[
**] Two plasmids encoding the full length of P450BM3 and the heme
domain of P450BM3 were kindly supplied by Prof. S. G. Sligar,
University of Illinois (USA). This research was supported by a Grant-
in-Aid for Scientific Research (S) to Y.W. (19105044) from the
Ministry of Education, Culture, Sports, Science, and Technology
Our strategy for the hydroxylation of gaseous alkanes and
a plausible active-site structure of P450BM3 containing both
perfluorodecanoic acid and propane are shown in Figure 1c.
We selected a series of PFs with alkyl chains containing 8–14
carbon atoms (PFC8–PFC14) as dummy substrates. We
expected that PFs with shorter alkyl chains than those of
(
(
Japan) and a Grant-in-Aid for Young Scientists (A) to O.S.
21685018).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007975.
Angew. Chem. Int. Ed. 2011, 50, 5315 –5318
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5315

Communications
Figure 1. a) General catalytic reaction mechanism of P450s. b) The natural and c) our proposed reaction of P450BM3. In (b) and (c), the
respective reaction model is shown on the left, and the image at the top right shows the active site of P450BM3 containing palmitoleic acid (PDB
accession No. 1FAG) and a plausible active-site structure containing manually docked propane and PFC10, respectively (O red, N dark blue, Fe
brown; C pale blue in (b); H white, C green, F aqua blue in (c)). The atoms of the heme group, substrates, and PFC10 are shown as spheres.
Hydrogen atoms are omitted for clarity, except those on the propane molecule. The respective substrate and hydroxylated product are shown at
the bottom right of (b) and (c); the oxygen atom inserted into the substrate is shown in red.
natural substrates would provide space for gaseous alkanes
because of the incomplete occupation of the heme cavity; the
substrate-binding site of P450BM3 naturally accommodates a
fatty acid containing 16 carbon atoms.
Table 1: Dissociation constants of the PFs and NADPH-consumption
rate in the presence of the PFs.
Dummy substrate
Kd
NADPH consumption
[min ]
À1 [a]
[mm]
Initially, we investigated whether a series of PFs would
serve as dummy substrates to remove the water molecule
coordinated to the heme iron atom of P450BM3 as the first
step of the catalytic cycle (Figure 1a) by examining the
change in the UV/Vis spectrum of P450BM3 on the addition
of PFs with alkyl chains containing 8–14 carbon atoms. A
spectral change would be indicative of binding to the dummy
substrate, and the dissociation constant of the PFs was
PFC8
PFC9
1900
980
290
91
30
1.8
13
72Æ19
190Æ6
310Æ8
330Æ20
430Æ20
440Æ20
390Æ4
PFC10
PFC11
PFC12
PFC13
PFC14
[
a] The unit for NADPH consumption is (nmol NADPH consumed)
À1
À1
estimated to be in the range K = 1.8–1900 mm (Table 1; see
d
min (nmol P450) . Values are averages Æstandard deviation calcu-
also Figure S1 in the Supporting Information).
lated from three different experiments.
5
316
www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 5315 –5318

NADPH to the heme iron atom was initiated by PFC9–
PFC14.
We next investigated the hydroxylation of gaseous alkane
molecules (methane, ethane, propane, and butane) and
cyclohexane with P450BM3 in the presence of a series of
PFs (Figure 3; see also Table S1). All gaseous alkanes were
supplied as a gas-saturated buffer maintained at a pressure of
5
–8 kPa; the mixed gas contained the gaseous alkane and
oxygen in an 8:2 ratio during the reaction. We examined the
rate of product formation and NADPH oxidation, as well as
the coupling efficiency (i.e., the ratio of product formation to
NADPH consumption), for a reaction time of 10 min
(
Figure 3). Propane, butane, and cyclohexane were hydroxy-
lated with P450BM3 (500 nm) in the presence of a PF
100 mm) to yield 2-propanol, 2-butanol, and cyclohexanol,
Figure 2. a) Reaction mechanism for the formation of a ferrous CO
complex. The terms E and EPF denote the P450BM3 and P450BM3/PF
complexes, respectively. b) UV/Vis spectra of P450BM3 in a buffer
saturated with CO gas before the addition of NADPH (black) and after
the addition of NADPH in the absence (blue) and presence (orange)
of PFC10.
(
respectively. The reactions with methane and ethane did not
lead to any detectable amount of the corresponding alco-
Although the UV/Vis spectral
changes induced by the PFs were
small, the elimination of the ligated
water molecule initiated the con-
sumption of NADPH (Table 1). This
NADPH oxidation, coupled with the
production of H O through the four-
2
electron reduction of the iron-atom-
bound oxygen molecule only in the
[
21]
presence of the PFs, indicated that
the binding of the PFs induces the
formation of compound I, followed
by its reduction by NADPH in the
absence of a gaseous substrate. The
reduction of the ferric heme of
P450BM3 with PFs (EPFferric) to
ferrous
heme
(EPFferrous
)
by
NADPH provided further confirma-
tion of the formation of a ferrous CO
complex (EPFferrousCO; Figure 2a).
The formation of a ferrous CO
complex of P450BM3 (EPFferrousCO
)
with a UV/Vis absorption maximum
at 448 nm was observed when
NADPH was added to a CO-satu-
rated reaction mixture containing
PFC9–PFC14, whereas no ferrous
CO complex was observed in the
absence of PFs (Figure 2b). In con-
trast to PFC9–PFC14, PFC8 did not
induce the formation of the ferrous
CO complex, although
a
small
amount of NADPH was consumed,
which implies that PFC8 is not effec-
tive for the elimination of the iron-
Figure 3. Histograms showing the formation rate of a) 2-propanol, b) 2-butanol, and c) cyclohexanol
produced by P450BM3 with various PFs. The square symbols denote the coupling efficiency. The error bars
denote the standard deviation calculated from three different experiments. The model on the right of each
graph shows the occupation of the heme cavity by a combination of the preferred PF with the corresponding
bound H O because of its short alkyl
2
chain. These results showed that the
removal of iron-bound H O and the
2
subsequent electron transfer from alkane.
Angew. Chem. Int. Ed. 2011, 50, 5315 –5318
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5317

Communications
[
22]
hols. In the absence of PFs, no product was detected for any
of these alkanes.
[
[
[
1] A. E. Shilov, G. B. Shulꢀpin, Chem. Rev. 1997, 97, 2879 – 2932.
2] M. S. Chen, M. C. White, Science 2007, 318, 783 – 787.
3] A. Company, L. Gꢁmez, X. Fontrodona, X. Ribas, M. Costas,
Chem. Eur. J. 2008, 14, 5727 – 5731.
4] E. McNeill, J. D. Bois, J. Am. Chem. Soc. 2010, 132, 10202 –
10204.
These results clearly show that the addition of the dummy
substrate to wild-type P450BM3 conferred the ability to
catalyze the hydroxylation of gaseous alkanes. Interestingly,
the rates of product formation and NADPH oxidation were
significantly influenced by the alkyl-chain length of the PFs.
[
[5] J. A. Labinger, J. E. Bercaw, Nature 2002, 417, 507 – 514.
6] M. Ayala, E. Torres, Appl. Catal. A 2004, 272, 1 – 13.
[
PFC10 showed the highest rate of product formation
À1
[7] K. Rabe, V. Gandubert, M. Spengler, M. Erkelenz, C. Niemeyer,
(
67 min ) and the highest coupling efficiency (18%) of the
Anal. Bioanal. Chem. 2008, 392, 1059 – 1073.
[
23]
PFs examined for propane hydroxylation.
The fact that
[
8] S. N. Daff, S. K. Chapman, K. L. Turner, R. A. Holt, S. Govin-
daraj, T. L. Poulos, A. W. Munro, Biochemistry 1997, 36, 13816 –
13823.
decanoic acid did not induce the formation of 2-propanol
clearly indicates that the substitution of hydrogen with
fluorine in the carboxylic acid is crucial for hydroxylation.
A decrease in the formation of 2-propanol was observed when
the length of the PF was changed. This result indicates that
the size of the heme cavity of P450BM3 depends on the alkyl-
chain length of the bound PF, and that the cavity size provided
by PFC10 is the most suitable for fixing propane molecules. In
accord with our hypothesis, the larger alkanes tended to
prefer the PFs with shorter alkyl chains for an efficient
reaction (Figure 3, right). PFC9 and PFC10 were almost
equally effective for butane hydroxylation and showed the
[9] B. G. Fox, W. A. Froland, D. R. Jollie, J. D. Lipscomb, Methods
Enzymol. 1990, 188, 191 – 202.
10] J. Shanklin, C. Achim, H. Schmidt, B. G. Fox, E. Mꢂnck, Proc.
Natl. Acad. Sci. USA 1997, 94, 2981 – 2986.
11] L. O. Narhi, A. J. Fulco, J. Biol. Chem. 1986, 261, 7160 – 7169.
12] S. S. Boddupalli, B. C. Pramanik, C. A. Slaughter, R. W. Esta-
brook, J. A. Peterson, Arch. Biochem. Biophys. 1992, 292, 20 –
28.
[13] S. G. Sligar, Biochemistry 1976, 15, 5399 – 5406.
14] M. B. Murataliev, R. Feyereisen, Biochemistry 1996, 35, 15029 –
5037.
[
[
[
[
1
À1
[15] T. W. Ost, J. Clark, C. G. Mowat, C. S. Miles, M. D. Walkinshaw,
maximum rate of formation of 2-butanol (100 and 113 min ),
G. A. Reid, S. K. Chapman, S. Daff, J. Am. Chem. Soc. 2003, 125,
whereas PFC9 showed the highest coupling efficiency of
15010 – 15020.
5
7%. In the hydroxylation of cyclohexane, PFC9 showed the
[
16] R. Fasan, M. M. Chen, N. C. Crook, F. H. Arnold, Angew. Chem.
2007, 119, 8566 – 8570; Angew. Chem. Int. Ed. 2007, 46, 8414 –
8418.
À1
highest rate of cyclohexanol formation (110 min ) with a
[
24]
coupling efficiency of 35%. Although PFC8 did not induce
the formation of the ferrous CO complex, PFC8 was effective
for the hydroxylation of cyclohexane with a coupling effi-
ciency of 45%. This result suggests that a cooperative effect
operates. In fact, the ferrous CO complex was formed in the
presence of both PFC8 and cyclohexane (see Figure S2).
These findings show that a combination of the alkyl-chain
length of the PFs and the size of the alkane governs the
efficiency of the hydroxylation reaction catalyzed by
P450BM3 with a dummy substrate.
[
[
[
17] P. Meinhold, M. W. Peters, M. M. Chen, K. Takahashi, F. H.
Arnold, ChemBioChem 2005, 6, 1765 – 1768.
18] D. C. Haines, B. Chen, D. R. Tomchick, M. Bondlela, A. Hegde,
M. Machius, J. A. Peterson, Biochemistry 2008, 47, 3662 – 3670.
19] M. A. Noble, L. Quaroni, G. D. Chumanov, K. L. Turner, S. K.
Chapman, R. P. Hanzlik, A. W. Munro, Biochemistry 1998, 37,
15799 – 15807.
[
[
20] R. E. Banks, J. C. Tatlow, J. Fluorine Chem. 1986, 33, 227 – 346.
21] N. Pons, S. Pipino, F. De Matteis, Biochem. Pharmacol. 2003, 66,
405 – 414.
[
22] The solubilities of methane, ethane, propane, and butane in
water at 293 K and 0.1 MPa are 1.4, 1.8, 1.6, and 1.2 mm,
respectively, which suggests that the solubility of a gaseous
alkane is not a major factor in determining whether it can be
Although we have focused herein on a series of perfluori-
nated fatty acids with a linear alkyl chain (PFC8–PFC14) as
the dummy substrate, PFs with a branched alkyl chain and/or
partially fluorinated carboxylic acids may also be potent
dummy substrates. Furthermore, our system is compatible
with reported protein-engineering techniques, such as muta-
genesis and chemical modification.
[
25]
hydroxylated.
[
[
23] P450BM3 with PFC10 catalyzed the hydroxylation of propane
for a period of more than 1 h, and the reaction stopped after all
of the added NADPH had been consumed (see the Supporting
Information).
24] We examined the origin of the oxygen atom in cyclohexanol in
Received: December 17, 2010
Published online: April 19, 2011
18
O labeling experiments and confirmed that, in most cases, the
oxygen atom in cyclohexanol was derived from molecular
oxygen (see the Supporting Information).
Keywords: cytochromes · enzyme catalysis · gaseous alkanes ·
hydroxylation · perfluorocarboxylic acids
[25] D. Wise, G. Houghton, Chem. Eng. Sci. 1966, 21, 999 – 1010.
.
5
318
www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 5315 –5318