Communications
DOI: 10.1002/anie.201006587
CÀH Activation
Tuning a P450 Enzyme for Methane Oxidation**
Felipe E. Zilly, Juan P. Acevedo, Wojciech Augustyniak, Alfred Deege, Ulrich W. Hꢀusig, and
Manfred T. Reetz*
Cytochrome P450 (CYP) enzymes are heme-dependent mon-
laboratory evolution, and showed for the first time notable
activity toward propane by formation of the respective
ooxygenases that catalyze the oxidation of CÀH bonds of
[
6a]
endogenous and exogenous organic compounds with forma-
alcohols (2-propanol/1-propanol = 9:1);
ethane to ethanol conversion remains problematic
however, the
[1]
[6b]
tion of the respective alcohols. The mechanism involves the
intermediacy of a high-spin oxyferryl porphyrin radical cation
which abstracts a hydrogen atom from the substrate, and the
short-lived alkyl radical then undergoes CÀO bond formation.
and
methane oxidation has not been achieved to date. Higher
activity in ethane oxidation was accomplished using mutants
[
6c]
of P450cam, but here again methane oxidation was not
reported. Our chemical approach involves a chemically inert
compound that serves as a guest in the binding pocket of P450
BM3, thereby filling the space and reducing the translational
freedom of small alkanes or of any other substrate. On the
basis of previous reports involving CYPs harboring various
The binding pockets of CYPs are relatively large, therefore
small compounds do not have a statistically high enough
probability of being properly oriented near the oxyferryl
moiety for rapid oxidation to occur; additionally there are
other effects that slow down or prevent catalysis. A notorious
challenge is the oxidation of methane to methanol by
[
1]
substrates, such guest/host interactions can be expected to
induce other modes of activation effects as well, specifically
water displacement at the Fe/heme site accompanied by a
change in the electronic state from the inactive low-spin state
[
2]
chemical catalysis or using enzymes of the type methane
[3]
monooxygenases (MMOs). It is not only the smallest
À1
alkane, but also has the strongest CÀH bond (104 kcalmol ).
[
1,5d]
Although CYPs represent a superfamily of monooxygenases,
none have been shown to accept methane, whereas MMOs
are complex enzymes (many membrane bound) that have not
been expressed in heterologous hosts in any significant
to the catalytically active high-spin states.
Moreover,
many studies have shown that P450 enzymes and mutants
thereof can harbor two different substrates simultaneously,
[1,7–9]
thus leading to cooperative effects;
one example is lauric
[3]
quantities, among other problems. Herein we show that
chemical tuning of a CYP, which is based on guest/host
activation using perfluoro carboxylic acids as chemically inert
guests, activates the enzyme for oxidation of not only
medium-sized alkanes such as n-hexane, but also of small
gaseous molecules such as propane and even methane as the
ultimate challenge.
acid and palmitic acid in which cooperativity has been
[
8]
demonstrated by isotope labeling experiments. In yet
another study regarding the metabolism of bilirubin, the
addition of lauric acid or the perfluorinated analogue was
reported to facilitate NADPH oxidation and substrate
degradation, a finding that has implications for the treatment
[9]
of jaundice, uroporphyria, and possibly cancer. It has also
been shown for the case of a distantly related H O -
In the present study we chose, for practical reasons, the
enzyme P450 BM3 (CYP102A1) from Bacillus megaterium,
which is a self-sufficient fusion protein composed of a P450
2
2
dependent P450 enzyme that its peroxidase activity can be
influenced by the addition of fatty acids, wherein increased or
decreased activity is observed depending upon their chain
[4]
monooxygenase and an NADPH diflavin reductase. Several
crystal structures of this CYP harboring a fatty acid or fatty
acid derived inhibitors, as well in the absence of such
[
10]
length.
In our endeavor we were guided by the binding mode of
the natural substrates, fatty acids, of P450 BM3. The binding
includes H-bonds originating from their carboxy function and
residues Arg 47 and Tyr 51, as well as hydro-
[
5]
compounds have been published. To engineer mutants of
P450 BM3 and of other CYPs for enhanced activity and
selectivity toward a variety of different compounds, including
such difficult substrates as small alkanes, rational design as
well as directed evolution have proven to be successful to
[
5]
phobic interactions. The use of perfluoro
carboxylic acids such as 1a–h as chemically
inert, yet activating guests was therefore envi-
sioned, because perfluoro alkyl groups are
known to be resistant to oxidation while having
[1c,6]
some extent.
For example, P450 BM3 variants character-
ized by numerous point mutations were obtained in extensive
[
11]
a hydrophobic character.
Moreover, it is
[*] Dr. F. E. Zilly, Dr. J. P. Acevedo, Dr. W. Augustyniak, A. Deege,
known that a CF residue is sterically compa-
3
U. W. Hꢀusig, Prof. M. T. Reetz
Max-Planck-Institut fꢁr Kohlenforschung
[11a]
rable to a CH(CH ) group,
which means
3
2
that a perfluoro fatty acid fills much more space in a P450
binding pocket than a traditional fatty acid, and can addi-
tionally induce the crucial low-spin to high-spin conversion of
Fe/heme.
Kaiser-Wilhelm-Platz 1, 45470 Mꢁlheim an der Ruhr (Germany)
reetz@mpiso-muelheim.mpg.de
[
**] We thank Heike Hinrichs, Frank Kohler, and Sylvia Ruthe for help
with the chromatographic measurements.
In exploratory studies, the oxidation of n-octane and
n-hexane as well as isomers thereof was studied using P450
2
720
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 2720 –2724