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| Binding pocket representation of P450 enzyme with activator (perfluoro chain in green) and docking poses of a methane cluster (carbon atoms in black and hydrogen atoms in white) hovering above Fe=O (red) in the porphyrin ring. Zilly et al. Click to enlarge. |
Researchers at the Max Planck Institute for Carbon Research in Mülheim (Germany), led by Manfred T. Reetz, have introduced a new approach for the enzymatic production of methanol from methane. Described in a paper in the journal Angewandte Chemie, their innovation is the inclusion of an inert guest in the enzyme’s binding pocket in order to make it smaller so that it can effectively bind methane.
Methanol is a useful starting material for many chemical syntheses, including fuels; it can also be added to conventional fuels to power fuel cell or used in combustion engines. Conventional processes for producing methanol from methane involve detours (synthesis gas), are complex and energy-intensive, and require high temperatures and pressures. By contrast, the enzyme methane monooxygenase does the job gently and efficiently. However, this is a very complex enzyme that cannot easily be produced and used in an artificial environment.
The cytochrome P450 (CYP) family of enzymes could represent an alternative starting point. The main job of these enzymes is the oxidation of various substances produced by the body or introduced to it. In the reaction, carbon-hydrogen bonds are oxidized to make alcohol groups (-OH). The active component of these enzymes is a heme, an iron-porphyrin complex similar to that in hemoglobin.
The problem is that the binding pocket of this enzyme is too big to bind and oxidize small molecules such as methane. Instead of trying to devise complex methods to create a suitable enzyme, Reetz and his co-workers chemically tuned a CYP enzyme by adding an additional guest into the binding pocket in order to make it smaller.
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
chemical catalysis or using enzymes of the type methane
monooxygenases (MMOs). It is not only the smallest
alkane, but also has the strongest C–H bond (104 kcal mol-1).
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
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.—Zilly et al.
The natural substrates for CYP enzymes are fatty acids. As a guest molecule, the researchers chose a compound that resembles a fatty acid, a carbonic acid in which all of the hydrogen atoms in the hydrocarbon chain have been replaced with fluorine atoms. This type of molecule is as water-repellent as the original, but takes up more room.
The fluorine atoms make it chemically inert so that it does not participate in any reactions. Like the molecule it is modeled on, this guest is able to bring the iron-heme complex of the enzyme into its catalytically active state (high-spin state). The significantly smaller binding pocket now allows methane to bind effectively so that it can be oxidized to methanol.
In contrast to time-consuming protein
engineering, the present approach simply requires the
addition of an appropriate chemically inert perfluoro fatty
acid to the enzyme, thereby triggering a catalytically activating
effect which originates from specific guest/host interactions
in the binding pocket. A shift from an inactive low-spin
state to a catalytically active high-spin state and a decrease in
the effective volume of the binding pocket appear to be the
crucial factors as shown by UV/Vis difference spectra as well
as a theoretical analysis based on MD simulations and
docking experiments.
The present approach not only allows
methane to be oxidized with notable enzyme activity, but also
opens the door for using perfluoro carboxylic acids, which can
be expected to bind to most CPYs, to influence the catalytic
profile of monooxygenases as catalysts in the functionalization
of more complex organic compounds, including the
control of regio- and stereoselectivity.—Zilly et al.
Resources
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Zilly, F. E., Acevedo, J. P., Augustyniak, W., Deege, A., Häusig, U. W. and Reetz, M. T. (2011) Tuning a P450 Enzyme for Methane Oxidation. Angewandte Chemie, doi: 10.1002/ange.201006587