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

• 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