How Do Perfluorinated Alkanoic Acids Elicit Cytochrome P450 to Catalyze Methane Hydroxylation? An MD and QM/MM Study

Abstract

Recent experimental studies show that usage of perfluoro decanoic acid (PFDA), as a dummy substrate, can elicit P450_BM3 to perform hydroxylation of small alkanes, such as methane (ref. 17) and propane (ref. 17 and ref. 18). To comprehend the mechanism whereby PFDA operates to potentiate P450_BM3 to catalyze the hydroxylation of small alkanes, we used molecular dynamics (MD) and hybrid quantum mechanical / molecular mechanical (QM/MM) calculations. The MD results show that without the PFDA, methane escapes the active site, while the presence of PFDA can potentially induce a productive Cpd I-Methane juxtaposition for rapid oxidation. Nevertheless, when only a single methane molecule is present near the PFDA, it still escapes the pocket within less than a nanosecond. However, when three methane molecules are present in the pocket, they alternate quasi-periodically such that at all times (within 10 ns), a molecule of methane is always present in the proximity of Cpd I in a reactive conformation. Our results further demonstrate that the PFDA does not exert any electrostatic catalysis, whether the PFDA is in the protonated or deprotonated forms. Taken together, we conclude that methane hydroxylation requires, in addition to PFDA, a high partial pressure of methane that will cause a high methane concentration in the active site. Further study of ethane and propane hydroxylations demonstrates that higher alkane concentration is helpful for all the three small alkanes. Thus for the smallest alkane, methane, at least three molecules are necessary whereas for the larger ethane, two molecules are needed to force one ethane to be closer to Cpd I. Finally, for propane a second molecule is helpful but not absolutely necessary; for this molecule the PFDA may well be sufficient to keep propane close to Cpd I for efficient oxidation. We therefore propose that high alkane pressure should assist small alkane hydroxylation by P450 in a manner inversely proportional to the size of the alkanes.

Figure 1

(a) The binding mode from docking results by Autodock 4.2, in which the carboxylic group of PFDA directly interacts with Ser72, and indirectly interacting with Tyr51 and Arg47. (b) The QM models (R1, R2 and R3) employed in QM/MM calculation.

Figure 2

The active site of P450_BM3 in the presence of PFDA and methane (a) with two additional water molecules, (b) with two additional methanes.

Figure 3

The mobility of methane in P450_BM3 binding pocket in the presence and absence of PFDA: (a) one methane without PFDA; (b) one methane with PFDA; (c) one methane and two additional water molecules with PFDA; (d) three methane molecules with PFDA; the molecules are colored differently to emphasize that one of them is close to Cpd I throughout the simulation.

Figure 4

The mobility of ethane and propane in P450_BM3 binding pocket in the presence of PFDA: (a) one ethane; (b) one propane; (c) two ethanes; (d) two propanes.

Figure 5

Energy profiles of methane hydroxylation by Cpd I. The relative energies are given as B3LYP/B2//B1(B3LYP/B2//B1+ZPE) in kcal mol⁻¹. The oxidation state of iron is given as Roman numerals in parentheses.

Figure 6

Key geometric features (geom), group spin densities (ρ), NBO charges (Q) of the hydrogen abstraction transition states in the C-H hydroxylation of methane by P450_BM3 Cpd I. The data are given in the order of ²TS_H(III)(²TS_H(IV))[⁴TS_H(III)]. Bond lengths are in angstroms and dihedral angles (D) are in degrees.

Figure 7

NBO charges of ²RC and ²TS_H(IV) in R1 and R2 models (in black). The distances between hydrogen atoms of methane and fluorine atoms of PFDA are shown as well (in red).

Figure 8

Energy profiles for ethane (a) and i-propane (b) hydroxylation by Cpd I. The relative energies are given as B3LYP/B2//B1(B3LYP/B2//B1+ZPE) in kcal mol⁻¹. The oxidation state of iron is given as Roman numerals in parentheses.

Scheme 1

Schematic representation of the rebound mechanism.