Regulation of ferryl reactivity by the cytochrome P450 decarboxylase OleT

Abstract

The cytochrome P450 OleT catalyzes the decarboxylation of long-chain fatty acid substrates to produce terminal alkenes using hydrogen peroxide as a co-substrate. The facile activation of peroxide to form Compound I in the first step of the reaction, and subsequent CC bond cleavage mediated by Compound II, provides a unique opportunity to visualize both ferryl intermediates using transient kinetic approaches. Analysis of the Arrhenius behavior yields activation barriers of ∼6 kcal/mol and ∼ 18 kcal/mol for the decay of Compound I and Compound II respectively. The influence of the secondary coordination sphere, probed through site-directed mutagenesis approaches, suggests that restriction of the donor-acceptor distance contributes to the reactivity of Compound I. The reactivity of Compound II was further probed using kinetic solvent isotope effect approaches, confirming that the large barrier owes to a proton-gated mechanism in the decarboxylation reaction coordinate. Hydrogen-bonding to an active-site histidine (H85) in the distal pocket plays a key role in this process.

Keywords: CYP152; Compound I; Cytochrome P450.

Fig. 1.

Proposed mechanisms and branchpoint of hydroxylation and decarboxylation in the CYP152 family. Following peroxide activation, hydrogen atom transfer (HAT) by Compound I (CYP-I) from the β-carbon of the fatty acid substrate yields Compound II (CYP-II) and a substrate radical. OleT enables C—C bond scission via proton-coupled electron transfer (PCET) and stabilization of Cpd-II, yielding an n-1 alkene and CO₂. In contrast, BSβ performs oxygen rebound, resulting in a distribution of hydroxylated fatty-acid products.

Fig. 2.

Temperature dependence of the reaction of OleT Compound I (Ole-I) with eicosanoic acid (EA). A) PDA spectra of d₃₉-EA-bound OleT upon rapid mixing with 5 mM H₂O₂. Inset: Single-wavelength PMT traces of Ole-I decay with d₃₉-EA measured at 690 nm at various temperatures. B) Arrhenius plots of Ole-I decay for h₃₉-EA (red) and d₃₉-EA (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Contribution of the distal pocket of F79A OleT on HAT efficiency. A) Crystal structure of OleT (PDB:4L40) showing F79 relative to the fatty acid substrate (pink). B) Comparison of timetraces for Ole-I decay with d₃₉-EA for WT (red) or F79A (blue) OleT. Residuals from fitting are shown at the bottom. Inset: Arrhenius plot of the temperature dependence of Ole-I decay for F79A (blue) compared to WT (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Temperature dependence of the reaction of OleT Compound II (Ole-II) with eicosanoic acid (EA). A) PDA spectra of d₃₉-EA-bound OleT upon rapid mixing with 5 mM H₂O₂. Inset: Single-wavelength PMT traces of Ole-I decay with d₃₉-EA measured at 440 nm at various temperatures. B) Arrhenius plots of Ole-II decay for h₃₉-EA (red) and d₃₉-EA (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Involvement of protons in the decay of Ole-II. A) PMT traces of Ole-II decay monitored at 440 nm in H₂O (red) or D₂O (blue) buffer. Residuals from fitting are shown at the bottom. B) Proton inventory plot for Ole-II decay in different mole fractions of D₂O. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

Reaction products of WT OleT and H85s mutant with C20, C16, and C12 chain-length fatty acid substrates. Reactions contained 100 equivalents of the fatty acid substrate and 1000 equivalents of H₂O₂ were slowly added over the course of one hour prior to analysis by gas chromatography.

Fig. 7.

A) Arrhenius plots of the decay of Ole-II as a function of temperature for WT (red) and H85Q (blue). B) Proton inventory plot for H85Q fit with a linear function. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8.

Comparison of the active site water network observed in the crystal structures of A) WT (PDB:4L40) and B) H85Q OleT (PDB:5MO0). H85 and Q85 are colored in light green. Waters are depicted as red spheres and the fatty acid substrate is colored in orange. Possible H-bonding networks for the respective OleT variants are depicted below the structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)