Catalytic strategy for carbon-carbon bond scission by the cytochrome P450 OleT

Abstract

OleT is a cytochrome P450 that catalyzes the hydrogen peroxide-dependent metabolism of Cn chain-length fatty acids to synthesize Cn-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe(4+)-oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C-H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe(4+)-OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon-carbon scission reaction catalyzed by OleT.

Keywords: compound II; cytochrome P450; hydrocarbon; metal−oxo; oxygen activation.

Fig. 1.

The decarboxylation reaction catalyzed by OleT. Using a hydrogen peroxide cosubstrate, OleT metabolizes a C_n chain-length fatty acid to produce a C_n-1 alkene and carbon dioxide coproduct.

Fig. 2.

Stopped-flow absorption evidence for a substrate ²H KIE in the OleT reaction. Representative single-wavelength time course for the reaction of 20 μM OleT−D₃₉-EA (red) or OleT−H₃₉-EA (blue, and Inset) with 10 mM H₂O₂ monitored at 370 nm. The superimposed white traces represent two-summed exponential fits to the data.

Fig. S1.

Representative time courses (black), superimposed fits (red), and residuals for the 20 μM OleT−EA + 10 mM H₂O₂ time course using single-wavelength detection for (A) D₃₉-EA at 370 nm, (B) H₃₉-EA at 370 nm, (C) D₃₉-EA at 440 nm, and (D) H₃₉-EA at 440 nm.

Fig. S2.

Time course for the 20 μM OleT−D₃₉-EA + 10 mM H₂O₂ reaction (black) using single-wavelength detection at 690 nm, single exponential fit (red), and residuals.

Fig. 3.

Identification of an additional intermediate (Int-2) that forms concomitant with OleT compound I (Ole-I) decay. PDA spectra of a single-turnover reaction of 20 μM OleT−D₃₉-EA with 10 mM H₂O₂ in (A) 2- to 40-ms and (B) 40- to 500-ms timeframes. (C) The OleT−H₃₉-EA + H₂O₂ reaction, from 2 ms to 500 ms. Insets represent kinetic difference spectra, 500 ms to 40 ms (in B) and 500 ms to 10 ms (in C).

Fig. 4.

Transient kinetics of Int-2 formation and decay. (A) Representative single-wavelength time course for the reaction of 20 μM OleT−D₃₉-EA (red) or OleT−H₃₉-EA (blue) with 10 mM H₂O₂ monitored at 440 nm. The superimposed white traces represent two-summed exponential fits to the data. (B) Plots of the dependence of the larger RRT on H₂O₂ concentration in respective colors. The solid lines represent nonlinear fitting to a hyperbolic expression (for the D₃₉-EA reaction) and linear fitting (H₃₉-EA). The Inset shows the decay rate of Ole-I at 370 nm and formation rate of Int-2 for the OleT–D₃₉-EA reaction.

Fig. S3.

Peroxide independence and isotopic insensitivity of the slower RRT associated with Int-2 decay at 440 nm.

Fig. 5.

Summary of the kinetic parameters for OleT compound I formation, and subsequent decay to Int-2 and the low-spin ferric enzyme. The decay rate constants for Ole-I with D₃₉-EA and H₃₉-EA are indicated as k_D and k_H, respectively. The formation of nonadecene and carbon dioxide was demonstrated in a previous study (35).

Fig. 6.

Optical spectroscopic features and reactivity of Int-2. (A) The pure component spectra of OleT Int-2 (orange) and compound I (green) as obtained from global fitting analysis of PDA data, and compared with the ferric H₂O low-spin enzyme (blue). (B) Plot of the apparent Int-2 decay rate constant with phenol (circle) and 3-chlorophenol (square). OleT−H₃₉-EA (40 μM) was mixed with 2 mM H₂O₂, aged 20 ms, and then mixed 1:1 with substituted phenols. The concentrations shown are after mixing. Error bars represent 1 SD.

Fig. S4.

Speciation plots for OleT reaction cycle intermediates computed using rate constants determined in single-wavelength studies with (A) D₃₉-EA and (B) H₃₉-EA. Rate constants are first-order (per second) or pseudo-first-order for the H₂O₂ concentration used.

Fig. S5.

(A) Comparison of the optical absorption spectra of OleT Int-2 (red), ferric H₂O (blue), and ferric OH (black) forms of the enzyme. The ferric hydroxide form was generated by a rapid pH jump to 12, and is stable for several seconds before decomposition. (B) Difference spectra of the ferric hydroxide (black) and Int-2 minus ferric H₂O (red) forms show that the two species are readily distinguishable.

Fig. S6.

Global analysis independent determination of Int-2 optical spectrum by linear combination. (A) The visible spectrum of the OleT–H₃₉-EA + H₂O₂ reaction at 50 ms (black) with a weighted ferric low-spin reference (blue) and Int-2 spectrum (red). (B) Overlay of the spectrum of Int-2 determined by SVD (red) and linear subtraction methods (black). (C) Speciation plots of Int-2 and the ferric low-spin species using rates determined from fitting single-wavelength data at 440 nm. A dashed line shows the predicted fraction of each species at the time point of interest. (D) The visible spectrum of the OleT–D₃₉-EA + H₂O₂ reaction at 50 ms (black) with a weighted ferric low-spin reference (blue) and Int-2 spectrum (red). (E) Overlay of the spectrum of Int-2 determined by SVD (red) and linear subtraction methods (black). (F) Speciation plots of Int-2 and the ferric low-spin species using rates determined from fitting single-wavelength data at 440 nm. (G) The visible spectrum of the OleT–D₃₉-EA + H₂O₂ reaction at 100 ms (black) with a weighted ferric low-spin reference (blue) and Int-2 spectrum (red). (H) Overlay of the spectrum of Int-2 determined by SVD (red) and linear subtraction methods (black). (I) Speciation plots of Int-2 and the ferric low-spin species using rates determined from fitting single-wavelength data at 440 nm.

Fig. S7.

Double-mixing studies to probe the reactivity of Int-2 toward phenols. Int-2 was generated by mixing 40 μM OleT−H₃₉-EA with 4 mM H₂O₂. After aging for 20 ms, the resulting solution was mixed 1:1 with buffered phenols at various concentrations. Representative time traces at 440 nm and superimposed single exponential fits (red) are shown for a reaction with no phenol (black) and with 10 mM 4-Cl-phenol (gray). The observed rates for these reactions are k_obs = 7.3 and 26.7 s⁻¹ respectively.

Fig. S8.

The pH dependence of the Int-2 decay rate. OleT−D₃₉-EA was prepared at several pHs using a 200 mM KH₂PO₄, 125 mM l-arginine buffer and was rapidly mixed with similarly buffered 500 μM H₂O₂. The spectral changes associated with Int-2 decay were monitored at 440 nm. The lnk_decay shows a linear dependence on pH, with a slope = 1.05 ± 0.07, consistent with a proton-coupled electron transfer involved in the conversion of Fe⁴⁺−OH to Fe³⁺−OH₂.