A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid

Abstract

Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+• termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.

Figure 1

Spectral characterization of WT CYP119 (5 µM) upon mixing with one equivalent of mCPBA (100 mM KPi, pH 6.2 at 10 °C): A) calculated spectra from the SVD analysis of the rapid scan absorbance traces for the initial 0.45 s: ferric protein (solid line) and compound I (dashed line); Inset, zoom in of the visible region. B) kinetic traces of the ferric (416 nm) and Cpd I (369 nm).

Figure 2

Spectral characterization of SeCYP119 (5 µM) upon mixing with one equivalent of mCPBA (100 mM KPi, pH 6.2 at 10 °C): A) calculated spectra from the SVD analysis of the rapid scan absorbance traces for the initial 0.9 s: A (ferric protein, blue); C (ferric like protein, green); D (ferric like protein with 2 nm blue shift, red), Inset, zoom in of the visible region. B) kinetic traces of the ferric (416 nm) and Cpd I (369 nm).

Figure 3

Spectral characterization of SeCYP119 (5 µM) upon mixing with five equivalents of mCPBA (100 mM KPi, pH 6.2 at 10 °C): A) traces of the rapid scan absorbance spectra obtained for the initial 0.9 s, Inset, traces of the rapid scan spectra obtained over 2.25 s. B) calculated spectra from the SVD analysis of the rapid scan data of the initial 0.9 s, traces: A (ferric protein, blue); C (ferric like protein, green); D (P406 species, red).

Figure 4

A) Oxidation of SeCYP119 (5 µM) with 10 equivalents of mCPBA (100 mM KPi, pH 6.2 at 24 °C). Spectra shown are native protein (416 nm); 5 min after the addition of 10× mCPBA (406 nm) and 2 min after the addition of ~100 µM 4-phenylimidazole (408 nm). B) Oxidation of WT CYP119 before and after the addition of 10x mCPBA. C) Fe(II)-CO difference spectrum of the P406 species, monitored over 10 min. D) Oxidation of SeCYP119 to generate P406 species followed by treatment with excess DTT (10 mM) to regenerate the native spectrum. Inset, zoom in of the visible region showing the α and β bands of the DTT treated P406.

Figure 5

Oxidation of SeCYP119 with excess PN. A) rapid scan absorbance traces for the oxidation with 1.5 mM PN over 45 s, showing the conversion of the ferric protein (416 nm) to a Cpd II like species (433 nm) that in turn reverts back to the starting ferric form. B) SVD analysis of the rapid scan data for the reaction with 0.5 mM PN over 1 s, showing the formation of Cpd II, traces: blue, native protein; green, SeCYP119 complex with PN; red, Cpd II like species. C) Kinetic traces of Cpd II for the WT (430 nm) and SeCYP119 (433 nm) monitored over 90 s. D) Kinetic traces for the PN decay over 90 s.

Scheme 1

Proposed pathway for the reaction of SeCYP119 with mCPBA to generate the P406 species