Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion

Abstract

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pK(a) of the ferric-peroxo anion, which favors its protonation and, therefore, Compound I formation.

Figure 1. Reverse-phase HPLC separation of CYP125-catalyzed oxidation products of (A) cholest-4-en-3-one, (B) 26-hydroxycholest-4-en-3-one and (C) cholest-4-en-3-one-26-aldehyde

The separation of the products was carried out using a reverse phase C8-A Polaris column (Varian), as described in the Experimental section. The elution was monitored at 240 nm. The retention times are: peak VI, 14.9 min; peak V, 15.8 min; peak IV, 17.2 min; peak III, 18.3 min; peak II, 20.8; peak I, 29.6 min. The m/z values of the parent and daughter ions are listed in Table S1. *, contaminant from the cholest-4-en-3-one-26-aldehyde preparation

Figure 2. Mass spectra of the CYP125 oxidation products

Black trace, cholest-4-en-3-one under an atmosphere of ¹⁶O₂; green trace, cholest-4-en-3-one under an atmosphere of ¹⁸O₂; red trace; 25,26,26,26,27,27,27-D₇-cholest-4-en-3-one under an atmosphere of ¹⁶O₂. For clarity only the m/z range showing the parent ion is presented. The proposed structures of the metabolites are also shown, where R represents the rest of the cholest-4-en-3-one structure.

Figure 3. UV-vis spectrophotometric characterization of SeCYP125

A, Comparison of the native ferric spectrum of the WT* and SeCYP125 proteins in 100 mM potassium phosphate buffer, pH 7.4. B, Fe²⁺-CO difference spectrum of the SeCYP125 measured over 12 min, at 1 min intervals, after the addition of 1 mM dithionite.

Figure 4. Resonance Raman spectra of Fe³⁺ WT* and SeCYP125

Room temperature RR spectra obtained with a 413 nm excitation.

Figure 5. EPR spectra of Fe³⁺ WT* and SeCYP125

EPR spectra of Fe³⁺ WT* and SeCYP125 at 10 K. Microwave frequency, 9.66 GHz; microwave power, 0.01 mW; modulation amplitude, 10 G.

Figure 6. Resonance Raman spectra of Fe³⁺-NO complexes

Room temperature RR spectra of the Fe³⁺-NO complexes of WT* and SeCYP125 prepared with ¹⁴NO and ¹⁵NO. All spectra were obtained with a 442 nm excitation. The low-frequency spectra were normalized on the ν₇ at 674 cm^-1. The inset shows ¹⁴NO minus ¹⁵NO high-frequency difference spectra obtained after normalization on the ν₄ mode at 1375 cm^-1.

Figure 7. Binding and catalytic activities of SeCYP125 toward cholest-4-en-3-one

(A) Binding of cholest-4-en-3-one to recombinant SeCYP125. The concentration dependence of ligand binding was deduced from the difference in absorption changes obtained from titration of the protein (2.5 μM P450) with increasing concentrations of cholest-4-en-3-one. The data was fit using a tight binding equation. (B) LC chromatograms with absorbance detection at 240 nm of the WT* and SeCYP125 oxidation products of cholest-4-en-3-one, separated using a reverse phase C-18 Xterra column (Waters), as described in the Experimental section. A control reaction carried out in the absence of NADPH is also included. Inset: zoom-in showing the HPLC elution of the minor products. S and I.S. stand for substrate and internal standard, respectively.

Figure 8

Comparison of the ratio of the 26-acid over the sum of the deformylation products produced during the oxidation of various substrates by WT* and SeCYP125.

Scheme 1

Partitioning of the ferric-peroxo anion into the Compound I-mediated monooxygenation (a) and peroxyhemiacetal-mediated deformylation (b) pathways in the oxidation of an aldehyde.

Scheme 2. Possible mechanisms for the deformylation reactions catalyzed by CYP125

The important steps involve addition of the ferric-peroxo anion of CYP125 to the C26 carbonyl and subsequent radical fragmentation of the peroxyhemiacetal adduct. The radical fragmentation of the peroxyhemiacetal adduct led to (a) the formation of an alkene (M1) or (b) the formation of a one-carbon deficient alcohol (M4). The Compound I-catalyzed oxidation of M1 generates a diol (M5) via the acid-catalyzed ring opening of an epoxide intermediate. A C25 cation may also derive from the single-electron oxidation of the C25 radical (c). Trapping of the cation by formate or water (e) results in the formation of the C25-oxyformyl (M2) and the one-carbon reduced alcohol (M4), respectively. Loss of a proton from the C25 cation may also generate M1 (d). The Compound I-catalyzed oxidation of M4 produces a gem-diol intermediate that dehydrates to a keto compound (M3).