Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products

Abstract

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography–mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.

1. Introduction

Members of the cytochrome P450 monooxygenase family play vital roles in the synthesis and degradation of many physiologically important compounds and xenobiotics [1,2]. Human Cytochrome P450 17A1 (CYP17A1), a steroidogenic cytochrome P450, found mainly in the adrenal glands and sex organs [1,3–5] is important in the production of androgens. This multifunctional enzyme catalyzes the hydroxylation of progesterone (PROG) or pregnenolone (PREG) to form 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) and then orchestrates the subsequent lyase reaction, where the C17-C20 bond is cleaved to produce DHEA from 17-OH PREG or AD from 17-OH PROG.

While there is consensus that the hydroxylation reaction proceeds through a Compound I intermediate [1,2] the precise mechanism associated with the remarkable lyase rection has been debated for decades [4–6] with arguments being made for either a Compound I-mediated reaction [1,2,7–10] or a peroxo-mediated scheme [1,2,10,11]. This proposed scheme derives from recent studies [12,13] where samples of CYP17 and active site mutants, prepared with the Nanodisc system [14,15], were studied in the presence of the natural substrates, 17-OH PROG and 17-OH PREG, by rR spectroscopy coupled with the cryoradiolysis method, first introduced by Symons [16,17] and refined by Hoffman and coworkers [18,19]. In the lyase cycle, an initially formed peroxo intermediate is converted to a new unstable peroxo-hemiketal intermediate, also documented by rR spectroscopic detection, which can decay upon annealing to room temperature to yield AD or DHEA as shown in Fig. 1 [10,12,13].

Fig. 1.

Cytochrome P450 enzymatic cycle and formation of a perexohemiketal intermediate with 17-OH PREG as a substrate.

Though application of this effective combination of innovative approaches provided definitive spectroscopic evidence for the peroxo-hemiketal intermediate within this scheme [12,13] it is reasonable to question if these documented species arise only under the cryogenic temperatures employed to trap and structurally characterize these unstable intermediates. The present work utilizes GC/MS analysis [20–22] to document product formation and show that, upon annealing to room temperature, these samples generate the expected lyase products, DHEA, and AD, consistent with the proposed peroxo-mediated lyase reaction [12,13].

2. Materials and methods

17-OH PROG, DHEA, and AD were purchased from Sigma Aldrich (Milwaukee, WI, USA). The sample of 17-OH PREG was purchased from Steraloids (Newport, RI, USA). All chemicals were used without further purification. The 9 mm inserts for the GC/MS autosampler were purchased from ThermoFisher Scientific (Milwaukee, WI, USA). The protein samples used in this study were incorporated in nanodiscs and detailed sources and how the samples were prepared was given in previously reported studies [12,13].

2.1. Instrumentation

Chromatographic analysis was performed using a Shimadzu GC/MS system (GC-2010Plus), coupled with a Shimadzu autosampler (AOC-20i + s), and GC/MS-QP2010 SE Single Quadrupole GC–MS. The software for acquisition and processing is GC/MS Solution which contains GC/MS analysis editor, GC/MS real time analysis and GC/MS Postrun analysis. A 1 μL sample was injected in the split-type injector [split ratio 1:5] for all 17-OH PROG and AD standards, while a 3 μL sample was used for the 17-OH PREG and DHEA standards. A 5 μL sample was used for the extracted CYP17A1 samples that were prepared with either 17-OH PROG or 17-OH PREG. The standard samples were contained in 1 mL vials and GC/MS spectra acquired while extracted samples were contained in 200 μL glass vial insert which was inserted into a 1 mL glass vial. The separation and resolution of metabolites was achieved using an SH-Rxi-5Sil MS (Fused silica) (30 m length × 0.25 mm column diameter × 0.25 μm film thickness) column (Restek, USA) which is a general-purpose low-polarity phase, Crossbond 1,4-bis(dimethylsiloxy)phenylene dimethyl polysiloxane with a temperature range of −60 to 350 °C. It is engineered to be a low-bleed GC–MS column, with excellent inertness for active compounds and only 5% polarity.

2.2. GC/MS analysis

A 1 μL blank sample of dichloromethane (DCM) was injected before running any set of samples to ensure there were no contaminating substances in the column by ensuring there were no spurious peaks in the blank run. Injection was performed in a split mode (split ratio 1: 5 at 250 °C) using an autosampler, Shimadzu [SPL1]. A temperature gradient was used, the oven being programmed to start at 150 °C for 0.7 min (min), ramped at 30 °C/ min to 280 °C and held for 15 min [20,21,23]. This program has been shown to be useful in identification of a sample of underivatized steroids [23]. The carrier gas was helium with a constant flow rate of 1.69 mL min⁻¹. The GC/MS interface (transfer line between gas chromatograph and mass spectrometer) and ion trap temperature were set at 250 and 200 °C, respectively. Mass spectra were obtained in full scan mode from m/z 90 to 500 Da mass range for qualitative analysis, using electron impact ionization mode, with the ionization voltage set at 70 eV.

2.3. Preparation of samples

The standard samples (commercial 17-OH PROG, 17-OH PREG) were dissolved in DCM to a final concentration of 450 μM while standards for products (commercial AD and DHEA) were dissolved to make final concentrations of 150 μM. GC/MS spectra of pure standard solutions were acquired. Standard mixtures were also prepared, i.e., a mixture of 450 μM 17-OH PROG with 150 μM AD and a mixture of 450 μM 17-OH PREG with 150 μM DHEA. The solutions were prepared by dissolving in DCM in a 1 mL glass vial. The mass spectra of all the compounds were consistent with mass spectra of the compounds in literature [23,24] The preparation of the cryoradiolyzed samples of CYP17A1 was described previously [12,13]; i.e., one sample contained 320 μM CYP17A1-ND in 100 mM potassium phosphate (pH 7.4), 250 mM sodium chloride, 30% (v/v) distilled glycerol, 6.24 μM methyl viologen, and 400 μM 17-OH PROG, while the other sample contained 280 μM CYP17A1-ND in 100 mM potassium phosphate (pH 7.4), 250 mM sodium chloride, 6.24 μM methyl viologen, and 400 μM 17-OH PREG; this solution was mixed with distilled glycerol, 30%(v/v). Cryoradiolysis of oxyferrous CYP17A1 was performed that generated the peroxo intermediate. Two “control” samples, with no CYP17A1 present, were prepared for irradiation; each contained glycerol, 30% (v/v), 400 μM 17-OH PREG (or 17-OH PROG) in 100 mM phosphate buffer (pH 7.4), 250 mM sodium chloride. One of these samples was saturated with oxygen gas (5 min bubbling with stirring); another sample was deaerated (5 min bubbling with argon gas with stirring). Both samples were quickly frozen by immersion into liquid nitrogen and then subjected to typical cryoradiolysis conditions, i. e., 3.5 Mrad dose of γ-rays (in Gammacell 220 ⁶⁰cobalt irradiation chamber at the University of Illinois at Urbana Champaign).

2.4. Sample extraction

Extraction and quantitation of the metabolites was accomplished by a modification of previously described methods [9,20,21,25–28] Briefly, an NMR tube containing frozen sample previously used for rR and cryoradiolysis was thawed and the (~0.1 mL) sample was pipetted into a 2 mL vial. A 1 mL aliquot of dichloromethane was added, and the vial shaken gently for about 2–5 min. The vial was left standing for about 3 min to allow separation of the organic (DCM) and aqueous layers. The bottom organic layer containing residual substrate and product was drained into a clean, new 5 mL glass vial. The extraction was repeated three more times, and the pooled organic layer was evaporated to dryness by flowing nitrogen gas gently over the solution. The sample was dissolved in 150 μL of DCM and was transferred to a 200 μL glass vial insert which was inserted into a GC/MS glass vial, placed in the auto-sampler and GC/MS spectra acquired.

3. Results and discussion

The GC/MS spectra obtained for a mixture of 450 μM 17-OH PROG and 150 μM AD in DCM solvent are shown in Fig. 2. The gas chromatogram (Fig. 2A) gave a retention time (RT) of 7.1 min for AD and 9.6 min for 17-OH PROG. The mass spectrum (Fig. 2B) shows the 17-OH PROG peak exhibits a parent ion peak [M⁺], with an m/z of 330 Da, and a fragmentation pattern that matches that reported in the literature [29] The mass spectrum of AD (Fig. 2C) shows the molecular ion peak [M⁺] of 286 Da, and a fragmentation pattern that matches the literature [24] mass spectrum. The symmetry of the GC peak is attributed to lack of hydroxyl groups, which can lead to peak tailing.

Fig. 2.

A. GC/MS of mixture of 450 μM 17-OH PROG (RT 9.6) and 150 μM AD (RT 7.1 min) and corresponding mass spectra with molecular ion peaks 330 Da (B) and 286 Da (C) respectively.

Fig. 3 shows GC/MS data obtained for a sample of 320 μM oxygenated CYP17A1-ND plus 400 μM 17-OH PROG, which was extracted from a sample that had been subjected to cryoradiolysis and annealing, followed by rR spectroscopic studies [12] The gas chromatogram (Fig. 3A) shows a peak corresponding to 17-OH PROG with RT 9.7 min, matching that observed for the standard of 17-OH PROG. This sample component exhibits a mass spectrum (Fig. 3B) matching the literature spectrum as well as the mass spectrum of the standard; [M⁺] = 330 Da. AD was also observed (Fig. 3A) with a RT of 7.2 min as in standards. SpectraBase reported that a m/z value of 286 Da is a signature fragmentation of androstenedione compound. Its full mass spectrum also matched the standards and literature data, i.e., [M⁺] = 286 Da. Note that some or all of the peaks including 281, 191, 207, 147, 133, 117 Da were observed especially in samples that had undergone cryoradiolysis and extraction. These peaks are caused by the background noise as concentration of samples got lower. These background peaks are due to septum and column bleeding and become more pronounced as concentration of samples decreases. As concentration of sample increases the background peaks become suppressed. We know these are background peaks as they appear at any point along the spectrum or on blank DCM or solvent blank spectrum.

Fig. 3.

A. GC/MS spectrum of the extract of an incubation with CYP17 bound to 17-OH PROG sample extracted after cryoradiolysis showing clearly the 17-OH PROG peak with RT 9.7 min and AD RT 7.2 min and the corresponding mass spectra with molecular ion peaks 330 Da (shown in B) and 286 Da (shown in C) respectively.

Fig. 4 shows GC/MS data acquired for a sample of underivatized mixture of 17-OH PREG and DHEA in DCM. The gas chromatogram (Fig. 4A) exhibits some broadening and peak tailing for both peaks, a consequence of the 3-OH substituent. The RT of 17-OH PREG was 10.3 min and the mass spectrum (Fig. 4B) revealed the presence of a parent ion m/z 332 Da [M⁺] with a variety of fragmentation products matching literature data [30] The peak assigned to DHEA had a RT 7.4 min and a mass spectrum that matches the literature mass spectrum [24], exhibiting a parent ion peak [M⁺] of 288 Da and appropriate fragmentation peaks (Fig. 4C).

Fig. 4.

A. GC/MS of 17-OH PREG 450 μM (RT 10.2 min) and DHEA 150 μM (RT 7.4 min) and corresponding mass spectra with molecular ion peaks at 332 Da (shown in B) and 288 Da (shown in C) respectively.

Fig. 5 shows GC/MS data obtained for a sample of 280 μM oxygenated CYP17A1-ND plus 450 μM 17-OH PREG, which was extracted from an NMR tube that had been subjected to cryoradiolysis and rR spectroscopic studies [31] followed by later annealing to room temperature. The gas chromatogram (Fig. 5A) shows a peak corresponding to 17-OH PREG with RT 10.3 min, matching that observed for the standard of 17-OH PREG, and exhibits a mass spectrum (Fig. 5B) matching the literature spectrum and the mass spectrum of standards; [M⁺] = 332 Da. DHEA was also observed with a RT of 7.6 min (Fig. 5A) as in standards. SpectraBase reported that a m/z value of 288 Da is a signature fragmentation of DHEA compound. Its mass spectrum matched the standards and literature data with [M⁺] = 288 Da. The results from this work is summarized in Table 1, clearly showing agreement of mass spectral data between standard mixtures and extracted cryoreduced samples.

Fig. 5.

A. GC/MS spectrum of the extract of an incubation with CYP17 bound to 17-OH PREG after irradiation and cryoradiolysis and the corresponding mass spectra with molecular ion peaks at 332 Da (shown in B) and 288 Da (shown in C) respectively.

Table 1.

RT and molecular masses from GC/MS spectra of steroids studied using the temperature program and conditions outlined above.

Steroid	Molecular ion peak [M+]/Da	RT/ Min
		Standard Mixture	Extracted CYP17A1 sample
17-OH PROG	330	9.6	9.7
17-OH PREG	332	10.3	10.3
AD	286	7.1	7.2
DHEA	288	7.4	7.6

4. Conclusions

Previous studies have shown analysis of steroids by GC/MS with or without derivatization. This is a report to detail the analysis of steroids from cryoradiolysis and annealing using GC/MS. The results demonstrate that the steroid metabolites AD and DHEA are obtained from oxygenated CYP17A1 after cryoradiolysis and annealing with 17-OH PROG, 17-OH PREG. No DHEA was observed in the control, showing that no DHEA was formed in the GC/MS column or during irradiation procedure. For the CYP17 sample, other peaks could be from other products including lipids and any other products formed. It has been established that cryoradiolysis products match those obtained in reconstituted system under physiological conditions.

Abbreviations:

17-OH PREG

17α-Hydroxypregnenolone

17-OH PROG

17α-Hydroxyprogesterone

AD

Androstenedione

CYP17A1

Cytochrome P450 CYP17A1

DHEA

Dehydroepiandrosterone

DCM

Dichloromethane

GC/MS

Gas chromatography/mass spectroscopy

M/Z

Mass to charge ratio

ND

Nanodiscs

CYP17A1-ND

Cytochrome P450 17A1 Nanodiscs

rR

Resonance Raman

RT

Retention time.

Data availability

Data will be made available on request.