Spectroscopic studies of the cytochrome P450 reaction mechanisms

Abstract

The cytochrome P450 monooxygenases (P450s) are thiolate heme proteins that can, often under physiological conditions, catalyze many distinct oxidative transformations on a wide variety of molecules, including relatively simple alkanes or fatty acids, as well as more complex compounds such as steroids and exogenous pollutants. They perform such impressive chemistry utilizing a sophisticated catalytic cycle that involves a series of consecutive chemical transformations of heme prosthetic group. Each of these steps provides a unique spectral signature that reflects changes in oxidation or spin states, deformation of the porphyrin ring or alteration of dioxygen moieties. For a long time, the focus of cytochrome P450 research was to understand the underlying reaction mechanism of each enzymatic step, with the biggest challenge being identification and characterization of the powerful oxidizing intermediates. Spectroscopic methods, such as electronic absorption (UV-Vis), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electron nuclear double resonance (ENDOR), Mössbauer, X-ray absorption (XAS), and resonance Raman (rR), have been useful tools in providing multifaceted and detailed mechanistic insights into the biophysics and biochemistry of these fascinating enzymes. The combination of spectroscopic techniques with novel approaches, such as cryoreduction and Nanodisc technology, allowed for generation, trapping and characterizing long sought transient intermediates, a task that has been difficult to achieve using other methods. Results obtained from the UV-Vis, rR and EPR spectroscopies are the main focus of this review, while the remaining spectroscopic techniques are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Keywords: Cytochrome P450; EPR spectroscopy; NMR spectroscopy; Nanodiscs; Resonance Raman spectroscopy; UV–Vis spectroscopy.

Figure 1

Left - the Nanodisc-CYP assembly. The cytochrome P450 molecule is shown in green, with heme presented in red sticks. The phospholipid bilayer is shown in orange with oxygen atoms in red, while the scaffold protein encompassing the lipid bilayer is shown as a blue cartoon representation. Right - CYP3A4 and CPR reconstituted into Nanodisc.

Figure 2

Electronic absorption spectra of human cytochrome P450 CYP3A4 in Nanodiscs without substrate (red line) and saturated with bromocryptine (blue line).

Figure 3

Titration curve calculated for non-cooperative binding to the protein with three binding sites and the same spectroscopic signal measured for all three sites (red) or from only second and third binding events with the first binding spectrally silent (blue). Populations of protein with one, two or three sites occupied with ligand are shown in black and marked (1), (2) and (3) correspondingly.

Figure 4

The high frequency rR spectra of cytochrome P450_cam in substrate-free (A) and substrate-bound (B) forms. Spectra were measured with 406.7 nm excitation line.

Figure 5

The high frequency rR spectra of 60 µM ferric cytochrome CYP3A4 in detergent without substrate (A), and with 600 µM testosterone (B), as well as rR spectra of ferric cytochrome 3A4 in nanodiscs (100 µM) without substrate (C), with 1200 µM of testosterone (D).

Figure 6

The low frequency rR spectra of cytochrome P450_cam in substrate-free (A) and substrate-bound (B) forms. Spectra were measured with 406.7 nm excitation line.

Figure 7

Low-frequency rR spectra of ferric CYP2B4: A) wild-type BHT-bound; B) F429H mutant BHT-bound. Spectra measured with 356 nm excitation line and normalized to the ν₇ mode at 676 cm⁻¹. Inserts show graphical representation of H-bond formation to CYP2B4 proximal cysteine upon Phe to His mutation.

Figure 8

The rR spectra of ¹⁶O₂ adducts of ND:CYP17 in H₂O buffer with PROG (panel A), 17-OH PROG (panel B), PREG (panel C) and 17-OH PREG (panel D). The lower section of each panel shows ¹⁶O₂–¹⁸O₂ difference plots in H₂O buffers in low (left) and high (right) frequency regions.

Figure 9

Electronic absorption spectra of CYP101A1 in its oxy (1), peroxy (2) and hydroperoxy (3) forms. [59]

Figure 10

EPR spectra of oxy-complex in CYP101A1, D251N mutant, cryoreduced in liquid nitrogen at 77K. Spectra were measured at 14 K after annealing for 1 minute at indicated temperatures, with g-values shown for each spectrum. The first spectrum with g₁ = 2.25 is typical for unprotonated peroxo-ferric intermediate, which is protonated to hydroperoxo-ferric complex after annealing at 173 – 190 K with g₁ = 2.29. Annealing at higher temperatures results in product hydroxyl-camphor formation with transient coordination complex of product and heme iron (g₁ = 2.62 and g₁ = 2.51) and gradual relaxation to the low-spin ferric CYP101A1 after warming up to the room temperature. Reproduced from reference [42] with permission from the American Chemical Society.

Figure 11

Resonance Raman spectral data for irradiated dioxygen adducts of CYP17A1. All spectra were measured with 442 nm excitation line at 77 K, total collection time of each spectrum was 6 hrs. The rR ¹⁶O₂ – ¹⁸O₂ difference traces in H₂O of irradiated oxy CYP17A1 samples (before annealing) with PREG (a) and 17-OH PREG (b) and corresponding samples after annealing to 165 K (c) and (d).

Figure 12

The ¹⁶O₂ – ¹⁸O₂ difference traces of irradiated dioxygen adducts of CYP17A1 samples with 17-OH PREG annealed at 190 K measured with 406 nm excitation line at 77 K.

Scheme 1

Cytochrome P450 catalytic cycle.

Scheme 2

Schematic representation of heme protein cryoreduction; glycerol (G).

Scheme 3

Proposed alternative mechanisms of C-C bond cleavage reaction catalyzed by CYP17.