Strength of axial water ligation in substrate-free cytochrome P450s is isoform dependent

Abstract

The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S(-)) → Fe(3+) transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well.

Figure 1

UV/vis absorbance of ligand-free CYPs at 298 K. The Soret and α,β bands at 520–680 nm are shown for CYP3A4, CYP2C9, CYP2E1, CYP51B1, and CYP125A1. The Soret of pure high spin heme is located at ∼390 nm and ∼417 for pure low spin. All spectra have been normalized to 1 μM CYP450.

Figure 2

Top: Near IR MCD spectra of CYP3A4, CYP125A1, CYP2C9, CYP51B1, and CYP2E1 at 4.2–4.7 K and 6 T. Bottom: 6 T near nIR MCD spectra of CYP3A4, CYP125A1, CYP51B1, and CYP2E1 at 298 K. Note that the predominantly high spin nature of CYP125A1 at 298 K (red) precludes measurement of the low spin transition. All samples were prepared in 100–200 mM KPi (pH = 7.4) + 55% glycerol-d₈.

Scheme 1. Schematic Free Energy Profile of the Macroscopic K_spin and the Differences in the Low Spin States Revealed by MCD

The right side includes the porphyrin HOMOs and the iron d-orbitals with ligand field parameters V and Δ indicated. The nIR charge transfer band energies for different CYPs correspond to different axial ligand field strengths in the relative order shown, with CYP2E1 and CYP125A1 having the strongest and weakest axial ligands, respectively. The lack of LFER between K_spin and these ligand field energies suggests a contribution of the high spin states to isoform-dependent differences in K_spin.

Figure 3

The 6 Tesla MCD spectra of the α,β region acquired at 298 K for CYP3A4, CYP2E1, CYP51B1, and CYP125A1 reveals large differences in high spin content. The negative MCD feature centered ∼650 (inset) is only present in high spin CYPs and represents thiolate → Fe(III) LMCT for the high spin enzyme.

Figure 4

CW EPR spectra of ligand-free CYPs 3A4, 2C9, 125A1, and 51B1. The EPR spectral g-values were fit using EasySpin as described in the text to extract accurate values that were used in the calculation of crystal field terms.

Figure 5

Measured MCD nIR CT energies for the CYPs studied here versus those predicted based on the EPR-derived axial (Δ/λ) and rhombic (V/λ) crystal field terms as described by Gadsby and Thomson. Note: no EPR data are available for CYP2E1. The results emphasize the inaccuracy of low spin ligand field energy calculations based on EPR correlations for these CYPs and the necessity for direct measurement of the nIR CT transition by MCD.