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–) → Fe3+ 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.

Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature

“…18 The higher population of high-spin heme observed by EPR upon rapid freezing is more consistent with the Type I spectral changes observed by UV–vis upon cYY binding at room temperature, where a blue-shift of the heme Soret band from 416 to 395 nm is observed, 14,15,27 which suggests displacement of the solvent-derived ligand upon substrate binding to yield a five-coordinate high-spin heme. 17,18 …”

“…Similar thermodynamic equilibria between the high- and low-spin hemes have been observed for different P450 isoforms. 17,32 …”

Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

“…The 2D pulsed EPR HYSCORE measurement can resolve the signals from hydrogens of the axial water ligand and can locate the hydrogens relative to the low-spin ferric heme iron. 1,9,19 The HYSCORE spectral region from hydrogens has a set of arcs from the axial water ligand (with simulated line shape in green) in drug-free CYP2C9d (Figure 4), and an additional pair of arcs from the β -hydrogens of cysteine. The water arcs disappear in D 2 O, but not those from the β -hydrogens (data not shown).…”

“…MCD intensities are the differential absorbance of left ( σ − ) and right ( σ + ) circularly polarized light (Δ A = A L − A R ), following the sign convention of Piepho and Schatz, 32 in units of θ (millidegrees) = 32982 × Δ A . Data collection and sample preparation were as previously described 1 except that 1 μ L (1.1%) of a DMSO stock solution of LP10 (100 mM) was added for a final inhibitor concentration of 1.1 mM ( K D = 1.7 μ M 11 ) to generate the reverse type I complex.…”

“…The equilibrium is isoform-dependent. 1 In the traditional paradigm, the low-spin state cannot be reduced by its physiological partner CYP reductase. Substrate binding displaces the water ligand, driving the equilibrium toward the reducible, high-spin form and allowing the catalytic cycle to start with reduction by CYP reductase.…”