CW EPR parameters reveal cytochrome P450 ligand binding modes

Abstract

Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-HO-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR.

Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases

“…This suggests that the substrate binds to CYP199A4 but does not displace the resting state aqua ligand in agreement with the crystal structure. 33,35 In the case of 4-(pyridin-3-yl)-benzoic acid all three g-values of the major component shift, with large gx and gz changes, which supports coordination of the pyridinyl nitrogen of the substrate to the ferric paramagnetic center (Figure 6). 29 However, to definitively determine the proximal ligand on the basis of the gvalue shifts alone carries uncertainty, and thus we sought to identify the proximal ligand using high-resolution pulsed EPR methods.…”

“…23,24,25,26 A ferric P450 heme can be high-spin (HS, S = 5/2), or low-spin (LS, S = 1/2), and these electronic ground states can be distinguished by optical and EPR spectra. [26][27][28][29][30][31][32][33] The UV-vis spectrum of a substrate-bound cytochrome P450 is dependent on the nature of heme ligation. A type I change (blue shift; 418 nm to 390 nm) is associated with an increase in HS character upon ligand binding to the enzyme, whereas a reverse type I spectral change (red shift; 390 nm to 418 nm) reflects a change from more HS to LS character.…”

“…23 This binding mode in various P450 enzymes has been investigated using continuous wave (CW) and pulse EPR spectroscopy. 26,28,32,33,[37][38][39][40] Given the importance of substrate/inhibitor binding to P450s and the different modes available we have used a set of biophysical measurements to characterize selected binding modes in a single enzyme system. The cytochrome P450 monooxygenase, CYP199A4 from Rhodopseudomonas palustris HaA2 (Uniprot ID; Q2IUO2), has a high affinity for para-substituted benzoate substrates.…”

Comparison of Antifungal Azole Interactions with Adult Cytochrome P450 3A4 versus Neonatal Cytochrome P450 3A7

“…The reverse type I spectral shifts associated with ligand binding reinforcing water interaction with the heme iron typically have a trough at ~390 nm and a peak at ~422 nm (Ouellet et al, 2011). EPR studies have shown that 1,2,4-triazoles do not form water-bridged complexes in CYP3A4 (Lockart et al, 2018). Type II difference spectra associated with ligand nitrogen binding to the heme iron have trough at ~410 nm and a peak at ~434.…”

“…Although direct azole coordination to iron is observed, there is also a second arrangement in which fluconazole forms a hydrogen bond to a water molecule that is in turn coordinated to the heme iron. This water-bridged binding mode has only been observed in a few other bacterial P450 enzyme structures (Poulos and Howard, 1987;Ouellet et al, 2011;Fonvielle et al, 2013), but EPR data suggests this binding mode may be more common in solution (Lockart et al, 2018). Thus, at least two different azole binding modes are documented, though both are inhibitory (Seward et al, 2006).…”