CW EPR parameters reveal cytochrome P450 ligand binding modes

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 s...

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Bibliographic Details
Published in:Journal of inorganic biochemistry 2018-06, Vol.183, p.157-164
Main Authors: Lockart, Molly M., Rodriguez, Carlo A., Atkins, William M., Bowman, Michael K.
Format: Article
Language:English
Subjects:
Cytochrome P-450 Enzyme System - chemistry
Cytochrome P-450 Enzyme System - metabolism
Cytochrome P450
Drug binding
Electron Spin Resonance Spectroscopy - methods
EPR
HYSCORE
Oxidation-Reduction
Principal Component Analysis
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Summary: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-H2O-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. Analysis of continuous-wave electron paramagnetic resonance parameters shows that several cytochrome P450-drug samples contain a mixture of directly-coordinated and water-bridged species. This suggests that the two binding modes are similar in energy and demonstrates that the water-bridged binding mode is more common than previously thought. [Display omitted] •Cytochrome P450 (CYP) binding modes are reflected in continuous-wave electron paramagnetic resonance (EPR) spectra.•EPR distinguishes between directly-coordinated and water-bridged complexes.•Many CYP-drug complexes contain a mixture of binding modes.•Water-bridged complexes are common for nitrogenous drugs and drug fragments.
ISSN:0162-0134
1873-3344
DOI:10.1016/j.jinorgbio.2018.02.021