CW EPR parameters reveal cytochrome P450 ligand binding modes

Area of Science:

• Biochemistry
• Enzymology
• Drug Metabolism

Background:

• Cytochrome P450 (CYP) enzymes are crucial for drug metabolism and interactions, utilizing heme cofactors.
• Drug binding to CYPs often involves alterations in heme iron's coordination and spin state, traditionally studied via UV-vis spectroscopy.
• Existing structural and spectroscopic data suggest some CYP-drug interactions involve water molecules bridging the heme and drug.

Purpose of the Study:

• To investigate the prevalence and characteristics of water-bridged heme complexes in Cytochrome P450 enzymes.
• To determine if pulsed and continuous wave electron paramagnetic resonance (EPR) can identify and differentiate water-bridged binding modes.
• To assess the underrepresentation of water-bridged complexes in current CYP structural databases.

Main Methods:

• Utilized pulsed and continuous wave (CW) EPR spectroscopy to study interactions of various nitrogenous drugs with CYP3A4 and CYP2C9.
• Employed principal component analysis to analyze EPR spectral parameters and identify distinct clusters.
• Compared EPR findings with existing UV-vis spectroscopic and crystallographic data.

Main Results:

• Demonstrated that water-bridged heme complexes are common among nitrogenous drugs binding to CYP3A4 and CYP2C9.
• CW EPR spectral parameters showed distinct clustering for water-bridged complexes, differentiating them from directly coordinated states.
• Identified heterogeneous mixtures of ligated states, including both directly coordinated and water-bridged complexes, in CYP enzymes.

Conclusions:

• Water-bridged binding modes are prevalent in CYP-drug interactions and can possess energies comparable to other ligation modes.
• CW EPR is a powerful tool for identifying and distinguishing water-bridged complexes from directly coordinated states.
• Current CYP structural databases may underrepresent the occurrence of water-bridged binding modes.