Intramolecular Heme Ligation of the Cytochrome P450 2C9 R108H Mutant Demonstrates Pronounced Conformational Flexibility of the B−C Loop Region: Implications for Substrate Binding

Abstract

A previous study (i.e. Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850.) revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In the present study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed in order to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme’s ability to regio- and stereo-specifically metabolize some ligands, while allosterically activating others.

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).…”

Exploration of the binding of curcumin analogues to human P450 2C9 based on docking and molecular dynamics simulation

“…This enzyme may be involved in potentially unfavorable drug-drug interactions or drug toxicity. Furthermore, CYP2C9 displays atypical kinetics with several substrates such as flurbiprofen, dapsone, naproxen [10][11][12]. Thus, it is necessary to explore how these drugs or substrates interact with 2 C9 so as to obtain some useful information for the drug's therapeutic efficacy or toxicity in clinical application.…”

Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism

“…The closed conformation is favored upon substrate binding (Williams et al, 2000(Williams et al, , 2003Yano et al, 2004) and alters the affinity toward enzyme partners Roberts et al, 2010). To account for the potential conformational changes on the modeling results, dimer formation was examined with an open and a closed form of CYP2C9.…”