scholarly journals Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase

2021 ◽  
Author(s):  
Julie Ducharme ◽  
Irina F. Sevrioukova ◽  
Christopher J. Thibodeaux ◽  
Karine Auclair
Keyword(s):  
Cytochrome P450 ◽  
Protein Interactions ◽  
Conformational Dynamics ◽  
Underlying Mechanism ◽  
Deuterium Exchange ◽  
Reduced Form ◽  
Cytochrome P450 3A4 ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
The Impact

AbstractCytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds, as well as by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects however is poorly understood. We have used hydrogen-deuterium exchange mass spectroscopy (HDX-MS) to investigate the impact of CPR and three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced form of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from a suppression of deuterium exchange in several regions of CYP3A4, including those known to be involved in protein-protein interactions (C-helix) as well as substrate binding and specificity (B’-, E-helices and K/β1-loop). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted by CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of allostery, ligand binding, and catalysis in CYP enzymes.

scholarly journals Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase-cytochrome P450 3A4 in nanodiscs

FEBS Journal ◽  
2017 ◽  
Vol 284 (14) ◽  
pp. 2302-2319 ◽  
Author(s):  
Kang-Cheng Liu ◽  
John M. X. Hughes ◽  
Sam Hay ◽  
Nigel S. Scrutton
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome P450 3A4 ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase

Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4

2010 ◽  
Vol 432 (3) ◽  
pp. 485-494 ◽  
Author(s):  
Yassar Farooq ◽  
Gordon C. K. Roberts
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Hill Coefficient ◽  
Molar Ratio ◽  
Cytochrome P450 3A4 ◽  
Cytochrome P450 Reductase ◽  
Nadph Cytochrome ◽  
P450 Reductase ◽  
Absorbance Change ◽  
Nadph Cytochrome P450 Reductase

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)–CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s−1. Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR–CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR–CYP3A4 complex can dissociate and reform between the first and second electron transfers.

scholarly journals Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

2011 ◽  
Vol 435 (1) ◽  
pp. 197-206 ◽  
Author(s):  
Louise Aigrain ◽  
Denis Pompon ◽  
Gilles Truan
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Reductase ◽  
Nadph Cytochrome ◽  
Catalytic Domains ◽  
P450 Reductase ◽  
Internal Electron ◽  
Nadph Cytochrome P450 Reductase ◽  
The Impact

CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.

Sign in / Sign up

Export Citation Format

Share Document