Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding

Abstract

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR’s degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP’s substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound.

The Central Role of Cytochrome P450 in Xenobiotic Metabolism—A Brief Review on a Fascinating Enzyme Family

“…This regards the genetic variability of CPR (encoded by the POR gene), the obligatory redox partner of microsomal CYPs for the reception of electron equivalents to sustain their activity. Recent data suggest that natural occurring genetic variants of POR may lead to altered CYP mediated drug metabolism [ 126 , 127 , 128 , 129 ].…”

“…Factors such as age, sex, hormone levels, and environment, as well as pathological conditions such as infection, inflammation, cholestasis, and cancer are aspects demonstrated to influence CYP expression and activity [ 25 , 130 , 131 ] ( Figure 4 ). Other biochemical factors such as protein-protein interaction—involving CYP interaction with redox partners and other proteins with allosteric regulatory effect [ 76 , 77 , 81 , 129 ], or substrate-substrate interaction—consisting in CYP activity inhibition due to substrate interference/competition mechanism, are also implicated in CYP function [ 25 , 100 , 140 , 141 , 142 ].…”

The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells

“…The microsomal contents of components of the CYP-enzyme complex system are shown in Table 1 . Microsomal CYP isoforms are strictly dependent on CPR in their activity [ 21 , 22 , 37 ]. In vivo, CPR:CYP contents are in favor of CYP, implying competition between individual CYP isoforms in binding to CPR in the endoplasmic reticulum [ 45 , 46 ].…”

“…Total membrane proteins were quantified using the Bradford method [ 35 ]. CYP quantification was performed by CO-difference spectrophotometry and CPR by cytochrome c reduction, similarly to what was previously described [ 36 , 37 , 38 ]. Due to the low CYP concentration in BC cell lines, a membrane protein fraction isolated as previously reported [ 39 ] from an in-house engineered bacterial cell model co-expressing CPR and CYP1A2 [ 40 , 41 ] was added to the MCF-7 cells derived membrane protein fractions in order to increase the signal-to-noise ratio.…”

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

“…Another possible reason for the low frequency of detecting CYP2E1-CPR crosslinks is a severe shortage of CPR compared to the concentration of its electron acceptor partners [ 47 ]. Low abundance of CPR in the microsomal membrane results in stiff competition between multiple P450 species and other CPR-dependent enzymes, such as heme oxygenase [ 48 , 49 ], to form their complexes with this universal electron donor [ 50 , 51 ]. Furthermore, the formation of mixed oligomers between multiple P450 species is hypothesized to result in selective barring of some cytochrome P450 species from interactions with CPR due to specific organization of the P450 heterooligomers [ 8 , 9 , 10 , 11 ].…”