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. 2020 Sep 11;21(18):6669.
doi: 10.3390/ijms21186669.

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

Francisco Esteves  1 , Philippe Urban  2 , José Rueff  1 , Gilles Truan  2 , Michel Kranendonk  1
Affiliations

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

Francisco Esteves et al. Int J Mol Sci. .

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.

Keywords: CPR-FMN-domain; CYP-substrate; NADPH-cytochrome P450 reductase; electron transfer; microsomal cytochrome P450; protein–protein interaction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Relative turnover rates (kcat) (x fold) of the CPRmut:CYP1A2 EROD (A), CPRmut:CYP2A6 C7H (B), and CPRmut:CYP3A4 DBODF (C) activities, normalized by the kcat demonstrated by CPRwt/CYP (determined in membrane fractions; technical replicates N = 3). CPR-FMN-domain wildtype (CPRwt:CYP, black stripes). CPR-FMN-domain mutants previously demonstrated to support a gain in CYP1A2-mediated EROD- (P117H and G144C: green), CYP2A6-mediated C7H- (G175D: blue), and CYP3A4-mediated DBODF-activity (N151D: red). kcat values of the CPRmut/CYP were compared with the ones of the CPRwt/CYP applying the unpaired t test (*** p < 0.0005; ** p < 0.005; * p < 0.05).
Figure 2
Figure 2
Relative turnover rates (kcat) (x fold) of the CPRmut:CYP1A2 CECOD (A), and DBODF (B) activities, normalized by the kcat demonstrated by CPRwt/CYP (determined in membrane fractions) (technical replicates N = 3). CPR-FMN-domain wildtype (CPRwt:CYP, black stripes). CPR-FMN-domain mutants previously demonstrated to support a gain in CYP1A2-mediated EROD- (P117H and G144C, green), CYP2A6-mediated C7H- (G175D, blue), and CYP3A4-mediated DBODF-activity (N151D, red). kcat values of the CPRmut/CYP1A2 were compared with the ones of the CPRwt/CYP1A2 applying the unpaired t test (** p < 0.005; * p < 0.05).
Figure 3
Figure 3
Relative CYP activities of the CPR-FMN-domain wildtype and four mutants plotted in function of the cyt c/CPR ratio. (A) CYP1A2 (EROD), (B) CYP2A6 (C7H), and (C) CYP3A4 (DBODF). Observed rate constants sustained by the CPR (kobs) were normalized by the CYP activity without cyt c [kobs (0 nM cyt c)]. FMN wildtype (CPRwt:CYP, black lines). FMN mutants previously demonstrated to support a gain in CYP1A2-mediated EROD- (P117H and G144C, green lines), CYP2A6-mediated C7H- (G175D, blue lines), and CYP3A4-mediated DBODF-activity (N151D, red lines). Normalized CYP activities represent the average of the three replicates and the error bars the standard deviation. Significant differences between the means of the activity profiles of CPR:CYP1A2 EROD (p = 0.015) (A) and CPR:2A6 C7H (p = 0.049) (B) were observed (ANOVA, one-way analysis of variance). (D) CPRwt supporting CYP1A2-mediated EROD- (green line), CYP2A6-mediated C7H- (blue line), and CYP3A4-mediated DBODF-activity (red line).
Figure 3
Figure 3
Relative CYP activities of the CPR-FMN-domain wildtype and four mutants plotted in function of the cyt c/CPR ratio. (A) CYP1A2 (EROD), (B) CYP2A6 (C7H), and (C) CYP3A4 (DBODF). Observed rate constants sustained by the CPR (kobs) were normalized by the CYP activity without cyt c [kobs (0 nM cyt c)]. FMN wildtype (CPRwt:CYP, black lines). FMN mutants previously demonstrated to support a gain in CYP1A2-mediated EROD- (P117H and G144C, green lines), CYP2A6-mediated C7H- (G175D, blue lines), and CYP3A4-mediated DBODF-activity (N151D, red lines). Normalized CYP activities represent the average of the three replicates and the error bars the standard deviation. Significant differences between the means of the activity profiles of CPR:CYP1A2 EROD (p = 0.015) (A) and CPR:2A6 C7H (p = 0.049) (B) were observed (ANOVA, one-way analysis of variance). (D) CPRwt supporting CYP1A2-mediated EROD- (green line), CYP2A6-mediated C7H- (blue line), and CYP3A4-mediated DBODF-activity (red line).
Figure 4
Figure 4
Electrostatics effects of the P117H and N151D mutations. Panels (A,C) present the variations of pKa of the surrounding aspartates and glutamates of P117H and N151D, respectively. pKas values were calculated on the average structures generated with four independent molecular dynamics simulations for wild-type (WT), P117H (P117H), or N151D (N151D) with the propka options of the pdb2pqr software, using a pH = 7.5 value and the AMBER force field. Theoretical pKa values of aspartate (3.8) or glutamate (4.5) were then subtracted from the simulated ones. Panels (B,D) present a mapping of the electrostatic potential onto the solvent excluded surface of P117H (Panel (B)) or N151D (Panel (D)) mutants compared to the wildtype FD. In Panel (B), the arrow represents the position of the H117 residue (hidden by the surface). In Panel (D), the magenta surface corresponds to the D151 residue. In Panels (B,D), the light orange oval shows the locations of the electrostatic potential and surface changes. Electrostatic potentials were calculated on the average structure of simulation 1 with the APBS module of Pymol using defaults parameters. The color scale is indicated below each surface and is in kT/e.
Figure 5
Figure 5
Effects of the G144C and G175D mutations. Panel (A) presents the wild-type (green, simulation 1) and G144C (magenta, simulation 1) average structures. Panel (B) presents a mapping of the electrostatic potential onto the solvent excluded surface of wild-type (left) or G144C mutant (right), the orange color corresponds to the surface of the flavin mononucleotide (FMN) residue. Panel (C,D) presents detailed view of the wild-type (green, simulation 1) and G175D (magenta, simulation 1) average structures.
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