Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Abstract

We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55+/-6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.

Figure 1

Formation of the CYP3A4 complexes with BMR studied by counter-flow (tandem cell) continuous variation titration. The interactions were monitored by the changes in the concentration of P450 low spin and high spin states. Panel a shows a series of spectra obtained in the experiment with CYP3A4 containing liposomes (LPS-76) at a concentration of 5 µM. The inset represents the spectrum of the first principal component obtained by PCA. The dashed line shows the approximation of this spectrum by the set of CYP3A4 absorbance standards. Panel b shows a series of titration curves obtained with LPS-76 and BMR taken at the concentration of 5 (circles), 2.6 (triangles), 1.1 (squares) and 0.8 µM (diamomds). Panel c represents a series obtained with CYP3A4-ND and BMR at a concentration of 5.3 (circles), 2.6 (triangles), and 1.3 µM (squares). Solid lines represent the results of the fitting of the data sets to the equation for the equilibrium of bimolecular association suited to the case of the counter-flow titration (eq. (3) in [50]).

Figure 2

Kinetics of NADPH-dependent reduction of BMR recorded with a rapid scanning stop-flow technique. Conditions: 20 µM BMR, 0.2 mM NADPH in 0.1 M Na-Hepes buffer, pH 7.4, 1 mM DTT, 1 mM EDTA containing NADPH-generating and oxygen-scavenging systems (see Materials and Methods), 5 °C. Spectra recorded in a stop-flow cell with 5 mm optical path length. a: Absorbance spectra of BMR taken during the reduction. The first spectrum corresponds to ~2 ms after addition of NADPH. b: The spectra of the first (solid line) and the second (dashed line) principal components found by PCA applied to a manifold of 5 series of spectra obtained in independent experiments. Panels c and d show the changes in absorbance at 455 and 385 nm respectively. Data points in circles correspond to the data set obtained at 5 °C shown in panel a. The data points shown in triangles in panel d represent a similar experiment at 25 °C. Solid lines show the results of fitting of the kinetic curves by a first order kinetic equation.

Figure 3

Kinetics of BMR-dependent reduction of CYP3A4-ND in the absence of substrate. Conditions: 2.6 µM 3A4, 5.2 µM BMR, 0.2 mM NADPH, CO-saturated 0.1 M Na-Hepes buffer, pH 7.4, 1 mM DTT, 1 mM EDTA containing oxygen-scavenging and NADPH-generating systems (see Materials and Methods), 25 °C. Spectra recorded in a stop-flow cell with 5 mm optical path length. a, b: Changes in absorbance in the Soret region during the reduction. The spectrum measured at time of origin is subtracted. Panel b scales up the spectra representing the first 14 seconds after mixing. c: The time course of the changes in the concentrations of the carbonyl complexes of CYP3A4(Fe²⁺) P450 (squares) and P420 states (diamonds), their total (triangles), as well as the low-spin (circles) and the high-spin (inverted triangles) states of CYP3A4(Fe³⁺). The dashed line indicates the total concentration of CYP3A4. Panel d shows the initial part of the kinetic curves for the low-spin (circles) and the high-spin (inverted triangles) states of CYP3A4(Fe³⁺).

Figure 4

Kinetics of BMR-dependent reduction of CYP3A4 incorporated in proteoliposomes at a 1:76 (LPS-76, a) or 1:910 (LPS-910, b) molar ratio of CYP3A4 to phospholipids. Reaction mixtures contained 2.95 (a) and 1.6 µM µM (b) CYP3A4 in proteoliposomes and a two-fold molar excess of BMR. Other conditions were as indicated for Fig. 3. The panels show the time course of the changes in the concentrations of the carbonyl complexes of CYP3A4(Fe²⁺) P450 (squares) and P420 states (diamonds), their total (triangles), as well as the low-spin (circles) and the high-spin (inverted triangles) states of CYP3A4(Fe³⁺). The dashed line indicates the total concentration of CYP3A4.

Figure 5

Kinetics of the changes in the intensity of fluorescence of CYP3A4(C468)-BODIPY upon its interactions with liposomes. Conditions: 1 µM CYP3A4(C468)-BODIPY in 0.1 M Na-HEPES buffer, 1 mM DTT, and 1 mM EDTA. Process was initiated by the addition of the liposomes to yield the L/P ratio of 100 (circles) and 1200 (triangles). Solid lines show the results of fitting of the kinetic curves with a bi-exponential equation, which gives the maximal amplitudes of the observed changes of 26% and 78% respectively.

Figure 6

Effect of the surface density of CYP3A4(C468)-BODIPY in proteoliposomes on the lifetime of fluorescence of the BODIPY probe. The preparations of the proteoliposomes were obtained by incubation of 1 µM CYP3A4-(C468)-BODIPY with various amounts of pre-formed liposomes for 1 hour in 0.1 M Na-HEPES buffer, 1 mM DTT, and 1 mM EDTA at 25 °C. Panel a: A series of normalized fluorescence decay traces (excitation an 405 nm, emission at 520 nm with the band with of 18 nm) obtained with L/P ratios of 100, 200, 400, 800, 1400 and 2000. All the curves of the set may be approximated by a sum of two exponents with characteristic times of 1.1 and 4.9 ns (ρ² > 0.95). The inset shows the traces obtained with L/P ratio of 100 (circles) and 2000 (triangles) in semi-logarithmic coordinates with fitting curves shown in solid lines. b: Dependence of F_fast on the concentration of CYP3A4(C468)-BODIPY in the membrane of proteoliposomes. Data points represent the results of two independent experiments. The solid line illustrates the fitting of the data set with the equation for the equilibrium of bimolecular association (K_D = 1.35 ± 0.06 pmol/cm²).