Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries

Abstract

Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl]>50mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type>E48G/E49G>D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4.

Keywords: Allostery; CYP3A4; Cytochrome P450; Cytochrome b(5); Drug oxidation; Testosterone.

FIGURE 1

Stimulation of CYP3A4-catalyzed progesterone 6β-hydroxylation by b₅ and b₅ mutations. (A) Effect of b₅ and b₅ mutations on catalytic activities of CYP3A4 in the presence of CYMS as lipid for reconstitution. Incubations contained 50 μM progesterone and a molar ratio of P450:POR:b₅ at 1:3:3. Results are shown as the percentage activity compared to wild-type b₅ values (=100%) from triplicate determinations, means ± standard deviations. (B) Comparison of wild-type b₅ and selected b₅ mutations on CYP3A4 catalysis with molar ratio of P450:POR:b₅ at 1:3:3 (black bars) or 1:3:10 (white bars). Results are shown as the percentage activity compared to wild-type b₅ values with 1:3:3 ratio of P450:POR:b₅ (=100%) from triplicate determinations, means ± standard deviations.

FIGURE 2

Stimulation of CYP3A4-catalyzed testosterone 6β-hydroxylation and nifedipine oxidation by b₅ and b₅ mutations. Purified CYP3A4 (30 pmol) was reconstituted with POR, various b₅ mutations, and CYMS as lipid for reconstitution. Catalytic activities toward (A) testosterone at 50 μM substrate concentration at a P450:POR:b₅ molar ratio of 1:2:3 and (B) nifedipine at 200 μM substrate concentration at a P450:POR:b₅ molar ratio of 1:2:4 were analyzed by HPLC. Results are shown as the percentage activity compared to wild-type b₅ values (=100%) from duplicate determinations, means ± standard deviations. (C) Titration of CYP3A4-catalyzed testosterone 6β-hydroxylation with human cytochrome b₅ mutation E48G/E49G. The stimulation of activity with P450:POR molar ratio of 1:2 reaches a maximum at a 3–5 molar excess of the b₅ mutation and does not increase further.

FIGURE 3

Circular dichroism (CD) spectra of cytochrome b₅ and mutations. CD spectra in (A) the far-UV, (B) the near UV, and (C) visible range for wild-type, D58G/D65G, and E48G/E49G double mutations are shown. Samples contained 10 μM b₅ in 100 mM potassium phosphate, pH 7.4.

FIGURE 4

(A) Steady-state kinetics of testosterone 6β-hydroxylation by CYP3A4 in the absence or presence of wild type b₅ or mutations. Incubations contained 20 to 600 μM testosterone and a molar ratio of P450:POR:b₅ at 1:2:3. The allosteric sigmoidal model was fit to the data points as described under Material and Methods. Kinetic parameters for CYP3A4 with wild-type b₅: the Hill coefficient h = 1.7 ± 0.2, V_max = 8.3 ± 1.0 nmol/min/nmol, and K_0.5 = 284 ± 52 μM; with b₅ mutation E48G/E49G, h = 1.4 ± 0.2, V_max = 3.2 ± 0.8 nmol/min/nmol, and K_0.5 = 467 ± 174 μM; with b₅ mutation D58G/D65G, h = 1.8 ± 0.2, V_max = 1.1 ± 0.1 nmol/min/nmol, and K_0.5 = 215 ± 26 μM; without b₅, h is 1.4 ± 0.3, V_max = 1.5 ± 0.3 nmol/min/nmol, and K_0.5 = 409 ± 170 μM. (B) Effect of [NaCl] on CYP3A4 testosterone 6β-hydroxylation in the presence of wild-type b₅ or b₅ mutation E48G/E49G. Reaction conditions were equivalent to those described in (A) with 200 μM testosterone.

FIGURE 5

Competition assays. CYP3A4-catalyzed formation of 6β-hydroxytestosterone in the presence of (A) wild-type b₅ and D58G/D65G double mutation or (B) wild-type b₅ and E48G/E49G double mutation. Reactions were conducted with a constant P450:POR molar ratio of 1:2 in the presence of constant wild-type b₅ and varying amounts of double mutation, or in the presence of constant double mutation and varying amounts of wild-type b₅. Competition experiments using b₅ mutations E48G (C) and E48G/E49G (D) show no inhibition by double mutation D58G/D65G. Error bars indicate means ± standard deviations of triplicate assays. Due to low conversion, experiments for panels C and D used tracer [³H]-testosterone as in ref. 21.

FIGURE 6

Effect of phospholipids on b₅-stimulated catalysis of CYP3A4. Purified CYP3A4 (30 pmol) and POR were reconstituted with wild-type b₅ or mutations D58G/D65G and E48G/E49G in the presence of various phospholipids at a P450:POR:b₅ molar ratio of 1:2:3. Activities with reconstituted assays using two types of purified phosphatidyl choline (DLPC and DOPC) and three types of natural phospholipids (PC from porcine brain, containing fatty acyl groups 18:1 (33%), 16:0 (31%) and 18:0 (17%); PE from bovine brain, containing fatty acyl groups 18:1 (24%), 20:4 (19%) and 18:0 (16%); and PS from bovine brain, containing fatty acyl groups 18:0 (40%) and 18:1 (29%)) are shown. Incubations were carried out with 50 μM testosterone, and the formation of 6β-hydroxytestosterone was determined by HPLC analysis. Results are shown as the percentage activity compared to wild-type b₅ values in PC+PS (=100%), means ± standard deviations from triplicate determinations. In the absence of b₅, conversion to 6β-hydroxytestosterone is 0.4±0.1% under these conditions.

FIGURE 7

Immunoblot analysis of CYP3A4 and b₅ cross-linked with EDC, and the effect of phospholipid on the complex formation. Reactions were performed in 50 μmM potassium phosphate buffer pH 7.0, containing 2 μM CYP3A4, 20 μM b₅, 200 μM phospholipid, and 2 mM EDC. (A) CYP3A4 (50 kDa) and wild-type b₅ or mutations (16 kDa) incubated with PS or PC+PE (1:1) in the absence of presence of EDC (B) CYP3A4 and b₅ incubated with PC+PS (1:1) or DLPC in the absence of presence of EDC. (C) The immunoreactive bands corresponding to CYP3A4-b₅ (1:1) complex were quantified by densitometric scanning. The bar graphs show the relative densities of bands for two experiments expressed as a percentage of the condition with wild-type b₅. Mean ± standard error, **, p < 0.01.