The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

Abstract

P450 family 4 fatty acid ω-hydroxylases preferentially oxygenate primary C-H bonds over adjacent, energetically favored secondary C-H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for ω-hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C-H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for ω-hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for ω-hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane ω-hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for ω-hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for ω-hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C-H bonds.

Keywords: X-ray crystallography; cytochrome P450; enzyme structure; fatty acid metabolism; heme; membrane protein; xenobiotic.

FIGURE 1.

Octane shifts the Soret absorption band of CYP4B1 to lower wavelengths. A, maximum absorption for the Soret band of CYP4B1 (red) exhibits a wavelength of 419 nm indicative of a predominantly low spin ferric heme. Incremental increases in the concentration of octane of 0.5 (orange), 1 (yellow), 2 (green), 4 (cyan), 8 (blue), 16 (dark blue), and 32 μm (violet) shift the Soret maximum to lower wavelengths indicative of a high spin ferric heme. B, concentration dependence of the difference in absorption was fit to the quadratic form of the one-site binding equation by non-linear regression and a protein concentration of 0.90 μm as described under “Experimental Procedures.” The estimated dissociation constant is 0.32 μm with a maximum absorbance change of 0.069 for the experiment shown. The mean and standard error for values of the dissociation constant obtained from five replicate experiments is 0.34 ± 0.10 μm.

FIGURE 2.

A, secondary and tertiary structure of rabbit cytochrome P450 4B1. The truncated N-terminal TMH domain is colored dark gray, the polar linker region is colored light gray, and the catalytic domain is colored from red beginning with residue 42 to blue at the C terminus. The heme prosthetic group is depicted as a stick figure with the iron shown as a sphere. The semi-transparent tubes illustrate tunnels rendered by Mole leading from the active-site cavity above the heme to the surface of the enzyme. B, linker region and TMH form a continuous A″ helix. Polar interactions between the linker region and the catalytic domain are depicted by dashed lines between the labeled amino acid residues shown as stick figures. Nitrogen, oxygen, and iron atoms are colored blue, red, and orange, respectively. C, trajectory of the A″ helix relative to the catalytic domain is compared with that of human P450 3A4 (PDB code 1TQN) and P450 51A1 from S. cerevisiae (PDB code 4LXJ). The 51A1 TMH is colored dark green; the relatively short polar linker is lime green, and a portion of the catalytic domain is depicted in pale green. Helix A″ in the linker region of 3A4 is colored copper, and a portion of the 3A4 catalytic domain is colored pale yellow.

FIGURE 3.

A, omit 2mF_o − DF_c electron density map contoured at 1.5 σ (gray mesh) and phased without Glu-310 and the heme define the covalent linkage of the heme to Glu-310. B, out-of-plane distortion of the 5-methyl and attached pyrrole relative to the plane of the heme macrocycle and the orientation of the heme 7-propionate into the active site. C, omit 2mF_o − DF_c electron density map contoured at 1.5 σ (gray mesh) and phased without octane defines the position of octane in the active site. D, hydrogen bonding and ionic interactions (dashed lines) that contribute to the observed position of the heme 7-propionate. The red and orange spheres represent a water molecule and the heme iron, respectively. Octane carbons are colored slate, and sulfur is colored yellow. Colors for other atoms correspond to those in Fig. 2A.

FIGURE 4.

Active-site cavity of rabbit P450 4B1 (transparent surface) with octane rendered as a space-filling model. The surface was truncated at the entrance to the solvent channel near Gln-377. A, view of the narrow dimension of the slot-like substrate-binding cavity with octane constrained in a planar conformation. B, view of the face of the slot displaying the alternating bond angles between the carbon atoms. Atom colors are described in legends to earlier figures. Residues indicated by bold text are conserved in other human P450 family 4 ω-hydroxylases (Fig. 5). C, portions of an amino acid sequence alignment for characterized P450 4B1 proteins from mammalian species with the active-site residues in rabbit 4B1 indicated by asterisks.

FIGURE 5.

Sequence alignment of functionally characterized human (h) P450 family 4A, 4F, 4V2, 4X1, and 4Z1 enzymes with rabbit (rb) 4B1. Regions containing amino acids residues in rabbit 4B1 that form the substrate-binding cavity are shown with the active-site residues of rabbit 4B1 designated by asterisks. The regions correspond to five of the six SRS regions defined by Gotoh (60). The residue numbering corresponds to rabbit 4B1. A consensus sequence is shown for ω-hydroxylases. Glu-310 is not conserved in the four non-ω-hydroxylases, and the latter show additional differences from the ω-hydroxylases for active-site residues. A complete sequence alignment is available as supplemental Fig. S1.

FIGURE 6.

Effects of substrate size and amino acid substitutions for Glu-310 on rates of oxygenation of primary (ω) and secondary (ω-1) C–H bonds. Decane exhibits significant amounts of (ω-2)-hydroxylated products that contribute to the total oxygenation of secondary C–H bonds. Means and standard deviations are provided in Table 2.

FIGURE 7.

A, homology model of human P450 4A11 with lauric acid (slate carbons) docked in the active-site cavity (transparent surface). Other protein and atom colors are as described in earlier figures. Arg-96 on strand 2 of sheet β1 can reside in the active site to interact with fatty acid substrates. Coordinates for the P450 4A11 homology model in PDB format are available as supplemental File P450–4A11-homology-model.pdb. B, amino acid sequence alignment of human 4A11 (residues 81–100) with rabbit, rat, and mouse 4A enzymes. 4A11 Arg-96 (red) is conserved in most 4A sequences with exceptions where an arginine is aligned with His-88 on the adjacent strand of β-sheet 1 which is also oriented into the active site.