Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics

Abstract

The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains a mystery in biology. It is widely believed that sterol 14α-demethylase (CYP51), an essential enzyme of sterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species in different biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamics simulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redox partner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterol-producing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanics in the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules in various orientations and the interactions are transient. Close proximity of ferredoxin, however, is required to complete the substrate-induced large-scale structural switch in the P450 domain that enables proton-coupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamental implications regarding the early evolution of electron transfer proteins and for the redox reactions in the early steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequent diversification of the P450 electron transfer machinery in nature.

Figure 1

(a) Cytochrome P450 catalytic cycle. The cycle begins with the binding of a substrate, which increases the redox potential of the ferric heme iron (1). The iron (FeIII) accepts the first electron from the redox partner protein [RP], and the redox complex dissociates (2). The ferrous iron (FeII) binds molecular oxygen (3). The dioxygen complex accepts the second electron from the RP producing the ferric-peroxo anion and the redox complex dissociates (4). The ferric-peroxo anion is then protonated to form the ferric-hydroperoxo state (5). The second protonation of the distal oxygen atom causes the O–O bond scission, release of a water molecule, and generation of a highly reactive electrophilic ferryl (FeIV)-oxo cation radical known as Compound I (6), leading to insertion of the second oxygen atom into the substrate (7). (b) CYP51 reaction includes three P450 cycles and thus requires six sequential electron transfer events.

Figure 2

Substrate-induced conformational switch is similar in bacterial and eukaryotic CYP51s. Overlaid structures of substrate-bound (grey) and substrate-free (khaki) CYP51 orthologs. M. capsulatus (7SNM and 6MI0, respectively, rmsd of Cα of 2.44 Å), human (6UEZ and 4UHI, rmsd of Cα of 1.98 Å), and T. cruzi (6FMO, molecules A and D, rmsd of Cα of 1.83 Å). The orientation is about the same (upper P450 view). The directions of the changes are outlined with blue arrows on the M. capsulatus CYP51 structure. The carbon atoms of lanosterol (M. capsulatus and human) and obtusifoliol (I105F T. cruzi) are colored in cyan. The ribbons of the parts of the molecules that are not involved in the conserved conformational change^, (7SNM: rmsd of Cα of 0.77) are transparent. For comparison, the ligand-free and detergent-bound M. capsulatus CYP51 structures (6MI0 vs. 6MCW) have RMSD of Cα of 0.37 Å.

Figure 3

Binding mode of the CYP51 substrate is conserved across phylogeny. (a) The 2Fo-Fc electron density map (at 2σ) for lanosterol, the heme, and the H-bond forming residues, Y186 and L324, in the structure of M. capsulatus CYP51. (b) Sterol substrates bound in the active center of bacterial (M. capsulatus—cyan, and eukaryotic CYP51s (human—rosy-brown, and T. cruzi—salmon). The residues preceding the β1-4 strand (L324, I379, and M358) whose main chain carbonyl oxygen forms the H-bond with the sterol hydroxyl are colored correspondingly. The distances and the H-bonds are depicted as magenta and green dashes, respectively.

Figure 4

Interactions between the CYP51 proteins and their substrates. Calculations were made in MOE using molecule A. Polar interactions are in plum; hydrophobic interactions are in green, and the arene–H interaction between Y103 and C19 atom of obtusiofoliol is indicated as a green dotted line.

Figure 5

Substrate binding cavities in (a) M. capsulatus, (b) human, and (c) T. cruzi CYP51. Distal P450 view. Calculated in Voidoo, active site cavity volumes are 913 ų, 865 ų, and 1203 ų, respectively. The substrate entrance^,– is marked with a blue arrow, the subpocket in the active site of I105F T. cruzi CYP51 is circled.

Figure 6

M. capsulatus CYP51-ferredoxin complex. Upper P450 view. (a,b) Five poses of the ferredoxin domain docked to the proximal surface of the P450 domain (grey), (a) docking in MOE, the heme iron to the Fe1 atom of the [2Fe-2S] cluster distance (|Fe–Fe|) range is 14.1–16.4 Å, (b) docking in Rosetta, |Fe–Fe| range is 13.3–16.7 Å. Lanosterol, heme, and [2Fe-2S] clusters are shown as stick models. (c,d) The MD snapshots of P450-superimposed complexes, showing changes in the distances/angles between (c) the heme iron/heme plane and Fe1 of ferredoxin and (d) the heme iron/heme plane and P63 Cγ atom of ferredoxin throughout the course of 600 ns simulations. The corresponding molecular dynamics trajectories can be seen as Supplementary Fig. S6.

Figure 7

Proximal P450 surface. (a) Positively charged residues in the lanosterol-bound M. capsulatus CYP51 molecule. The ribbon of helix K (306–318) is colored in dim grey, the meander (370–385) is in black. The heme and lanosterol are shown as black and cyan stick models, respectively. K99 (colored in black) is not exposed to the surface, its side chain retains the H-bond with the heme ring D propionate. (b–f) Electrostatic potential mapped onto the surface of bacterial (b–d) versus eukaryotic microsomal (e,f) and mitochondrial P450s (g,h). (b) M. capsulatus CYP51 [7SNM], (c) Tepidiphilus thermophiles CYP116B46 (P450_TT, 6LAA), (d) M. tuberculosis CYP51 [1E9X], (e) T. cruzi CYP51 [6FMO], (f) human CYP51 [6UEZ], (g) human CYP11A1 [P450scc, 3N9Y], (h) CYP11B2 [7M8I]. Red for positive and blue for negative charge, white for neutral. The view of the superimposed structures is the same as in (a).

Figure 8

Crystal structures of P450-redox partner complexes. (a) Bacterial (Pseudomonas putida) P450cam with putidaredoxin [4JWS, tan] and human mitochondrial P450s with adrenodoxin: CYP11A1 [3N9Y, cyan] and CYP11B2 [7M8L, plum]. P450 orientation is the same. The distances between the Fe1 and the heme are marked. (b,c) Overlaid 3N9Y/7M8I (b) and 3N9Y/4JWS (c).