Choreography of the Reductase and P450_BM3 Domains Toward Electron Transfer Is Instigated by the Substrate

The driving force for the electron transfer (ET) step in the catalytic cycle of cytochrome P450_BM3 is investigated using three sets of 1 μs molecular dynamic simulations for the resting state of P450 in complex with the flavin (FMN) in the reductase domain. These sets involve the following: (i) substrate-free (SF), (ii) substrate (N-palmitoyl glycine, i.e., NPG)-bound (SB), and (iii) SB with the semiquinone radical anion (SQ^-) of FMN. Starting from the X-ray structure of the SF heme domain, we observe that the α1-helix (of the reductase) and the C-helix (of the heme) undergo reorientation to a parallel orientation, which is the thermochemically stable form. The reorientation of the helices pushes away the FMN to a distance of 18.4 Å from the heme's center. When the substrate binds it causes the I-helix of the heme domain to kink and push the C-helix toward the α1-helix, thereby locking the latter two into a stabilized perpendicular conformation, wherein the FMN-heme distance is 12 Å. The distance drops further in the SQ^- form, and upon QM/MM geometry optimization the two moieties approach 8.8 Å, which enhances the ET rate (by 10⁴-10⁶ fold) to the heme's Fe³⁺ ion. These motions are driven by hydrogen bond strengthening between the C- and the α1-helices. Finally, substrate binding leads to formation of an organized water chain connecting the FMN and heme moieties. The water channel assists the ET and couples it to the proton transfer steps that should activate O₂ and create the oxo-iron active species.