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  • Functional implications notwithstanding the intermolecular c

    2023-12-08

    Functional implications notwithstanding, the intermolecular contact appears to shield the Y361 side chain (Fig. 3) from being accessible for regulation by phosphorylation/dephosphorylation as has been proposed [18,19]. It is, therefore, likely that Src kinase and PTP1B phosphatase bind to an AROM monomer with open proximal sites and an accessible Y361. This scenario is consistent with the notion that a dynamic equilibrium between AROM monomers and oligomers exists in solution, and that oligomerization of AROM is concentration dependent – the lower order is favored at lower concentrations [15]. The catalytic residues of the kinase and the phosphatase would probably have closer interactions with the second compartment adjacent to Y361, while the FMN moiety of CPR could bind at the first, near the PEG5 site in close proximity to heme. Again, a direct one-to-one association between AROM and any of these functional partners using most of the proximal interface cannot be ruled out. A PEG pentamer at the proximal ligand site is consistent with the EGTA synthesis density map as well as binding interactions. Not only all human placental pAROM crystals examined to date exhibit the density, the crystals of rAROM expressed in bacteria [15] do as well. PEG used in crystallization and as a solvent for steroidal ligands is the only logical choice that explains the experimental data. Given the location of the PEG binding site that borders the bulk solvent boundary, it is highly plausible that only a short section of the polymer chain lodged within the cavity will be relatively “static” and have defined electron density, while the rest will be dynamically disordered within the bulk solvent-detergent continuum. Although the data suggests that PEG at best is a weak inhibitor of pAROM (maximum inhibition of about 20% at 182 mM), the time-dependent nature of non-covalent binding at a non-substrate site could be indicative of conformational changes and/or allostery, as others previously noted [35,36]. A ligand at this site could prevent or destabilize the formation of the redox complex, and/or interfere with the transfer of electrons, unlike a self-contained catalytic system. PEG5 makes polar contacts with the K440 side chain and G433 backbone NH (Supplemental Fig. S1). We have shown that K440Q mutation nearly abolishes the enzyme activity and the presence of the positive charge here could be critical to the stability of the region [15]. K440 is the first residue of helix L, at the end of a long loop between residues 419 and 439 connecting the K” and L helices. This loop contains several key residues including R435, involved in heme coordination, and C437, the ligand to heme iron. The loop is stabilized by an intra-strand hydrogen bond F430COHNC437 and a hydrogen bond between the K440 side chain and G431 carbonyl. Therefore, PEG binding here could directly influence AROM activity by introducing structural/chemical instability and by shielding the positive electrostatic potential that drives the AROM-CPR coupling [37]. Nevertheless, whether a ligand at the EGTA synthesis proximal cavity of AROM can allosterically influence catalysis at the distal substrate site is only a matter of conjecture at this point. A high-affinity ligand at this PEG binding site is necessary in order to address this question. On a related note, the proximal cavity region has shown modest fluctuations in our previous computational work [14]. Therefore, some conformational flexibility of this area is likely, and possibly is conducive to its interaction and binding with molecular partners via “induced fit” mechanisms that could result in alterations of conformation of the proximal cavity region depending on the ligand. All P450 catalysis requires water molecules in and out of the active site. Water channels or aqueducts have thus been identified in many structures of P450s [38,39]. We have previously located an “active site access” or “front door” channel, presumably for the passage of substrate/product to/from the active site [10] (Fig. 6). This channel also accommodates water molecules and a proton relay network for the protonation of D309 as well as enolizalation of 3-keto [10]. The newly discovered “backdoor” channel is located on the opposite side of the catalytic cleft and starts near S314 of I-helix (Fig. 2, Fig. 5). The channel is linked to S314 side chain by a hydrogen bond while donating the proton to the T310 carbonyl (Fig. 5). The backdoor channel could, thus, be involved in the passage of previously postulated “catalytic water’ between T310-OγH and the Fe-peroxy/hydroperoxy intermediate for H2β extraction and aromatization [10], and egress of water molecules and formic acid, the reaction byproducts. The other end of the channel is directed towards the lipid interface as depicted in Fig. 6. The channel appears to serve as an aqueduct that connects the water reservoir at the lipid-protein interface to the catalytic site, and is topographically somewhat similar to the previously described “Channel 5” [39]. Ideally, the water molecules in the channel should be in dynamic equilibrium with an active enzyme and ought not to be “trapped” by strong interaction with protein atoms inside the channel. Accordingly, modeling of a continuous electron density inside the channel as discrete water molecules is an approximate representation of the experimental data that suggest it is a water-filled channel.