The study uses MD simulations and QM/MM calculations to address the preparatory half of the catalytic cycle for two cytochrome P450 (CYP450) enzymes, CYP450CAM and CYP450BM3. We focus on two unexplored/"gray"events: (a) the O2 entrance and binding to form the oxyferrous species as a function of the O2 concentration/pressure and (b) the protonation steps that eventually lead to the formation of the ultimate active species, compound I (cf. 7). Two fundamental and paradigm-shifting findings emerge: (a) the O2 concentration/pressure required for O2 binding must be neither high nor low. The binding is ushered by substrate choreography, which is guided by the moderate O2 pressure and strategic protein residues (Arg112 and Phe87) that block the substrate approach to the heme, thus enabling O2 accumulation near iron. (b) The formation of water channels, which elicit that the protonation steps are different for 5 → 6 and 6 → 7. The process 5 → 6, generating the ferric hydroperoxide, occurs via the channels that are formed by breakage of the salt bridges holding the propionate side chains of the porphyrin, while the formation of compound I, 6 → 7, transpires via the traditional acid-alcohol pairs (Asp251/Thr252 in CAM; Glu267/Thr268 in BM3). The location of acidic ammonium groups (Lys69 in CYP450BM3 and Arg299 in CYP450CAM) near the propionate side chains is generally conserved in the CYP450 family. Therefore, our study highlights the evolutionary need for at least two alternative water channels to tune the P450 nanomachine, so it can complete the two steps of the protonation and form ultimately compound I. Copyright © 2020 American Chemical Society.