The conserved thiolate axial ligand in Cytochrome P450 enzymes has been attributed to the ability to selectively and efficiently cleave unactivated C-H bonds (BDE ~100 kcal/mol). The thiolate is electron rich, serving as a strong donor into the FeO π* orbitals. Only thiolate ligated heme enzymes are known to oxidize hydrocarbons by two electrons, while those with weaker ligands (histidine) have only been shown to perform one-electron oxidations. The thiolate ligand in P450 substantially increases basicity of the rebound intermediate, Compound II, relative to histidine ligated heme peroxidases, enhancing the favorability of on-pathway substrate oxidations and the control of the oxidative power of Compound I. However, a more comprehensive mechanistic picture of how the thiolate gears P450-I/II (Compounds I & II) towards C-H bond activation is lacking. Developing isoelectronic replacements of the thiolate ligand to directly correlate donation strength, reactivity, and changes in the properties of P450-I/II would reveal the role played by the thiolate in tuning intermediates for catalysis. Selenocysteine (SeP450) represents the only reported ligand substitution within the P450 framework to retain monooxygenase activity. We have previously demonstrated a ~10-fold increase in reactivity towards C-H bonds for SeP450-I. Here, we report a link between increased ferryl basicity in SeP450-II and reactivity, alongside structural characterization of the SeP450 Compound II intermediates in the protonated and unprotonated states, representing the most basic ferryl species reported to date.