Cytochrome P450 (P450) enzymes are responsible for the phase I metabolism of ~75% of known pharmaceuticals. Since their discovery over four decades ago, chemists have been enamored with P450’s ability to selectively functionalize a wide range of hydrocarbons. Two enigmatic aspects of P450 chemistry are i) the enzyme’s use of an electron-rich thiolate-ligated heme to catalyze the oxidation of inert hydrocarbons and ii) the enzyme’s ability to perform these demanding oxidations without damage to its own relatively fragile protein superstructure. It has been suggested that the electron-donating thiolate-ligand promotes C-H bond activation at biologically viable reduction potentials through the generation of basic iron(IV)oxo (or ferryl) species. The metal-oxo pKa is thought to be a key thermodynamic parameter in C-H bond activation, with a unit increase in ferryl pKa allowing for a 59 mV drop in the one-electron reduction potential of compound I (the active intermediate in P450 catalysis). It has been proposed that this drop in reduction potential decreases the driving force for deleterious oxidations of the protein superstructure, biasing the system for C-H bond activation. But while experiments have confirmed the basic nature of ferryl P450s, the full magnitude of the ferryl pKa’s role in hydrocarbon oxidations has proven difficult to assess. Previous investigations of P450s have only set a lower limit, pKa > 8, on the thermodynamic quantity. Here we report the identification of a P450 whose ferryl form can be prepared in high (≥90%) yield over a wide pH range. This discovery has allowed for the first determination of a ferryl pKa. Additionally, we have found this system amenable to characterization of its iron(IV/III) reduction potential. These results and their implications for C-H bond activation will be discussed.