Cytochrome P450 enzymes play critical roles in hormone synthesis and xenobiotic metabolism. Since the discovery of P450s over four decades ago, chemists have been enamored with their ability to selectively functionalize a wide range of organic compounds. 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 autooxidations, biasing the system for C-H bond activation. However, 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. Recently we identified a P450 (CYP158) whose ferryl form can be prepared in ≥ 90% yield over a wide pH range. Using Mössbauer and UV/Visible spectroscopies (in conjunction with X-ray absorption measurements) we determined a ferryl pKa of 11.9. This value is remarkable. It is at least 8.5 units higher than the ferryl pKas of histidine-ligated peroxidases and globins, allowing for more than a 0.5V drop in the one-electron reduction potential of compound I. Arguments based on Marcus theory indicate that this change in potential results in over a 10,000 fold reduction in the rate constant for oxidations of the protein superstructure, making these processes noncompetitive with substrate oxidation.

Cytochrome P450 oxidations: A controlled burn of inert organic compounds

249th National Meeting (2015)

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