Reduction Potentials of P450 Compounds I and II: Insight into the Thermodynamics of C–H Bond Activation

We present a mixed experimental/theoretical determination of the bond strengths and redox potentials that define the ground-state thermodynamics for C–H bond activation in cytochrome P450 catalysis. Using redox titrations with [Ir­(IV)­Cl6]2–, we have determined the compound II/ferric (or Fe­(IV)­OH/Fe­(III)­OH2) couple and its associated D(O–H)Ferric bond strength in CYP158. Knowledge of this potential as well as the compound II/ferric (or Fe­(IV)­O/Fe­(III)­OH) reduction potential in horseradish peroxidase and the two-electron compound I/ferric (or Fe­(IV)­O­(Por•)/Fe­(III)­OH2(Por)) reduction potential in aromatic peroxidase has allowed us to gauge the accuracy of theoretically determined bond strengths. Using the restricted open shell (ROS) method as proposed by Wright and co-workers, we have obtained O–H bond strengths and associated redox potentials for charge-neutral H-atom reductions of these iron­(IV)-hydroxo and -oxo porphyrin species that are within 1 kcal/mol of experimentally determined values, suggesting that the ROS method may provide accurate values for the P450-II O–H bond strength and P450-I reduction potential. The efforts detailed here indicate that the ground-state thermodynamics of C–H bond activation in P450 are best described as follows: E 0′ Comp‑I = 1.22 V (at pH 7, vs NHE) with D(O–H)Comp‑II = 95 kcal/mol and E 0′ Comp‑II = 0.99 V (at pH 7, vs NHE) with D(O–H)Ferric = 90 kcal/mol.