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)Cl-6](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 CYP1S8. 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(center dot))/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 coworkers, 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 the best describes as follows: E-comp-I(0,) = 1.22 V (at pH 7, vs NHE) with D(O-H)(comp-II )= 95 kcal/mol and E-comp-II(0,) = 0.99 V (at pH 7, vs NHE) with D(O-H)(ferric) =90Kcal/mol.