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

Abstract

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.

Figure 1.

Bonds strengths that define the ground state thermodynamics of P450 catalysis. D(O-H)_Comp-II is the strength of the O-H bond in P450 compound II. D(O-H)_Ferric is the O-H bond strength in the ferric water-bound form of the enzyme.

Figure 2.

Time trace at 430 nm of reaction between CYP158 and [Ir(IV)Cl₆]²⁻ at pH 8.3. The reaction can be divided into three distinct phases. It begins with Phase I, dominated by the forward reaction (i.e. formation of P450-II). This is followed by a brief period of pseudo-equilibrium, denoted by Phase II. This is the phase where the potentials are determined from the concentration of each species. This is followed by Phase III, which is dominated by decay of P450-II back to ferric enzyme.

Figure 3.

Experimental and simulated spectra from the pseudo-equilibrium region (490 ms, Fig. 2) using pure spectra of ferric CYP158, CYP158-II, and [Ir(IV)Cl₆]²⁻. The residual shows the difference between the experimental and simulated data.

Figure 4.

E^0’_Comp-II versus pH.

Figure 5.

General illustration depicting how a rapid rebound step provides a mechanism through which an increase in the forward rate constant for H-atom abstraction creates a more reactive hydroxylating intermediate irrespective of the equilibrium constant for H-atom transfer. k_f is the forward rate constant for H-atom transfer. k_r is the reverse rate constant for H-atom transfer. k_R is the rate constant for rebound.