Oxygen activation by cytochrome P450: A thermodynamic analysis

Electrode potentials for every intermediate in the cytochrome P450 cycle were estimated and evaluated by means of an oxidation state diagram. By this approach, and within the uncertainties of the approximations, the superoxide complex of cytochrome P450 at pH 7 is oxidizing: E degrees' (P450FeO(2)(2+), H+/P450FeOOH(2+)) = +0.93 V, and the Gibbs energy for the reaction of the hydroperoxo complex of cytochrome P450 to form compound I and water, P450FeOOH(2+) + H+ = P450FeO(2+)por(center dot+) + H2O, is 0 kJ/mol. Although cytochrome P450FeOOH(2+) and cytochrome P450FeO(2+)por(center dot+) are approximately isoenergetic, they are likely to react at different rates with substrates and may yield different products. Homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical, P450FeOOH(2+) = P450FeO(2+) + HO center dot, is unfavorable (Delta G degrees' = +92 kJ/mol), as is the dissociation into HOO- and cytochrome P450Fe(3+) (+73 kJ/mol). It is shown that the sum of the Gibbs energy of association for cytochrome P450Fe(3+) with the hydroperoxo anion and the Gibbs energy for the one-electron reduction of cytochrome P450FeOOH(2+), relative to NHE, is constant (-203 kJ/mol). While the estimated E degrees' (P450FeO(2)(2+), H+/P450FeOOH(2+)) = +0.93 V at pH 7 is larger than necessary to effect reduction of cytochrome P450FeO(2)(2+), the magnitude of this electrode potential implies that the binding constant for cytochrome P450Fe(3+) with hydrogen peroxide is ca. 3 x 10(6) M-1 at pH 7. An association constant of this magnitude ensures that a fraction of cytochrome P450FeOOH(2+) is available to form compound I or to react with substrates directly, while a larger one would imply that compound I is too weak an oxidant. In general, the energetics of the reduction of dioxygen to water determines the energetics of catalysis of hydroxylations by cytochrome P450. These results enable calibration of energy levels obtained for intermediates in the cytochome P450 reaction cycle obtained by ab initio calculations and provide insights into the catalytic efficiency of cytochrome P450 and guidelines for the development of competent hydroxylation catalysts.