Ab initio dynamics of the cytochrome P450 hydroxylation reaction

Abstract

The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

FIG. 1.

Cpd I-catalyzed substrate hydroxylation. Hydrogen atom abstraction by Cpd I (a) leads to formation of the hydroxyiron(IV) intermediate and a substrate-centered radical. This complex undergoes hydroxy-radical rebound to the substrate (b), yielding the hydroxylated product. The resultant species coordinates to the Fe(III) metal center until dissociation of the complex and release of product from the enzyme.

FIG. 2.

Free energy surface for Cpd I mediated propane hydroxylation in the S = 1/2 state at 300.0 K (gold) and 1.0 K (blue) as a function of FeO–H distance. Labeled points correspond to the reactant complex (a) and trapped hydroxyiron(IV) porphine (b), and the 2-propanol product state (c). Activation barriers ΔA_B and exothermicities ΔA_X are indicated at both simulation temperatures.

FIG. 3.

Free energy surface for Cpd I mediated propane hydroxylation in the S = 3/2 state at 300.0 K (gold) and 1.0 K (blue) as a function of FeO–H distance. Labeled points correspond to the reactant complex (a) and trapped hydroxyiron(IV) porphine (b), (c) on both surfaces. Activation barriers ΔA_B and exothermicities ΔA_X are indicated at both simulation temperatures.

FIG. 4.

Rebound trajectory for hydroxyiron(IV) porphine in the S = 1/2 state as initiated from the trapped geometry at d(O–H) = 1.10 Å  (a). Initial rotation of the Fe–OH moiety (green arrow; (a) and (b)) prepares the system for hydroxylation by presenting electron density to the substrate. The radical center of propane approaches the bound OH, which proceeds to rebound as a hydroxy radical (c) and (d). The resultant 2-propanol coordinates to Fe(III) porphine.

FIG. 5.

Distribution of outcomes in the S = 1/2 state following 3.60 ps of simulation time for each trajectory of the 101 member ensemble. The number of instances observed for each reactive outcome is indicated numerically.

FIG. 6.

Histogram of hydroxyiron(IV) porphine lifetimes, defined as the time from hydrogen atom abstraction to substrate hydroxylation in the S = 1/2 state following 3.60 ps of simulation time. A total 46 simulations within the ensemble exhibited this behavior.

FIG. 7.

Thermal scaling of the potential of mean force at d(O–H) = 1.10 Å  on the hydrogen atom abstraction surface, as initiated from the S = 1/2 Cpd I doublet. Note the presence of two distinct fits corresponding to reactant (dashed line) and product states (dotted line).

FIG. 8.

Representative trajectory for substrate desaturation by hydroxyiron(IV) porphine in the S = 1/2 spin state. The reaction is initiated through an initial hydrogen atom abstraction by Cpd I (a) and subsequent rotation of the Fe–OH moiety of hydroxyiron(IV) porphine (b). The substrate then translocates with respect to the catalytic center as opposed undergoing hydroxy radical rebound (c), leaving the complex poised for a second hydrogen atom abstraction (d). This yields the desaturated product and a water molecule coordinated to Fe(III) (e).