No abstract

A two-state rebound mechanism of alkane hydroxylation by a model active species of the enzyme cytochrome P450 is studied using density functional theoretic calculations. Theory corroborates Groves's rebound mechanism (Groves, J. T. J. Chem. Educ. 1985, 62, 928), with a key difference, namely that in the two-state rebound the reactivity and product distribution result from the interplay of two reactive states of the active ferryl-oxene (Por+•FeO) species of the enzyme:  one state is low-spin (doublet) and the other high-spin (quartet). Transition-state structures, intermediates, and product complexes are identified for the two states. The bond activation in either one of the two states involves a hydrogen abstraction-like transition structure. However, while in the high-spin state there forms a radical that has a significant barrier for rebound, in the low-spin state the rebound is virtually barrierless. Even though one cannot ignore incursion of a small amount of radicals in the low-spin state, it is clear that the radical has a significant lifetime mainly on the high-spin surface. The results are used to gain insight into puzzling experimental data which emerge from studies of ultrafast radical clocks (e.g., Toy, P. H.; Newcomb, M.; Hollenberg, P. F., J. Am. Chem. Soc. 1998, 120, 7719), vis à vis the nature the transition state, deduced from kinetic isotope effect measurements (Manchester, J. I.; Dinnocenzo, J. P.; Higgins, L. A.; Jones, J. P. J. Am. Chem. Soc. 1997, 119, 5069) and stereochemical scrambling patterns (Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J. Biochem. Biophys. Res. Commun. 1978, 81, 154). Understanding the electronic structure of the various species leads to a predictive structure−reactivity picture, based on the two-state reactivity scenario (Shaik, S.; Filatov, M.; Schröder, D.; Schwarz, H. Chem. Eur. J. 1998, 4, 193). The model makes it possible to predict the dependence of the relative rates of the two states, and of the corresponding steps as a function of the nature of the alkane, the resulting alkyl radical, and the binding capability of the thiolate proximal ligand of the active species.

The primary oxidant of cytochrome P450 enzymes, Compound I, is hard to detect experimentally; in the case of cytochrome P450(cam), this intermediate does not accumulate in solution during the catalytic cycle even at temperatures as low as 200 K (ref 4). Theory can play an important role in characterizing such elusive species. We present here combined quantum mechanical/molecular mechanical (QM/MM) calculations of Compound I of cytochrome P450(cam) in the full enzyme environment as well as density functional studies of the isolated QM region. The calculations assign the ground state of the species, quantify the effect of polarization and hydrogen bonding on its properties, and show that the protein environment and its specific hydrogen bonding to the cysteinate ligand are crucial for sustaining the Fe-S bond and for preventing the full oxidation of the sulfur.

The stereospecific cytochrome P450-catalyzed hydroxylation of the C(5)-H((5-exo)) bond in camphor has been studied theoretically by a combined quantum mechanical/molecular mechanical (QM/MM) approach. Density functional theory is employed to treat the electronic structure of the active site (40-100 atoms), while the protein and solvent environment (ca. 24,000 atoms) is described by the CHARMM force field. The calculated energy profile of the hydrogen-abstraction oxygen-rebound mechanism indicates that the reaction takes place in two spin states (doublet and quartet), as has been suggested earlier on the basis of calculations on simpler models ("two-state reactivity"). While the reaction on the doublet potential energy surface is nonsynchronous, yet effectively concerted, the quartet pathway is truly stepwise, including formation of a distinct intermediate substrate radical and a hydroxo-iron complex. Comparative calculations in the gas phase demonstrate the effect of the protein environment on the geometry and relative stability of intermediates (in terms of spin states and redox electromers) through steric constraints and electronic polarization.