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The present work is concerned with theoretical studies on the cytochrome P450-catalyzed hydroxylation of C–H bonds. To describe the heme-enzyme, substrate, and solvent, a combined quantum mechanical/molecular mechanical (QM/MM) approach is adopted. Density functional theory is employed to treat the electronic structure of the active site (40–84 atoms), while the protein and solvent environment (ca. 24000 atoms) is approximated at the classical force field level.
The calculations characterize the electronic and geometric features of the elusive active oxidant of P450cam (compound I), i.e. the oxoferryl species [FeIV(O)(porph·+)(SR)] (SR = cysteine-357, porph = protoporphyrin IX). We analyze how the explicit consideration of the protein environment by the QM/MM treatment influences the results with respect to simplified compound I models in the gas phase. We find that a porphyrin π-cation radical (A2u state) is stabilized in the enzyme, while the gas phase models are mainly sulfur-centered radicals. A hydrogen bonding network around the proximal cysteine-357 favors localization of charge density at the coordinating sulfur atom, which shortens and strengthens the Fe–S bond with respect to the gas phase complex.
The calculated energy profile of the “rebound” mechanism of C–H hydroxylation 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 non-synchronous, 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 that the polarizing effect of the enzyme environment affects the relative stability of spin states and redox electromers.
Calculations on the substrate-free resting form of P450cam correctly predict the ground state multiplicity and are in good accord with experimentally known bond lengths and EPR hyperfine coupling constants on ligand atoms. Furthermore, these investigations offer insights into the factors that govern the binding properties of the axial water ligand in the enzyme environment.
Additional density functional-based calculations address the spectroscopic parameters of intermediates in the protein environment and corresponding model complexes. The comparison of computational results on the complex [FeO(TPP)]+ (TPP = meso-tetraphenylporphyrin) and experimental data of its derivatives shows that Heisenberg exchange coupling constants, Mößbauer isomer shifts, and quadrupole splittings are obtained with satisfactory accuracy. Corresponding predictions for the spectroscopic parameters of the high-valent oxoferryl intermediate (compound I) in P450cam are presented, to facilitate its spectroscopic detection.
