Quantum Mechanical/Molecular Mechanical Investigation of the Mechanism of C−H Hydroxylation of Camphor by Cytochrome P450_cam:  Theory Supports a Two-State Rebound Mechanism

Abstract

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.

QM/MM Study of the Active Species of the Human Cytochrome P450 3A4, and the Influence Thereof of the Multiple Substrate Binding

“…The results of QM(DFT)/MM studies and appropriately designed QM ones, [31][32][33][34] show good agreement with experimental data 16,17,35-37 on a variety of Cpd I species. This agreement involves the calculated 23-25,30-34 energy proximity of the doublet and quartet spin states, as is commonly observed in Cpd I species, 35-37 the structural parameters, 23-25 the spin density distribution 23-25,33,34 and the Mössbauer spectroscopic parameters, 30 which resemble the same parameters observed for the known Cpd I species of the analogous CPO enzyme.…”

Recent density functional theory model calculations of drug metabolism by cytochrome P450

“…ab initio, DFT, or semiempirical method) treatment of atoms involved in the active site with a MM description of the native protein environment [135][136][137]. QM/MM approach is becoming increasingly popular for the study of biochemical reactions [138][139][140].…”

The Effect of Heme Environment on the Hydrogen Abstraction Reaction of Camphor in P450cam Catalysis: A QM/MM Study

“…Lower values of 11.7 and 4.5 kcal/mol are found in the restricted open-shell B3LYP (ROB3LYP)/OPLS-AA study of G/F at the R2s/Bg* level;3 , 4 the A-propionate (Aprop) heme side chain carries significant spin density which decreases during the Habstraction reaction, with concomitant increase of negative charge at the A-prop carboxylate that is proposed to provide a differential stabilization of TS H and HYD through electrostatic interactions with Arg299.3 , 4 By contrast, single-point UB3LYP/CHARMM calculations by S/T on their snapshots (with extended QM regions) and UB3LYP calculations on corresponding model systems do not give such spin density, neither in the enzyme nor in the gas phase, as long as the propionates are screened by neighboring Arg residues.2 , 8 In view of these significant differences in relative energies, spin densities, and role of the propionates, both groups decided to pinpoint the source of these discrepancies, by comparing the species common to their previous studies, namely, the lowest-energy electromers on the quartet surface, RC(IV), TS H (III), and HYD(III), during H-abstraction in the enzyme environment. [2][3][4] For this purpose new UB3LYP/CHARMM calculations were carried out at Mülheim to treat the G/F and S/T systems on equal footing. The MM region was represented by the CHARMM22 force field.…”