The Effect of Heme Environment on the Hydrogen Abstraction Reaction of Camphor in P450_cam Catalysis:  A QM/MM Study

Abstract

The discrepancies between the published QM/MM studies (Schöneboom, J. C.; Cohen, S.; Lin, H.; Shaik, S.; Thiel, W. J. Am. Chem. Soc. 2004, 126, 4017 / Guallar, V.; Friesner, R. A. J. Am. Chem. Soc. 2004, 126, 8501) on H-abstraction of camphor in P450 cam have largely been resolved. The crystallographic water molecule 903 situated near the oxo atom of Compound I acts as a catalyst for H-abstraction, lowering the barrier by about 4 kcal/mol. Spin density at the A-propionate side chain of heme can occur in the case of incomplete screening, but has no major effect on the computed barrier.Cytochrome P450 monooxygenases catalyze regio-and stereoselective C-H bond hydroxylation in bioorganisms. 1 The primary reactive species of P450 enzymes is deemed to be an elusive high-valence oxyferryl-porphyrin π-cation species called Compound I (Cpd I). In the consensus rebound mechanism (see Scheme 1), 1 an initial rate-determining hydrogen abstraction from the substrate yields an iron-hydroxo intermediate radical pair HYD via a transition structure TS H which then recombines to give the ferric-alcohol complex PC. P450 cam hydroxylates camphor at the C 5 position leading to 5-exo-hydroxycamphor as the sole product.Combined quantum mechanical/molecular mechanical (QM/MM) studies on biohydroxylation of camphor by Cpd I of P450 cam have been performed by two groups (Shaik/Thiel [S/T]2 and Guallar/Friesner [G/F]3 , 4) recently. These studies followed the same general strategy but differed in scope, system setup, and QM/MM methodology. S/T investigated the full rebound mechanism for four different snapshots of a molecular dynamics (MD) trajectory,2 , 5 while G/F addressed the initial H-abstraction step in the quartet state starting from a geometry close to the X-ray structure (pdb code: 1DZ9). The S/ T system includes a water layer of 16 Å and has 24394 atoms with a net charge of −10e, whereas the G/F system is neutral and has 7448 atoms (no solvent layer, polar surface residues neutralized). The heme side chains were incorporated into the QM region by G/F, but not by S/T in their standard setup. The QM/MM boundary was handled by frozen . 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...

Mapping protein electron transfer pathways with QM/MM methods

“…Thus, the third unpaired electron partially delocalizes into the propionate groups. These results generated quite a controversy and produced several additional studies by us and other groups (Schoneboom et al 2004;Altun et al 2006Altun et al , 2007Guallar & Olsen 2006;Zheng et al 2006;Zurek et al 2006).…”

The Role of Equatorial and Axial Ligands in Promoting the Activity of Non‐Heme Oxidoiron(IV) Catalysts in Alkane Hydroxylation

“…A classical example of the influence of the axial ligand on the activity of metal centres in enzymes is the push effect in the dioxygen cleavage reaction catalysed by cytochrome P450, [47][48][49][50] to yield the elusive Compound I. [3,13,43,[51][52][53] A strong electron donation from the thiolate ligand in the proximal site of heme was first proposed in 1976 [47] to be solely responsible for the highly efficient O-O breaking ability of P450. Although experimental findings have provided some support for this hypothesis, [48,50] a complete model for the catalytic activity of P450 is currently still lacking, [54] and various additional factors (e.g.…”

Understanding the determinants of selectivity in drug metabolism through modeling of dextromethorphan oxidation by cytochrome P450

“…Previous QM/MM studies of cytochrome P450s have concentrated on the metabolism of model compounds such as propene (22,33), cyclohexene (22), or benzene (21), or on camphor metabolism by the bacterial P450cam enzyme (34). These studies gave important insights into the effect of enzyme environment on the mechanism of metabolism, but due to the small size of the ligands, specific interactions between the ligands and protein residues are not crucial determinants of reactivity.…”