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.

Quantum mechanical/molecular mechanical (QM/MM) methods have been used in conjunction with density functional theory (DFT) and correlated ab initio methods to predict the electron paramagnetic resonance (EPR) and Mossbauer (MB) properties of Compound I in P450(cam). For calibration purposes, a small Fe(IV)-oxo complex [Fe(O)(NH(3))(4)(H(2)O)](2+) was studied. The (3)A(2) and (5)A(1) states (in C(4)(v)() symmetry) are found to be within 0.1-0.2 eV. The large zero-field splitting (ZFS) of the (FeO)(2+) unit in the (3)A(2) state arises from spin-orbit coupling with the low-lying quintet and singlet states. The intrinsic g-anisotropy is very small. The spectroscopic properties of the model complex [Fe(O)(TMC)(CH(3)CN)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) are well reproduced by theory. In the model complexes [Fe(O)(TMP)(X)](+) (TMP = tetramesitylporphyrin, X = nothing or H(2)O) the computations again account for the observed spectroscopic properties and predict that the coupling of the (5)A(1) state of the (FeO)(2+) unit to the porphyrin radical leads to a low-lying sextet/quartet manifold approximately 12 kcal/mol above the quartet ground state. The calculations on cytochrome P450(cam), with and without the simulation of the protein environment by point charges, predict a small antiferromagnetic coupling (J approximately -13 to -16 cm(-)(1); H(HDvV) = - 2JS(A)S(B)) and a large ZFS > 15 cm(-)(1) (with E/D approximately 1/3) which will compete with the exchange coupling. This leads to three Kramers doublets of mixed multiplicity which are all populated at room temperature and may therefore contribute to the observed reactivity. The MB and ligand hyperfine couplings ((14)N, (1)H) are fairly sensitive to the protein environment which controls the spin density distribution between the porphyrin ring and the axial cysteinate ligand.