Does compound I vary significantly between isoforms of cytochrome P450?

The cytochrome P450 (CYP) enzymes are important in many areas, including pharmaceutical development. Subtle changes in the electronic structure of the active species, Compound I, have been postulated previously to account partly for the experimentally observed differences in reactivity between isoforms. Current predictive models of CYP metabolism typically assume an identical Compound I in all isoforms. Here we present a method to calculate the electronic structure and to estimate the Fe-O bond enthalpy of Compound I, and apply it to several human and bacterial CYP isoforms. Conformational flexibility is accounted for by sampling large numbers of structures from molecular dynamics simulations, which are subsequently optimized with density functional theory (B3LYP) based quantum mechanics/molecular mechanics. The observed differences in Compound I between human isoforms are small: They are generally smaller than the spread of values obtained for the same isoform starting from different initial structures. Hence, it is unlikely that the variation in activity between human isoforms is due to differences in the electronic structure of Compound I. A larger difference in electronic structure is observed between the human isoforms and P450(cam) and may be explained by the slightly different hydrogen-bonding environment surrounding the cysteinyl sulfur. The presence of substrate in the active site of all isoforms studied appears to cause a slight decrease in the Fe-O bond enthalpy, apparently due to displacement of water out of the active site, suggesting that Compound I is less stable in the presence of substrate.     

Toward identification of the compound I reactive intermediate in cytochrome P450 chemistry: a QM/MM study of its EPR and Mössbauer parameters

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.     

Is the ruthenium analogue of compound I of cytochrome p450 an efficient oxidant? A theoretical investigation of the methane hydroxylation reaction

High-valent metal-oxo complexes catalyze C-H bond activation by oxygen insertion, with an efficiency that depends on the identity of the transition metal and its oxidation state. Our study uses density functional calculations and theoretical analysis to derive fundamental factors of catalytic activity, by comparison of a ruthenium-oxo catalyst with its iron-oxo analogue toward methane hydroxylation. The study focuses on the ruthenium analogue of the active species of the enzyme cytochrome P450, which is known to be among the most potent catalysts for C-H activation. The computed reaction pathways reveal one high-spin (HS) and two low-spin (LS) mechanisms, all nascent from the low-lying states of the ruthenium-oxo catalyst (Ogliaro, F.; de Visser, S. P.; Groves, J. T.; Shaik, S. Angew. Chem. Int. Ed. 2001, 40, 2874-2878). These mechanisms involve a bond activation phase, in which the transition states (TS's) appear as hydrogen abstraction species, followed by a C-O bond making phase, through a rebound of the methyl radical on the metal-hydroxo complex. However, while the HS mechanism has a significant rebound barrier, and hence a long lifetime of the radical intermediate, by contrast, the LS ones are effectively concerted with small barriers to rebound, if at all. Unlike the iron catalyst, the hydroxylation reaction for the ruthenium analogue is expected to follow largely a single-state reactivity on the LS surface, due to a very large rebound barrier of the HS process and to the more efficient spin crossover expected for ruthenium. As such, ruthenium-oxo catalysts (Groves, J. T.; Shalyaev, K.; Lee, J. In The Porphyrin Handbook; Biochemistry and Binding: Activation of Small Molecules, Vol. 4; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 2000; pp 17-40) are expected to lead to more stereoselective hydroxylations compared with the corresponding iron-oxo reactions. It is reasoned that the ruthenium-oxo catalyst should have larger turnover numbers compared with the iron-oxo analogue, due to lesser production of suicidal side products that destroy the catalyst (Ortiz de Montellano, P. R.; Beilan, H. S.; Kunze, K. L.; Mico, B. A. J. Biol. Chem. 1981, 256, 4395-4399). The computations reveal also that the ruthenium complex is more electrophilic than its iron analogue, having lower hydrogen abstraction barriers. These reactivity features of the ruthenium-oxo system are analyzed and shown to originate from a key fundamental factor, namely, the strong 4d(Ru)-2p(O,N) overlaps, which produce high-lying pi(Ru-O), sigma(Ru-O), and sigma(Ru-N) orbitals and thereby to lead to a preference of ruthenium for higher-valent oxidation states with higher electrophilicity, for the effectively concerted LS hydroxylation mechanism, and for less suicidal complexes. As such, the ruthenium-oxo species is predicted to be a more robust catalyst than its iron-oxo analogue.     

What factors influence the ratio of C-H hydroxylation versus C=C epoxidation by a nonheme cytochrome P450 biomimetic?

Density functional calculations on a nonheme biomimetic (Fe=O(TMCS+) have been performed and its catalytic properties versus propene investigated. Our studies show that this catalyst is able to chemoselectively hydroxylate C=H bonds even in the presence of C=C double bonds. This phenomenon has been analyzed and found to occur due to Pauli repusions between protons on the TMCS ligand with protons attached to the approaching substrate. The geometries of the rate determining transition states indicate that the steric hindrance is larger in the epoxidation transition states than in the hydroxylation ones with much shorter distances; hence the hydroxylation pathway is favored over the epoxidation. Although, the reactant experiences close lying triplet and quintet spin states, the dominant reaction mechanism takes place on the quintet spin state surface; i.e., Fe=O(TMCS)+ reacts via single-state reactivity. Our calculations show that this spin state selectivity is the result of geometric orientation of the transition state structures, whereby the triplet ones are destabilized by electrostatic repulsions between the substrate and the ligand while the quintet spin transition states are aligned along the ideal axis. The reactivity patterns and geometries are compared with oxoiron species of dioxygenase and monoxygenase enzymes. Thus, Fe=O(TMCS)+ shows some similarities with P450 enzyme reactivity: it chemoselectively hydroxylates C=H bonds even in the presence of a C=C double bond and therefore is an acceptable P450 biomimetic. However, the absolute barriers of substrate oxidation by Fe=O(TMCS)+ are higher than the ones obtained with heme enzymes, but the chemoselectivity is lesser affected by external perturbations such as hydrogen bonding of a methanol molecule toward the thiolate sulfur or a dielectric constant. This is the first oxoiron complex whereby we calculated a chemoselective hydroxylation over epoxidation in the gas phase.     

Can ferric-superoxide act as a potential oxidant in P450(cam)? QM/MM investigation of hydroxylation, epoxidation, and sulfoxidation

In view of recent reports of high reactivity of ferric-superoxide species in heme and nonheme systems (Morokuma et al. J. Am. Chem. Soc. 2010, 132, 11993-12005; Que et al. Inorg. Chem. 2010, 49, 3618-3628; Nam et al. J. Am. Chem. Soc. 2010, 132, 5958-5959; J. Am. Chem. Soc. 2010, 132, 10668-10670), we use herein combined quantum mechanics/molecular mechanics (QM/MM) methods to explore the potential reactivity of P450(cam) ferric-superoxide toward hydroxylation, epoxidation, and sulfoxidation. The calculations demonstrate that P450 ferric-superoxide is a sluggish oxidant compared with the high-valent oxoiron porphyrin cation-radical species. As such, unlike heme enzymes with a histidine axial ligand, the P450 superoxo species does not function as an oxidant in P450(cam). The origin of this different behavior of the superoxo species of P450 vis-a?-vis other heme enzymes like tryptophan 2, 3-dioxygenase (TDO) is traced to the ability of the latter superoxo species to make a stronger FeOO-X (X = H,C) bond and to stabilize the corresponding bond-activation transition states by resonance with charge-transfer configurations. By contrast, the negatively charged thiolate ligand in the P450 superoxo species minimizes the mixing of charge transfer configurations in the transition state and raises the reaction barrier. However, as we demonstrate, an external electric field oriented along the Fe-O axis with a direction pointing from Fe toward O will quench Cpd I formation by slowing the reduction of ferric-superoxide and will simultaneously lower the barriers for oxidation by the latter species, thereby enabling observation of superoxo chemistry in P450. Other options for nascent superoxo reactivity in P450 are discussed.     

NR transfer reactivity of azo-compound I of P450. How does the nitrogen substituent tune the reactivity of the species toward C-H and C=C activation?

We studied electronic structures and reactivity patterns of azo-compound I species (RN-Cpd I) by comparison to O-Cpd I of, e.g., cytochrome P450. The study shows that the RN-Cpd I species are capable of C=C aziridination and C-H amidation, in a two-state mechanism similar to that of O-Cpd I. However, unlike O-Cpd I, here the nitrogen substituent (R) exerts a major impact on structure and reactivity. Thus, it is demonstrated that Fe=NR bonds of RN-Cpd I will generally be substantially longer than Fe=O bonds; electron-withdrawing R groups will generate a very long Fe=N bond, whereas electron-releasing R groups should have the opposite effect and hence a shorter Fe=N bond. The R substituent controls also the reactivity of RN-Cpd I toward C=C and C-H bonds by exerting steric and electronic effects. Our analysis shows that an electron-releasing substituent will lower the barriers for both bond activation reactions, since the electronic factor makes the reactions highly exothermic, while an electron-withdrawing one should raise both barriers. The steric bulk of the substituent is predicted to inhibit more strongly the aziridination reactions. It is predicted that electron-releasing substituents with small bulk will create powerful aziridination reagents, whereas electron-withdrawing substituents like MeSO(2) will prefer C-H bond activation with preference that increases with steric bulk. Finally, the study predicts (i) that the reactions of RN-Cpd I will be less stereospecific than those of O-Cpd I and (ii) that aziridination will be more stereoselective than amidation.     

Why are the Elemental Nonmetals (F2, Cl2, Br2, I2, S8, P4) of so Many Hues or of Any Hues and Where is the Chromophore? Insight into Intera-X–X Bonds

A unique approach is used to relate the HOMO-LUMO energy difference to the difference between the ionization potential (IP) and electron affinity (EA) to assist in deducing not only the colors, but also chromophores in elemental nonmetals. Our analysis focuses on compounds with lone pair electrons and σ electrons, namely X₂ (X = F, Cl, Br, I), S₈ and P₄. For the dihalogens, the [IP – EA] energies are found to be: F₂ (12.58 eV), Cl₂ (8.98 eV), Br₂ (7.90 eV), I₂ (6.78 eV). We suggest that the interahalogen X–X bond itself is the chromophore for these dihalogens, in which the light absorbed by the F₂, Cl₂, Br₂, I₂ leads to longer wavelengths in the visible by a π → σ* transition. Trace impurities are a likely case of cyclic S₈ which contains amounts of selenium leading to a yellow color, where the [IP – EA] energy of S₈ is found to be 7.02 eV. Elemental P₄ with an [IP – EA] energy of 9.09 eV contains a tetrahedral and σ aromatic structure. In future work, refinement of the analysis will be required for compounds with π electrons and σ electrons, such as polycyclic aromatic hydrocarbons (PAHs).