John C. Hackett

Peroxo−Iron Mediated Deformylation in Sterol 14α-Demethylase Catalysis

The mechanisms of cytochrome P450 (CYP) catalyzed C-C bond cleavage have been strongly debated and difficult to unravel. Herein, deformylation mechanisms of the sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis are elucidated using molecular dynamics simulation, density functional theory, and hybrid quantum mechanics/molecular mechanics methods. These results provide strong theoretical support for the operation of the peroxo intermediate in CYP-catalyzed deformylation. Molecular dynamics simulations support the lanosterol carboxaldehyde intermediate diverts the hydrogen-bonded network of water putatively involved in proton delivery to peroxo and compound 0 (Cmpd 0) away from the O(2) ligand. In the presence of the aldehyde substrate, the peroxo intermediate is trapped as the peroxohemiacetal without an apparent barrier, which may then be protonated in the active site. The unprotonated peroxohemiacetal provides a branch point for a concerted deformylation mechanism; however, a stepwise mechanism initiated by cleavage of the C-C bond was found to be more energetically feasible. Population analyses of the peroxoformate/deformylated substrate complex indicate that heterolytic cleavage of the C-C bond in the enzyme environment generates a carbanion at C14. Conversely, in the absence of the protein electrostatic background, the C-C cleavage reaction proceeds homolytically, indicating that the active site environment exerts a strong modulatory effect on the electronic structure of this intermediate. If the peroxohemiacetal is protonated, this species preferentially expels formic acid through an O-O cleavage transition state. After expulsion of the formyl unit, both proton-independent and -dependent pathways converge to a complex containing compound II, which readily abstracts the 15alpha-hydrogen, thereby inserting the 14,15 double bond into the steroid skeleton. Parallel studies considering nucleophilic addition of Cmpd 0 to the aldehyde intermediate indicated that this reaction proceeds with high energetic barriers. Finally, the hydrogen atom abstraction and proton coupled electron transfer mechanism (J. Am. Chem. Soc. 2005, 127, 5224-5237) for compound I (Cmpd I) mediated deformylation of the geminal diol was considered in the context of the protein environment. In contrast to gas phase calculations, triradicaloid and pentaradicaloid Cmpd I states failed to initiate a concerted deformylation of the geminal diol. This study provides a unified mechanistic view consistent with decades of experiments aimed at understanding the deformylation reaction. Additionally, these results provide general mechanistic insight into the catalytic mechanisms of several biosynthetic and xenobiotic-oxidizing CYP enzymes of biomedical importance.

Coupled electron transfer and proton hopping in the final step of CYP19-catalyzed androgen aromatization.

Aromatase (CYP19) catalyzes the terminal step in estrogen biosynthesis, which requires three separate oxidation reactions, culminating in an enigmatic aromatization that converts an androgen to an estrogen. A stable ferric peroxo (Fe(3+)O(2)(2-)) intermediate is seen by electron paramagnetic resonance, but its role in this complex reaction remains controversial. Combining molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics, we show that ferric peroxo addition to the 19-aldehyde initiates the reaction. Stepwise cleavage of the C10-C19 and O-O bonds of the peroxohemiacetal extrudes formate and yields Compound II, which in turn desaturates the steroid through successive abstraction of the 1β-hydrogen atom and deprotonation of the 2β-position. Throughout the transformation, a proton is cyclically relayed between D309 and the substrate to stabilize reaction intermediates. This mechanism invokes novel oxygen intermediates and provides a unifying interpretation of past experimental mechanistic studies.

Tetrahydrofolate Recognition by the Mitochondrial Folate Transporter

A mitochondrial carrier family (MCF) of transport proteins facilitates the transfer of charged small molecules across the inner mitochondrial membrane. The human genome has ∼50 genes corresponding to members of this family. All MCF proteins contain three repeats of a characteristic and conserved PX(D/E)XX(K/R) motif thought to be central to the mechanism of these transporters. The mammalian mitochondrial folate transporter (MFT) is one of a few MCF members, known as the P(I/L)W subfamily, that have evolved a tryptophan residue in place of the (D/E) in the second conserved motif; the function of this substitution (Trp-142) is unclear. Molecular dynamics simulations of the MFT in its explicit membrane environment identified this tryptophan, as well as several other residues lining the transport cavity, to be involved in a series of sequential interactions that coordinated the movement of the tetrahydrofolate substrate within the transport cavity. We probed the function of these residues by mutagenesis. The mutation of every residue identified by molecular dynamics to interact with tetrahydrofolate during simulated transit into the aqueous channel severely impaired folate transport. Mutation of the subfamily-defining tryptophan residue in the MFT to match the MCF consensus at this position (W142D) was incompatible with cell survival. These studies indicate that MFT Trp-142, as well as other residues lining the transporter interior, coordinate tetrahydrofolate descent and positioning of the substrate in the transporter basin. Overall, we identified residues in the walls and at the base of the transport cavity that are involved in substrate recognition by the MFT.

Solvatochromism and the solvation structure of benzophenone.

Many complex molecular phenomena, including macromolecular association, protein folding, and chemical reactivity, are determined by the nuances of their electrostatic landscapes. The measurement of such electrostatic effects is nonetheless difficult, and is typically accomplished by exploiting a spectroscopic probe within the system of interest, such as through the vibrational Stark effect. Raman spectroscopy and solvatochromism afford an alternative to this method, circumventing the limitations of infrared spectroscopy, providing a lower detection limit, and permitting measurement in a native chemical environment. To explore this possibility, the solvatochromism of the C=O and aromatic C-H stretching modes of benzophenone are investigated using Raman spectroscopy. In conjunction with density functional theory calculations, these observations are sufficient to determine the probe electrostatic environment as well as contributions from halogen and hydrogen bonding. Further analysis using a detailed Kubo-Anderson lineshape model permits the detailed assignment of distinct hydrogen bonding configurations for water in the benzophenone solvation shell. These observations reinforce the use of benzophenone as an effective electrostatic probe for complex chemical systems.

A GGA+U approach to effective electronic correlations in thiolate-ligated iron-oxo (IV) porphyrin

High-valent oxo-metal complexes exhibit correlated electronic behavior on dense, low-lying electronic state manifolds, presenting challenging systems for electronic structure methods. Among these species, the iron-oxo (IV) porphyrin denoted Compound I occupies a privileged position, serving a broad spectrum of catalytic roles. The most reactive members of this family bear a thiolate axial ligand, exhibiting high activity toward molecular oxygen activation and substrate oxidation. The default approach to such systems has entailed the use of hybrid density functionals or multi-configurational/multireference methods to treat electronic correlation. An alternative approach is presented based on the GGA+U approximation to density functional theory, in which a generalized gradient approximation (GGA) functional is supplemented with a localization correction to treat on-site correlation as inspired by the Hubbard model. The electronic structure of thiolate-ligated iron-oxo (IV) porphyrin and corresponding Coulomb repulsion U are determined both empirically and self-consistently, yielding spin-distributions, state level splittings, and electronic densities of states consistent with prior hybrid functional calculations. Comparison of this detailed electronic structure with model Hamiltonian calculations suggests that the localized 3d iron moments induce correlation in the surrounding electron gas, strengthening local moment formation. This behavior is analogous to strongly correlated electronic systems such as Mott insulators, in which the GGA+U scheme serves as an effective single-particle representation for the full, correlated many-body problem.