WOS:000373395200004International audienceSemiclassical simulations of spectrum and dynamics of complex molecules require statistical sampling of coordinates and momenta. The effects of using thermal and quantum samplings are analyzed taking pyrrole as a test case. It is shown that there are significant differences in the results obtained with each of these two approaches. Overall, quantum sampling based on a Wigner distribution renders superior results, comparing well to the experiments. Dynamics simulations based on surface hopping and ADC(2) reveal that pyrrole internal conversion to the ground state occurs not only through H-elimination path, but also through ring-distortion paths, which have been systematically neglected by diverse experimental setups. The analysis of the reaction paths also shows that the ionization potential varies by more than 5 eV between ionization of the excited state at the Franck-Condon region and at the intersections with the ground state. This feature may have major implications for time-resolved photoelectron spectroscopy. (c) 2015 Wiley Periodicals, Inc

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.

Donor-acceptor heterojunctions composed of thiophene oligomers and C60 fullerene were investigated with computational methods. Benchmark calculations were performed with time-dependent density functional theory. The effects of varying the density functional, the number of oligomers, the intermolecular distance, the medium polarization, and the chemical functionalization of the monomers were analyzed. The results are presented in terms of diagrams where the electronic states are classified as locally excited states, charge-transfer states, and delocalized states. The effects of each option for computational simulations of realistic heterojunctions employed in photovoltaic devices are evaluated and discussed.

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 (Fe3+O22−) 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.

The CYP51 lanosterol 14alpha-demethylases are evolutionarily ancient enzymes ubiquitously distributed throughout the biological domains. The experimental X-ray crystal structure of Mycobacterium tuberculosis (Mtb) CYP51 is the first of an enzyme capable of catalyzing inert C-C bond cleavage. Amino acid sequence comparisons of CYP51 family members with other members of the CYP superfamily reveal the almost universally conserved "acid-alcohol" pair, putatively involved in proton transport and O(2) activation, is replaced with a His-Thr dyad. In this study, extended molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics calculations (QM/MM) are applied to characterize reactive oxygen intermediates and to unravel mechanisms of O(2) activation vis-a-vis proton transport for this important enzyme. MD confirms stable His259deltaH(+)-Thr260OH-O(2) (Mtb numbering) hydrogen bonding early in the simulations, suggesting these amino acids could function similarly to the Asp251-Thr252 pair in CYP101. QM/MM calculations support this dyad competently catalyzes the peroxo to Compound 0 (Cmpd 0) reaction, albeit an endothermic homolytic O-O scission mechanism affording Compound I (Cmpd I) was identified. Disruption of the His259H(+)-Thr260OH hydrogen bond in MD simulation divulges a second previously unidentified hydrogen-bond network, including three water molecules linking Glu173 in the CYP51 F-helix to the distal O(2) atom. Expansion of the QM region to contain these atoms unveils an unprecedented triradicaloid electronic structure of the peroxo intermediate characterized by spin polarization to the Glu173 side chain, attributable to the protein electrostatic environment. This amino acid, in concert with an active-site water network, catalyzes a facile protonation of the peroxo intermediate and offers a series of redundant heterolytic and homolytic mechanisms, affording exothermic formation of the ultimate oxidant Cmpd I. In summary, this study highlights the importance of the protein electrostatic environment to tune the electronic structure of CYP catalytic intermediates in addition to Cmpd I and illustrates the diversity of proton transport pathways available to these enzymes to drive catalysis.

Classical molecular dynamics (MD) simulations and combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the origin of the enantioselectivity of the Candida antarctica lipase B (CalB) catalyzed O-acetylation of (R,S)-propranolol. The reaction is a two-step process. The initial step is the formation of a reactive acyl enzyme (AcCalB) via a tetrahedral intermediate (TI-1). The stereoselectivity originates from the second step, when AcCalB reacts with the racemic substrate via a second tetrahedral intermediate (TI-2). Reaction barriers for the conversion of (R)- and (S)-propranolol to O-acetylpropranolol were computed for several distinct conformations of TI-2. In QM/MM geometry optimizations and reaction path calculations the QM region was described by density functional theory (B3LYP/TZVP) and the MM region by the CHARMM force field. The QM/MM calculations show that the formation of TI-2 is the rate-determining step. The energy barrier for transformation of (R)-propranolol to O-acetylpropranolol is 4.5 kcal/mol lower than that of the reaction of (S)-propranolol. Enzyme–substrate interactions were identified that play an important role in the enantioselectivity of the reaction. Our QM/MM calculations reproduce and rationalize the experimentally observed enantioselectivity in favor of (R)-propranolol. Furthermore, in contrast to what is commonly suggested for lipase-catalyzed reactions, our results indicate that the tetrahedral intermediate is not a good approximation of the corresponding transition states

PDB references: copper nitrite reductase, 190 K data set 1, 5of5; 190 K data set 2, 5of6; 190 K data set 10, 5of7; 190 K data set 18, 5of8; 190 K data set 50, 5ofc; 190 K data set 69, 5ofd; 190 K data set 75, 5ofe; RT data set 1, 5off; RT data set 2, 5ofg; RT data set 3, 5ofh; RT data set 4, 5og2; RT data set 5, 5og3; RT data set 6, 5og4; RT data set 7, 5og5; RT data set 8, 5og6; RT data set 9, 5ogf; RT data set 10, 5ogg High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c′ (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe–NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of hemenitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.