Experimental Documentation of the Structural Consequences of Hydrogen‐Bonding Interactions to the Proximal Cysteine of a Cytochrome P450

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Does Compound I Vary Significantly between Isoforms of Cytochrome P450?

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QM/MM modeling of the hydroxylation of the androstenedione substrate catalyzed by cytochrome P450 aromatase (CYP19A1)

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three-step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone-dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (∼10) overpassing the semiclassical limit for the C[BOND]H, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects.We acknowledge the Servei d'Informàtica of the Universitat Jaume I, GENCI-CINES, and BSC-Marenostrum for providing us with computer capabilities. The authors thank V. Moliner for valuable comments and discussion

A Systematic Account on Aromatic Hydroxylation by a Cytochrome P450 Model Compound I:A Low-Pressure Mass Spectrometry and Computational Study

Cytochrome P450 enzymes are heme containing mono-oxygenases that mainly react through oxygen atom transfer. Specific features of substrate and oxidant that determine the reaction rate constant for oxygen atom transfer are still poorly understood and, therefore, we did a systematic gas-phase study on reactions by iron(IV)-oxo porphyrin cation radical structures with arenes. We present here the first results obtained by using Fourier transform-ion cyclotron resonance mass spectrometry and provide rate constants and product distributions for the assayed reactions. Product distributions and kinetic isotope effect studies implicate a rate determining aromatic hydroxylation reaction that correlates with the ionization energy of the substrate and no evidence of aliphatic hydroxylation products is observed. To further understand the details of the reaction mechanism, a computational study on a model complex was performed. These studies confirm the experimental hypothesis of dominant aromatic over aliphatic hydroxylation and show that the lack of an axial ligand affects the aliphatic pathways. Moreover, a two parabola valence bond model is used to rationalize the rate constant and identify key properties of the oxidant and substrate that drive the reaction. In particular, the work shows that aromatic hydroxylation rates correlate with the ionization energy of the substrate as well as with the electron affinity of the oxidant

Hydrogen atom versus hydride transfer in cytochrome P450 oxidations: A combined mass spectrometry and computational study

Biomimetic models of short-lived enzymatic reaction intermediates can give useful insight into the properties and coordination chemistry of transition metal complexes. In this work we investigate a high-valent iron(IV)-oxo porphyrin cation radical complex, namely [FeIV(O)(TPFPP+•)]+ where TPFPP is the dianion of 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin. The [FeIV(O)(TPFPP+•)]+ ion was studied by ion-molecule reactions in a Fourier transform-ion cyclotron resonance mass spectrometer through reactivities with 1,3,5-cycloheptatriene, 1,3-cyclohexadiene and toluene. The different substrates give dramatic changes in reaction mechanism and efficiencies, whereby cycloheptatriene leads to hydride transfer, while cyclohexadiene and toluene react via hydrogen atom abstraction. Detailed computational studies point to major differences in ionization energy as well as C–H bond energies of the substrates that influence the hydrogen atom abstraction versus electron transfer pathways. The various variables that determine the pathways for hydride transfer versus hydrogen atom transfer are elucidated and discussed

Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme