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.     

Radical clock substrates, their C-H hydroxylation mechanism by cytochrome P450, and other reactivity patterns: what does theory reveal about the clocks' behavior?

There is an ongoing and tantalizing controversy regarding the mechanism of a key process in nature, C-H hydroxylation, by the enzyme cytochrome P450 (Auclaire, K.; Hu, Z.; Little, D. M.; Ortiz de Montellano, P. R.; Groves, J. T. J. Am. Chem. Soc. 2002, 124, 6020-6027. Newcomb, M.; Aebisher, D.; Shen, R.; Esala, R.; Chandrasena, P.; Hollenberg, P. F.; Coon, M. J. J. Am. Chem. Soc. 2003, 125, 6064-6065). To definitely resolve this controversy, theory must first address the actual systems that have been used by experiment, and that generated the controversy. This is done in the present paper, which constitutes the first extensive theoretical study of such two experimental systems, trans-2-phenylmethyl-cyclopropane (1) and trans-2-phenyl-iso-propylcyclopropane (4). The theoretical study of these substrates reveals that the only low energy pathway for C-H hydroxylation is the two-state rebound mechanism described originally for methane hydroxylation (Ogliaro, F.; Harris, N.; Cohen, S.; Filatov, M.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 8977-8989). The paper shows that the scenario of a two-state rebound mechanism accommodates much of the experimental data. The computational results provide a good match to experimental results concerning the very different extents of rearrangement for 1 (20-30%) vs 4 (virtually none), lead to product isotope effect for the reaction of 1, in the direction of the experimental result, and predict as well the observed metabolic switching from methyl to phenyl hydroxylation, which occurs upon deuteration of the methyl group. Furthermore, the study reveals that an intimate ion pair species involving an alkyl carbocation derived from 4 gives no rearranged products, again in accord with experiment. This coherent match between theory and experiment cannot be merely accidental; it comes close to being aproof that the actual mechanism of C-H hydroxylation involves the two-state reactivity revealed by theory. Analysis of the rearrangement modes of the carbocations derived from 1 and 4 excludes the participation of free carbocations during the hydroxylation of these substrates. Finally, the mechanistic significance of product isotope effect (different isotope effects for the rearranged and unrearranged alcohol products) is analyzed. It is shown to be a sensitive probe of two-state reactivity; the size of the intrinsic product isotope effect and its direction reveal the structural differences of the hydrogen abstraction transition states in the low-spin vs high-spin reaction manifolds.     

The directive of the protein: how does cytochrome P450 select the mechanism of dopamine formation?

Dopamine can be generated from tyramine via arene hydroxylation catalyzed by a cytochrome P450 enzyme (CYP2D6). Our quantum mechanical/molecular mechanical (QM/MM) results reveal the decisive impact of the protein in selecting the 'best' reaction mechanism. Instead of the traditional Meisenheimer-complex mechanism, the study reveals a mechanism involving an initial hydrogen atom transfer from the phenolic hydroxyl group of the tyramine to the iron-oxo of the compound I (Cpd I), followed by a ring-π radical rebound that eventually leads to dopamine by keto-enol rearrangement. This mechanism is not viable in the gas phase since the O-H bond activation by Cpd I is endothermic and the process does not form a stable intermediate. By contrast, the in-protein reaction has a low barrier and is exothermic. It is shown that the local electric field of the protein environment serves as a template that stabilizes the intermediate of the H-abstraction step and thereby mediates the catalysis of dopamine formation at a lower energy cost. Furthermore, it is shown that external electric fields can either catalyze or inhibit the process depending on their directionality.     

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.     

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.     

Can a single oxidant with two spin states masquerade as two different oxidants? A study of the sulfoxidation mechanism by cytochrome p450

Density functional calculations were performed on the sulfoxidation reaction by a model compound I (Cpd I) of cytochrome P450. By contrast to previous alkane hydroxylation studies, which exhibit a dominant low-spin (LS) pathway, the sulfoxidation follows a dominant high-spin (HS) reaction. Thus, competing hydroxylation and sulfoxidation processes as observed for instance by Jones et al. (Volz, T. J.; Rock, D. A.; Jones, J. P. J. Am. Chem. Soc. 2002, 124, 9724) are the result of a two-state reactivity scenario, whereby the hydroxylation originates from the LS pathway and the sulfoxidation from the HS pathway. In this manner, two spin states of a single oxidant (Cpd I) can be disguised as two different oxidants. The calculations rule out the possibility that a second oxidant (the ferric peroxide, Cpd 0 species) interferes in the observed results of Jones et al.     

Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents: evidence for interactions between a C-F bond and aromatic π systems

We employed the water-soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent Fe(IV)=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF(2) group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound.     

How does mechanism of biosorption determine the differences between the initial and equilibrium adsorption states?

The analysis of various models assumed to represent the influence of pH on heavy metals biosorption equilibrium is presented. It shows that all of them lead to the same mathematical expressions (e.g. the Langmuir or the Flory adsorption isotherm equations) when the pH effects are neglected. Even if considering the pH effects, some of them (competitive adsorption and ion-exchange models, for instance) still lead to analogical expressions for sorption isotherm equations. The accepted mechanism of biosorption may, however, influence strongly the differences between the initial and equilibrium states of biosorption system.     

Does substrate oxidation determine the regioselectivity of cyclohexene and propene oxidation by cytochrome p450?

DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and propene by Compound I of P450cam demonstrate that the relative barriers for the oxidative processes themselves are not good predictors of the observed selectivity. However, a kinetic expression previously developed (Kozuch, S.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 3355) for catalytic cycles under steady-state conditions, predicts, in accord with experiment, that propene will undergo exclusive C=C epoxidation, while cyclohexene will undergo both reactions with a small preference for epoxidation. The model expression for the effective barrier of the cycle forms a general basis for understanding and predicting the selectivity of P450 isozymes.     

Thermal equilibrium of high- and low-spin forms of cytochrome P450 BM-3: repositioning of the substrate?

We demonstrate that cytochrome P450 BM-3 in complex with N-palmitoylglycine undergoes a spin state change between room temperature, where optimal activity is seen, and low temperatures, where X-ray diffraction characterization has been carried out. On the basis of NMR measurements of the full-length protein, this spin state change is likely to be accompanied by a general structural rearrangement in the enzyme pocket. The substrate remains bound at all temperatures. We propose that the substrate may "slide" from a position directly atop the heme (thus displacing the ligating water) to the more distant position (thus restoring the ligating water) as the temperature is lowered. This proposal is evaluated on the basis of computational modeling of the protein-ligand complex, using a novel induced fit methodology. We thereby generate a structure with the ligand in close contact with the heme, similar in energy to the experimental structure. With this combination of theory and experiment we provide a specific proposal of how ligands may be positioned for chemistry for this enzyme.     

How does surface modification aid in the dispersion of carbon nanofibers?

Small-angle light scattering is used to assess the dispersion behavior of vapor-grown carbon nanofibers suspended in water. These data provide the first insights into the mechanism by which surface treatment promotes dispersion. Both acid-treated and untreated nanofibers exhibit hierarchical morphology consisting of small-scale aggregates (small bundles) that agglomerate to form fractal clusters that eventually precipitate. Although the morphology of the aggregates and agglomerates is nearly independent of surface treatment, their time evolution is quite different. The time evolution of the small-scale bundles is studied by extracting the size distribution from the angle-dependence of the scattered intensity, using the maximum entropy method in conjunction with a simplified tube form factor. The bundles consist of multiple tubes possibly aggregated side-by-side. Acid oxidation has little effect on this bundle morphology. Rather acid treatment inhibits agglomeration of the bundles. The time evolution of agglomeration is followed by fitting the scattering data to a generalized fractal model. Agglomerates appear immediately after cessation of sonication for untreated fibers but only after hours for treated fibers. Eventually, however, both systems precipitate.     

What is the active species of cytochrome P450 during camphor hydroxylation? QM/MM studies of different electronic states of compound I and of reduced and oxidized iron-oxo intermediates

We have investigated C-H hydroxylation of camphor by Compound I (Cpd I) of cytochrome P450cam in different electronic states and by its one-electron reduced and oxidized forms, using QM/MM calculations in the native protein/solvent environment. Cpd I species with five unpaired electrons (pentaradicaloids) are ca. 12 kcal/mol higher in energy than the ground state Cpd I species with three unpaired electrons (triradicaloids). The H-abstraction transition states of pentaradicaloids lie ca. 21 (9) kcal/mol above the triradicaloid (pentaradicaloid) reactants. Hydroxylation via pentaradicaloids is thus facile provided that they can react before relaxing to the ground-state triradicaloids. Excited states of Cpd I with an Fe(V)-oxo moiety lie more than 20 kcal/mol above the triradicaloid ground state in single-point gas-phase calculations, but these electronic configurations are not stable upon including the point-charge protein environment which causes SCF convergence to the triradicaloid ground state. One-electron reduced species (Cpd II) show sluggish reactivity compared with Cpd I in agreement with experimental model studies. One-electron oxidized species are more reactive than Cpd I but seem too high in energy to be accessible. The barriers to hydrogen abstraction for the various forms of Cpd I are generally not affected much by the chosen protonation states of the Asp297 and His355 residues near the propionate side chains of the heme or by the appearance of radical character at Asp297, His355, or the propionates.     

Oxygen economy of cytochrome P450: what is the origin of the mixed functionality as a dehydrogenase-oxidase enzyme compared with its normal function?

The economy of dioxygen consumption by enzymes constitutes a fundamental problem in enzymatic chemistry (ref 1). Sometimes, the enzyme converts ALL the oxygen into water, without affecting the organic substrate, thereby acting as an "oxidase" (ref 1). Other times, the enzyme converts all the oxygen into water and causes desaturation in the substrate, thus exhibiting a mixed function as both "oxidase" and "dehydrogenase" (refs 2-5). The present paper describes density functional calculations demonstrating that the oxidase-dehydrogenase mixed activity occurs from the cationic intermediate species and requires electro-steric inhibition of the rebound process. Furthermore, the calculations reveal that the carbocation is formally nascent from an excited state of the active species of the enzyme (2Cpd I), in which the Fe=O moiety is singlet coupled as in the 1Deltag state of dioxygen! Thus, our results resolve an important mechanism and reveal the factors that underlie its observability.     

How does cross-linking affect the stability of block copolymer vesicles in the presence of surfactant?

Block copolymer vesicles are conveniently prepared directly in water at relatively high solids by polymerization-induced self-assembly using an aqueous dispersion polymerization formulation based on 2-hydroxypropyl methacrylate. However, dynamic light scattering studies clearly demonstrate that addition of small molecule surfactants to such linear copolymer vesicles disrupts the vesicular membrane. This causes rapid vesicle dissolution in the case of ionic surfactants, with nonionic surfactants proving somewhat less destructive. To address this problem, glycidyl methacrylate can be copolymerized with 2-hydroxypropyl methacrylate and the resulting epoxy-functional block copolymer vesicles are readily cross-linked in aqueous solution using cheap commercially available polymeric diamines. Such epoxy-amine chemistry confers exceptional surfactant tolerance on the cross-linked vesicles and also leads to a distinctive change in their morphology, as judged by transmission electron microscopy. Moreover, pendent unreacted amine groups confer cationic character on these cross-linked vesicles and offer further opportunities for functionalization.     

Origin of activation barriers in the dimerization of neutral radicals: a "nonperfect synchronization" effect?

Dimerizations of delocalized neutral radicals may be endowed with quite significant activation barriers. The origin of these barriers is discussed in terms of a model that emphasizes the role of localization of the unpaired radical upon bond formation. Several examples are given in which the model is compared with the results of quantum chemical calculations including the coupling of allyl radicals and of benzyl radicals at various possible carbon sites. The dimerization behavior of radicals in the NADH family is also examined. The connection between the reasons that underlay the existence of the activation barrier and the principle of "nonperfect synchronization" is discussed. The dimerization of conjugated radicals indeed offers a precious example that can be used to decipher the reasons behind these behaviors, being devoid of the ambiguities arising from the simultaneous involvement of ionic and covalent states, significant solvent reorganization, and the contribution of extensive proton tunneling, in the mostly discussed case of proton transfer at carbon.     

How Do Metal Ions Modulate the Rate-Determining Electron-Transfer Step in Cytochrome P450 Reactions?

Cytochrome P450 (CYP450) enzymes play important roles in maintaining human health and their reaction rates are dependent on the first electron transfer from the reduction partner. Interestingly, experimental work has shown that this step is highly influenced by the addition of metal ions. To understand the effect of external perturbations on the CYP450 first reduction step, we have performed a computational study with model complexes in the presence of metal and organic ions, solvent molecules, and an electric field. The results show that these medium-range interactions affect the driving force as well as electron-transfer rates dramatically. Based on the location, distance, and direction of the ions/electric field, the catalytic reaction rates are enhanced or impaired. Calculations on a large crystal structure with bonded alkali metal ions indicated inhibition patterns of the ions. Therefore, we predict that the active forms of the natural CYP450 isozymes will not have more than one alkali metal ion bound in the second-coordination sphere. As such, this study provides an insight into the activity of CYP450 enzymes and the effects of ions and electric field perturbations on their activity.     

How Does the Axial Ligand of Cytochrome P450 Biomimetics Influence the Regioselectivity of Aliphatic versus Aromatic Hydroxylation?

How deep is your orbital? Density functional theory studies on the axial ligand effect of aliphatic versus aromatic hydroxylation of ethylbenzene by iron–oxo complexes with a variable axial ligand show that strong (anionic) ligands pull the metal inside the plane of the haeme and destabilise cationic intermediates through orbital interactions (see picture).

     

How does the mobility of phospholipid molecules at a water/oil interface reflect the viscosity of the surrounding oil?

The mobility of phospholipid molecules at a water/oil interface on cell-sized phospholipid-coated microdroplets was investigated through the measurement of diffusion constants by fluorescence recovery after photobleaching. It is found that the diffusion constant of phospholipids showed the relation D approximately (eta water + eta oil) -0.85, where D is the diffusion constant, eta water is the viscosity of water, and eta oil is the viscosity of oil. This observation indicates that the viscosity of the surrounding oil is the primary factor that determines the diffusibility of phospholipids at a water/oil interface.