The "somersault" mechanism for the p-450 hydroxylation of hydrocarbons. The intervention of transient inverted metastable hydroperoxides

A series of model theoretical calculations are described that suggest a new mechanism for the oxidation step in enzymatic cytochrome P450 hydroxylation of saturated hydrocarbons. A new class of metastable metal hydroperoxides is described that involves the rearrangement of the ground-state metal hydroperoxide to its inverted isomeric form with a hydroxyl radical hydrogen bonded to the metal oxide (MO-OH --> MO....HO). The activation energy for this somersault motion of the FeO-OH group is 20.3 kcal/mol for the P450 model porphyrin iron(III) hydroperoxide [Por(SH)Fe(III)-OOH(-)] to produce the isomeric ferryl oxygen hydrogen bonded to an *OH radical [Por(SH)Fe(III)-O....HO(-)]. This isomeric metastable hydroperoxide, the proposed primary oxidant in the P450 hydroxylation reaction, is calculated to be 17.8 kcal/mol higher in energy than the ground-state iron(III) hydroperoxide Cpd 0. The first step of the proposed mechanism for isobutane oxidation is abstraction of a hydrogen atom from the C-H bond of isobutane by the hydrogen-bonded hydroxyl radical to produce a water molecule strongly hydrogen bonded to anionic Cpd II. The hydroxylation step involves a concerted but nonsynchronous transfer of a hydrogen atom from this newly formed, bound, water molecule to the ferryl oxygen with a concomitant rebound of the incipient *OH radical to the carbon radical of isobutane to produce the C-O bond of the final product, tert-butyl alcohol. The TS for the oxygen rebound step is 2 kcal/mol lower in energy than the hydrogen abstraction TS (DeltaE() = 19.5 kcal/mol). The overall proposed new mechanism is consistent with a lot of the ancillary experimental data for this enzymatic hydroxylation reaction.     

A theoretical study of the dynamic behavior of alkane hydroxylation by a compound I model of cytochrome P450

Dynamic aspects of alkane hydroxylation mediated by Compound I of cytochrome P450 are discussed from classical trajectory calculations at the B3LYP level of density functional theory. The nuclei of the reacting system are propagated from a transition state to a reactant or product direction according to classical dynamics on a Born-Oppenheimer potential energy surface. Geometric and energetic changes in both low-spin doublet and high-spin quartet states are followed along the ethane to ethanol reaction pathway, which is partitioned into two chemical steps: the first is the H-atom abstraction from ethane by the iron-oxo species of Compound I and the second is the rebound step in which the resultant iron-hydroxo complex and the ethyl radical intermediate react to form the ethanol complex. Molecular vibrations of the C-H bond being dissociated and the O-H bond being formed are significantly activated before and after the transition state, respectively, in the H-atom abstraction. The principal reaction coordinate that can represent the first chemical step is the C-H distance or the O-H distance while other geometric parameters remain almost unchanged. The rebound process begins with the iron-hydroxo complex and the ethyl radical intermediate and ends with the formation of the ethanol complex, the essential process in this reaction being the formation of the C-O bond. The H-O-Fe-C dihedral angle corresponds to the principal reaction coordinate for the rebound step. When sufficient kinetic energy is supplied to this rotational mode, the rebound process should efficiently take place. Trajectory calculations suggest that about 200 fs is required for the rebound process under specific initial conditions, in which a small amount of kinetic energy (0.1 kcal/mol) is supplied to the transition state exactly along the reaction coordinate. An important issue about which normal mode of vibration is activated during the hydroxylation reaction is investigated in detail from trajectory calculations. A large part of the kinetic energy is distributed to the C-H and O-H stretching modes before and after the transition state for the H-atom abstraction, respectively, and a small part of the kinetic energy is distributed to the Fe-O and Fe-S stretching modes and some characteristic modes of the porphyrin ring. The porphyrin marker modes of nu(3) and nu(4) that explicitly involve Fe-N stretching motion are effectively enhanced in the hydroxylation reaction. These vibrational modes of the porphyrin ring can play an important role in the energy transfer during the enzymatic process.     

A theoretical study of reactivity and regioselectivity in the hydroxylation of adamantane by ferrate(VI)

The conversion of adamantane to adamantanols mediated by ferrate (FeO(4)(2)(-)), monoprotonated ferrate (HFeO(4)(-)), and diprotonated ferrate (H(2)FeO(4)) is discussed with the hybrid B3LYP density functional theory (DFT) method. Diprotonated ferrate is the best mediator for the activation of the C-H bonds of adamantane via two reaction pathways, in which 1-adamantanol is formed by the abstraction of a tertiary hydrogen atom (3 degrees ) and 2-adamantanol by the abstraction of a secondary hydrogen atom (2 degrees ). Each reaction pathway is initiated by a C-H bond cleavage via an H-atom abstraction that leads to a radical intermediate, followed by a C-O bond formation via an oxygen rebound step to lead to an adamantanol complex. The activation energies for the C-H cleavage step are 6.9 kcal/mol in the 1-adamantanol pathway and 8.4 kcal/mol in the 2-adamantanol pathway, respectively, at the B3LYP/6-311++G level of theory, whereas those of the second reaction step corresponding to the rebound step are relatively small. Thus, the rate-determining step in the two pathways is the C-H bond dissociation step, which is relevant to the regioselectivity for adamantane hydroxylation. The relative rate constant (3 degrees )/(2 degrees ) for the competing H-atom abstraction reactions is calculated to be 9.30 at 75 degrees C, which is fully consistent with an experimental value of 10.1.     

Catalytic mechanism of dopamine beta-monooxygenase mediated by Cu(III)-oxo

Mechanisms of dopamine hydroxylation by the Cu(II)-superoxo species and the Cu(III)-oxo species of dopamine beta-monooxygenase (DBM) are discussed using QM/MM calculations for a whole-enzyme model of 4700 atoms. A calculated activation barrier for the hydrogen-atom abstraction by the Cu(II)-superoxo species is 23.1 kcal/mol, while that of the Cu(III)-oxo, which can be viewed as Cu(II)-O*, is 5.4 kcal/mol. Energies of the optimized radical intermediate in the superoxo- and oxo-mediated pathways are 18.4 and -14.2 kcal/mol, relative to the corresponding reactant complexes, respectively. These results demonstrate that the Cu(III)-oxo species can better mediate dopamine hydroxylation in the protein environment of DBM. The side chains of three amino acid residues (His415, His417, and Met490) coordinate to the Cu(B) atom, one of the copper sites in the catalytic core that plays a role for the catalytic function. The hydrogen-bonding network between dopamine and the three amino acid residues (Glu268, Glu369, and Tyr494) plays an essential role in substrate binding and the stereospecific hydroxylation of dopamine to norepinephrine. The dopamine hydroxylation by the Cu(III)-oxo species is a downhill and lower-barrier process toward the product direction with the aid of the protein environment of DBM. This enzyme is likely to use the high reactivity of the Cu(III)-oxo species to activate the benzylic C-H bond of dopamine; the enzymatic reaction can be explained by the so-called oxygen rebound mechanism.     

Importance of cytochromes in cyclization reactions: Quantum chemical study on a model reaction of proguanil to cycloguanil

Proguanil, an anti‐malarial prodrug, undergoes cytochrome P450 catalyzed biotransformation to the pharmacologically active triazine metabolite (cycloguanil), which inhibits plasmodial dihydrofolate reductase. This cyclization is catalyzed by CYP2C19 and many anti‐malarial lead compounds are being designed and synthesized to exploit this pathway. Quantum chemical calculations were performed using the model species (Cpd I for active species of cytochrome and N4‐isopropyl‐N6‐methylbiguanide for proguanil) to elucidate the mechanism of the cyclization pathway. The overall reaction involves the loss of a water molecule, and is exothermic by approximately 55 kcal/mol, and involves a barrier of approximately 17 kcal/mol. The plausible reaction pathway involves the initial H‐radical abstraction from the isopropyl group by Cpd I, followed by two alternative paths‐ (i) oxygen rebound to provide hydroxyl derivative and (ii) loss of additional H‐radical to yield 1,3,5‐triazatriene, which undergoes cyclization. This study helped in understanding the role of the active species of cytochromes in this important cyclization reaction. © 2014 Wiley Periodicals, Inc.     

A density functional study on a biomimetic non-heme iron catalyst: insights into alkane hydroxylation by a formally HO-FeV=O oxidant

The reactivity of [HO-(tpa)Fe(V)=O] (TPA=tris(2-pyridylmethyl)amine), derived from O-O bond heterolysis of its [H(2)O-(tpa)Fe(III)-OOH] precursor, was explored by means of hybrid density functional theory. The mechanism for alkane hydroxylation by the high-valent iron-oxo species invoked as an intermediate in Fe(tpa)/H(2)O(2) catalysis was investigated. Hydroxylation of methane and propane by HO-Fe(V)=O was studied by following the rebound mechanism associated with the heme center of cytochrome P450, and it is demonstrated that this species is capable of stereospecific alkane hydroxylation. The mechanism proposed for alkane hydroxylation by HO-Fe(V)=O accounts for the experimentally observed incorporation of solvent water into the products. An investigation of the possible hydroxylation of acetonitrile (i.e., the solvent used in the experiments) shows that the activation energy for hydrogen-atom abstraction by HO-Fe(V)=O is rather high and, in fact, rather similar to that of methane, despite the similarity of the H-CH(2)CN bond strength to that of the secondary C-H bond in propane. This result indicates that the kinetics of hydrogen-atom abstraction are strongly affected by the cyano group and rationalizes the lack of experimental evidence for solvent hydroxylation in competition with that of substrates such as cyclohexane.     

The catalytic mechanism of peptidylglycine alpha-hydroxylating monooxygenase investigated by computer simulation

The molecular basis of the hydroxylation reaction of the Calpha of a C-terminal glycine catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) was investigated using hybrid quantum-classical (QM-MM) computational techniques. We have identified the most reactive oxygenated species and presented new insights into the hydrogen abstraction (H-abstraction) mechanism operative in PHM. Our results suggest that O(2) binds to Cu(B) to generate Cu(B)(II)-O(2)(.-) followed by electron transfer (ET) from Cu(A) to form Cu(B)(I)-O(2)(.-). The computed potential energy profiles for the H-abstraction reaction for Cu(B)(II)-O(2)(.-), Cu(B)(I)-O(2)(.-), and [Cu(B)(II)-OOH](+) species indicate that none of these species can be responsible for abstraction. However, the latter species can spontaneously form [Cu(B)O](+2) (which consists of a two-unpaired-electrons [Cu(B)O](+) moiety ferromagnetically coupled with a radical cation located over the three Cu(B) ligands, in the quartet spin ground state) by abstracting a proton from the surrounding solvent. Both this monooxygenated species and the one obtained by reduction with ascorbate, [Cu(B)O](+), were found to be capable of carrying out the H-abstraction; however, whereas the former abstracts the hydrogen atom concertedly with almost no activation energy, the later forms an intermediate that continues the reaction by a rebinding step. We propose that the active species in H-abstraction in PHM is probably [Cu(B)O](+2) because it is formed exothermically and can concertedly abstract the substrate HA atom with the lower overall activation energy. Interestingly, this species resembles the active oxidant in cytochrome P450 enzymes, Compound I, suggesting that both PHM and cytochrome P450 enzymes may carry out substrate hydroxylation by using a similar mechanism.     

Oxygen activation by the noncoupled binuclear copper site in peptidylglycine alpha-hydroxylating monooxygenase. Reaction mechanism and role of the noncoupled nature of the active site

Reaction thermodynamics and potential energy surfaces are calculated using density functional methods to investigate possible reactive Cu/O(2) species for H-atom abstraction in peptidylglycine alpha-hydroxylating monooxygenase (PHM), which has a noncoupled binuclear Cu active site. Two possible mononuclear Cu/O(2) species have been evaluated, the 2-electron reduced Cu(II)(M)-OOH intermediate and the 1-electron reduced side-on Cu(II)(M)-superoxo intermediate, which could form with comparable thermodynamics at the catalytic Cu(M) site. The substrate H-atom abstraction reaction by the Cu(II)(M)-OOH intermediate is found to be thermodynamically accessible due to the contribution of the methionine ligand, but with a high activation barrier ( approximately 37 kcal/mol, at a 3.0-A active site/substrate distance), arguing against the Cu(II)(M)-OOH species as the reactive Cu/O(2) intermediate in PHM. In contrast, H-atom abstraction from substrate by the side-on Cu(II)(M)-superoxo intermediate is a nearly isoenergetic process with a low reaction barrier at a comparable active site/substrate distance ( approximately 14 kcal/mol), suggesting that side-on Cu(II)(M)-superoxo is the reactive species in PHM. The differential reactivities of the Cu(II)(M)-OOH and Cu(II)(M)-superoxo species correlate to their different frontier molecular orbitals involved in the H-atom abstraction reaction. After the H-atom abstraction, a reasonable pathway for substrate hydroxylation involves a "water-assisted" direct OH transfer to the substrate radical, which generates a high-energy Cu(II)(M)-oxyl species. This provides the necessary driving force for intramolecular electron transfer from the Cu(H) site to complete the reaction in PHM. The differential reactivity pattern between the Cu(II)(M)-OOH and Cu(II)(M)-superoxo intermediates provides insight into the role of the noncoupled nature of PHM and dopamine beta-monooxygenase active sites, as compared to the coupled binuclear Cu active sites in hemocyanin, tyrosinase, and catechol oxidase, in O(2) activation.     

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.     

The final catalytic step of cytochrome p450 aromatase: a density functional theory study

B3LYP density functional theory calculations are used to unravel the mysterious third step of aromatase catalysis. The feasibility of mechanisms in which the reduced ferrous dioxygen intermediate mediates androgen aromatization is explored and determined to be unlikely. However, proton-assisted homolysis of the peroxo hemiacetal intermediate to produce P450 compound I and the C19 gem-diol likely proceeds with a low energetic barrier. Mechanisms for the aromatization and deformylation sequence which are initiated by 1beta-hydrogen atom abstraction by P450 compound I are considered. 1beta-Hydrogen atom abstraction from substrates in the presence of the 2,3-enol encounters strikingly low barriers (5.3-7.8 kcal/mol), whereas barriers for this same process rise to 17.0-27.1 kcal/mol in the keto tautomer. Transition states for 1beta-hydrogen atom abstraction from enolized substrates in the presence of the 19-gem-diol decayed directly to the experimentally observed products. If the C19 aldehyde remains unhydrated, aromatization occurs with concomitant decarbonylation and therefore does not support dehydration of the C19 aldehyde prior to the final catalytic step. On the doublet surface, the transition state connects to a potentially labile 1(10) dehydrogenated product, which may undergo rapid aromatization, as well as formic acid. Ab initio molecular dynamics confirmed that the 1beta-hydrogen atom abstraction and deformylation or decarbonylation occur in a nonsynchronous, coordinated manner. These calculations support a dehydrogenase behavior of aromatase in the final catalytic step, which can be summarized by 1beta-hydrogen atom abstraction followed by gem-diol deprotonation.     

细胞色素P450催化4-氯-N-环丙基-N-异丙基苯胺Ca-H羟基化反应机理的理论研究

采用密度泛函理论的B3LYP方法,研究了细胞色素P450催化4-氯-N-环丙基-N-异丙基苯胺Ca-H羟基化的反应机理,该反应包含环丙基的羟基化和异丙基的羟基化两个反应途径,且这两个反应路径都是包含氢原子传递的协同过程,二重态的能垒明显低于四重态,反应主要在二重态上进行.通过比较这两个反应路径中Ca-H羟基化反应的活化...     

A predictive pattern of computed barriers for C-h hydroxylation by compound I of cytochrome p450

The communication presents DFT calculations of 10 different C-H hydroxylation barriers by the active species of the enzyme cytochrome P450. The work demonstrates the existence of an excellent barrier-bond energy correlation. The so-obtained equation of the straight line is demonstrated to be useful for predicting barriers of related C-H activation processes, as well as for assessing barrier heights within the protein environment. This facility is demonstrated be estimating the barrier of camphor hydroxylation by P450cam.     

A valence bond modeling of trends in hydrogen abstraction barriers and transition states of hydroxylation reactions catalyzed by cytochrome P450 enzymes

The paper outlines the fundamental factors that govern the mechanisms of alkane hydroxylation by cytochrome P450 and the corresponding barrier heights during the hydrogen abstraction and radical rebound steps of the process. This is done by a combination of density functional theory calculations for 11 alkanes and valence bond (VB) modeling of the results. The energy profiles and transition states for the various steps are reconstructed using VB diagrams (Shaik, S. S. J. Am. Chem. Soc. 1981, 103, 3692-3701. Shaik, S.; Shurki, A. Angew. Chem. Int. Ed. 1999, 38, 586-625.) and the DFT barriers are reproduced by the VB model from raw data based on C-H bond energies. The model explains a variety of other features of P450 hydroxylations: (a) the nature of the polar effect during hydrogen abstraction, (b) the difference between the activation mechanisms leading to the Fe(IV) vs the Fe(III) electromers, (c) the difference between the gas phase and the enzymatic reaction, and (d) the dependence of the rebound barrier on the spin state. The VB mechanism shows that the active species of the enzyme performs a complex reaction that involves multiple bond making and breakage mechanisms by utilizing an intermediate VB structure that cuts through the high barrier of the principal transformation between reactants and products, thereby mediating the process at a low energy cost. The correlations derived in this paper create order and organize the data for a process of a complex and important enzyme. This treatment can be generalized to the reactivity patterns of nonheme systems and synthetic iron-oxo porphyrin reagents.     

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? 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. © 2015 Wiley Periodicals, Inc.     

Monooxygenation mechanism by cytochrome p-450

The substrate oxygenation mechanism by an ultimate species in monooxygenation by cytochrome P-450 (compound I) was investigated by the density functional theory method. An initial model compound was constructed from a structure obtained by 300-ps molecular dynamics simulation of compound I-formed P-450cam under physiologic conditions, and it consisted of porphine for protoporphyrin IX, S(-)-CH(3) for the side chain of Cys357 of the fifth ligand of heme, a methane molecule for the substrate, a heme iron, and an oxygen atom of the sixth ligand of heme. The results of the calculation revealed that the substrate oxygenation mechanism had four elementary processes, i.e., (1) formation of [FeOH](3+) and a substrate radical by hydrogen atom abstraction from the substrate caused by [FeO](3+), (2) rotation of the OH group of the sixth ligand of [FeOH](3+) produced by process 1, (3) substrate radical binding with the [FeOH](3+), and (4) elimination of the oxygenated substrate formed at the sixth ligand binding site. The rate-determining step is process 1, hydrogen atom abstraction from the substrate, and the activation energy was determined to be about 15 kcal/mol. For this reason, it is thought that this reaction occurs in vivo.     

Influence of collision energy on the dynamics of the reaction H (2S) + NH (X3Σ−) → N (4S) + H2 (X1Σ g + ) by the state-to-state quantum mechanical study

The conversion of cholesterol to pregnenolone is a physiologically essential process which initiates with two sequential hydroxylation processes catalyzed by cytochrome P450 side-chain cleavage enzyme (P450_SCC). Extensive efforts have been exerted; however, the mechanistic details remain obscure. In this work, we employed the dispersion-corrected density functional theoretical (DFT-D) calculations to investigate the mechanistic details of such hydroxylation processes. Calculated results reveal that the active intermediate Compound I (CpdI) of P450_SCC hydroxylates cholesterol efficiently, which coincides with previous spectrometric observations. The hydrogen bond effect of water molecule within the active site lowers the energy barrier significantly. Intriguingly, the adjacent hydrogen bond (H-bond) between the hydroxyl group of the substrate and the oxo group of CpdI in the second hydroxylation affects the H-abstraction significantly. Such H-bond was weakened during the C–H bond activation process, increasing the energy barriers by approximately 2 kcal/mol, which is different to the intermolecular H-bond effect of water₉₀₃ found by Shaik et al. that decreases the barrier by about 4 kcal/mol. Such adjacent H-bond also affects the transition state by bending the alignment of the C–H–O moiety, and consequently lowering the kinetic isotope effect values. Besides, a series of DFT-D calculations (Grimme’s D2, D3-zero, and D3-BJ methods) were performed and accessed to find out an appropriate protocol for H-bond containing hydroxylation process. Our results show that DFT-D single-point energies (SPE) based on geometries optimized with non-dispersion-corrected DFT varies drastically and sometime presents unreasonable results. DFT-D SPE calculations on DFT-D optimized geometries present stable and reasonable results.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C?H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C?H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Regioselectivity of aliphatic versus aromatic hydroxylation by a nonheme iron(II)-superoxo complex

Many enzymes in nature utilize molecular oxygen on an iron center for the catalysis of substrate hydroxylation. In recent years, great progress has been made in understanding the function and properties of iron(IV)-oxo complexes; however, little is known about the reactivity of iron(II)-superoxo intermediates in substrate activation. It has been proposed recently that iron(II)-superoxo intermediates take part as hydrogen abstraction species in the catalytic cycles of nonheme iron enzymes. To gain insight into oxygen atom transfer reactions by the nonheme iron(II)-superoxo species, we performed a density functional theory study on the aliphatic and aromatic hydroxylation reactions using a biomimetic model complex. The calculations show that nonheme iron(II)-superoxo complexes can be considered as effective oxidants in hydrogen atom abstraction reactions, for which we find a low barrier of 14.7 kcal mol(-1) on the sextet spin state surface. On the other hand, electrophilic reactions, such as aromatic hydroxylation, encounter much higher (>20 kcal mol(-1)) barrier heights and therefore are unlikely to proceed. A thermodynamic analysis puts our barrier heights into a larger context of previous studies using nonheme iron(IV)-oxo oxidants and predicts the activity of enzymatic iron(II)-superoxo intermediates.     

Is the bound substrate in nitric oxide synthase protonated or neutral and what is the active oxidant that performs substrate hydroxylation?

We present here results of a series of density functional theory (DFT) studies on enzyme active site models of nitric oxide synthase (NOS) and address the key steps in the catalytic cycle whereby the substrate (L-arginine) is hydroxylated to N(omega)-hydroxo-arginine. It has been proposed that the mechanism follows a cytochrome P450-type catalytic cycle; however, our calculations find an alternative low energy pathway whereby the bound L-arginine substrate has two important functions in the catalytic cycle, namely first as a proton donor and later as the substrate in the reaction mechanism. Thus, the DFT studies show that the oxo-iron active species (compound I) cannot abstract a proton and neither a hydrogen atom from protonated L-arginine due to the strength of the N-H bonds of the substrate. However, the hydroxylation of neutral arginine by compound I and its one electron reduced form (compound II) requires much lower barriers and is highly exothermic. Detailed analysis of proton transfer mechanisms shows that the basicity of the dioxo dianion and the hydroperoxo-iron (compound 0) intermediates in the catalytic cycle are larger than that of arginine, which makes it likely that protonated arginine donates one of the two protons needed during the first catalytic cycle of NOS. Therefore, DFT predicts that in NOS enzymes arginine binds to the active site in its protonated form, but is deprotonated during the oxygen activation process in the catalytic cycle by either the dioxo dianion species or compound 0. As a result of the low ionization potential of neutral arginine, the actual hydroxylation reaction starts with an initial electron transfer from the substrate to compound I to create compound II followed by a concerted hydrogen abstraction/radical rebound from the substrate. These studies indicate that compound II is the actual oxidant in NOS enzymes that performs the hydroxylation reaction of arginine, which is in sharp contrast with the cytochromes P450 where compound II was shown to be a sluggish oxidant. This is the first example of an enzyme where compound II is able to participate in the reaction mechanism. Moreover, arginine hydroxylation by NOS enzymes is catalyzed in a significantly different way from the cytochromes P450 although the active sites of the two enzyme classes are very similar in structure. Detailed studies of environmental effects on the reaction mechanism show that environmental perturbations as appear in the protein have little effect and do not change the energies of the reaction. Finally, a valence bond curve crossing model has been set up to explain the obtained reaction mechanisms for the hydrogen abstraction processes in P450 and NOS enzymes.